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THE SALMONELLA FLAGELLAR ANTIGEN ^ ... AS
A POTENTIAL CARRIER OF "FOREIGN EPITOPES"
by
BARBARA JEAN MASTEN, B.S., B.S.
A DISSERTATION
IN
MEDICAL MICROBIOLOGY
Submitted to the Graduate Faculty of Texas Tech University Health Sciences Center
in Partial Fulfillment of the Requirements for
the Degree of
DOCTOR OF PHILOSOPHY
Approved
December, 1993
©1993
BARBARA JEAN MASTEN
All Rights Reserved
ACKNOWLEDGMENTS
I give my thanks to Dr. Terence Joys for chairing my committee and for
providing me the opportunity to engage in this research. I am indebted for his support,
criticisms, and encouragement. My thanks also to Drs. La Jean Chaffin, Charles Faust,
Joe Fralick and Abdul Hamood for serving on my committee. I am likewise grateful to
my Graduate School committee representative, Dr. Randall Jeter, for his participation.
Every member of my committee has stimulated me scientifically and philosophically.
My graduate education encompassed not only science, but also comradeship. I
would like to thank my fellow graduate students, departmental faculty and staff for their
goodwill and lighthearted rapport - thanks especially to Dr Jackie Brown, Dr Lisa
Bums-Keliher and Cathy McVay.
I would like to thank my family for all the encouragement and support they have
provided. To my husband and best friend, Gordon, whom I hold in the highest regards,
I am grateful. He blessed me with his enduring understanding, patience and
encouragement.
Finally, in the words of Daniel 2:23, I record my thanks and acknowledgment of
my heart.
V TABLE OF CONTENTS
ACKOWLEDGEMENTS ii
LIST OF TABLES vi
LIST OF FIGURES viii
I. INTRODUCTION 1
II. MATERIALS AND METHODS 9
General 9
Bacterial Strains 9
Plasmid Vectors 11
Media 11
Ethanol Precipitation of DNA 11
Agarose Gel Electrophoresis 12
Chromosomal Amplification by PCR 13
Genomic DNA Isolation 12
Phenol:Chlorofonm Extraction of Genomic DNA 13
PCR Amplification 14
Purification of PCR Products 14
Sequencing in General 16
Cleaning Sequencing Plates 16
Treatment of Cleaned Sequencing Plates 16
Sandwiching of Treated Sequencing Plates 17
Preparation of Sequencing Gel-Forming Solutions 17
Pouring Sequencing Gels 18
Electrophoresis of Sequencing Gels 18
Developing Exposed Autoradiograms 19
Data Interpretation 19
Data Handling 19
Sequencing of Purified PCR Products 20
Primer Radiolabeling 21
Extension/Termination Reactions 22
Cloning PCR-Generated Products 22
Amplification of ff/C by PCR 22
Annealing and Transformation 23
Screening for Plasmld-lnsert-Containing Colonies 23
Transformation of LC2a 24
Sequencing Cloned Plasmid DNA in LC2a 25
Isolation of Clone Plasmid DNA 26
Primer Radiolabeling 26
Sequencing Reactions 27
Serology 27
Preparation of Agglutinable H Suspension 28
Preparation of Antisera 28
Sera Titration Using H Suspensions 29
Preparation of Absortsing Suspension 29
Preparation of Absorbed H Antisera 30
Motility Inhibition Test 30
Epitope Mapping on Polyethylene Pins 32
Generating Schedules for Synthesis of Peptides 32
Purification of N,N-Dimethylfonnamide with Molecular Sieves 32
Synthesis of Peptides 34
Fmoc-Deprotection and Washing 34
Addition of Fmoc-Amino Acid Active Esters 34
Acetylation of Terminal Amino Groups 35
Side Chain Deprotection and Neutralization 35
ELISA Testing 35
Dismption and Sonication Procedures 36
ELISA Testing Procedure 36
Epitope Mapping by the Novatope System 37
PCR Amplification 38
DNase Shotgun Cleavage 38
Single dA Tailing 39
Ligation to pTOPE T-Vector 40
Transformation Using NovaBlue (DE3) Cells 40
Colony Screening by Protein Expression 41
iv
7
Transformation Using NovaBlue Cells 42
Colony Screening by Hybridization 43
Colony Lysis 43
Oligonucleotide Probe Labeling 43
Hybridization 44
Plasmid Preparation and Screening for Plasmid-lnsert-Containing Colonies 45
Sequencing Clone Plasmid DNA 46
III. RESULTS 47
PCR Amplification, Cloning and Sequencing of fliQ 47
Comparative Analyses of Salmonella g... series serovars 47
Comparative Analyses of Salmonella fliC a,g... series, c, d, i, r, and E.
coli fliC flagelWns 56
Hydropathic Character of Flagellin 56
Evolutionary Relatedness of Salmonella fliC a, c, d, i, r, and g... series and E. co//flagellin 64
Preparation and Titration of Absorbed H Antisera 66
Serological Identities of /C Antigens 74
Epitope Mapping on Polyethylene Pins 82
Epitope Mapping by the Novatope System 82
IV DISCUSSION 86
LITERATURE CITED 92 APPENDICES
A. NUCLEOTIDE ALIGNMENT OF 17 MEMBERS OF THE SALMONELLA fliC FLAGELLIN g... SERIES AND c, d, /, AND r AND THE E. COLI K-12 fliC FLAGELLIN WITH fliC FLAGELLIN a 102
B. AMINO ACID ALIGNMENT OF 17 MEMBERS OF THE SALMONELLA fliC FLAGELLIN g... SERIES AND c, d, i, AND r AND THE E. COLI K-12 fliC FLAGELLIN WITH fliC FLAGELLIN a 114
C. AMINO ACID ALIGNMENT OF 17 MEMBERS OF THE S^LA^OA/ELM/7/C FLAGELLIN g... SERIES 119
ABSTRACT
Salmonella flagella filaments are polymers of a protein termed, 'flagellin'. We
are interested in expressing medically important epitopes at the site(s) of naturally
occurring B-cell epitopes expressed by flagellin. To determine the molecular basis for
expression of the epitopes by which the Salmonella phase-1 g... series flagellin
antigens are distinguished, 17 members (S. adelaide [fliC9^, S. berta [fliC9% S.
budapest [fliC9^, S. califomia [fUCd^rJt]^ S. chaco [fliCdrrt]^ S. danysz [fliCd"^, S. derby
[fliC9f\, S. dublin [fliC9P], S. enteritidis [fliCQrrf], S. essen [fliCd'T^, S. jena [fiiCdn^, S.
monschaui [fliC^^, S. montevideo [fliCd'T^^, S. moscow [fliC9Q], S. oranienberg
[ftiC^, S. mstock [fliC9P% and S. senftenberg [fliC9S^) of this series were selected
and their fliC (the stmctural gene for phase-1 flagellin) genes sequenced. Comparison
of the flagellin amino acid sequences showed complete homology in the N-tenminal
(region I, II and III) and C-terminal (region VIII) segments of the proteins. Differences In
amino acids were found throughout the central portion (regions IV, V, VI and VII) of the
flagellins. No localized area substituted to specify subfactor epitopes could be
identified, suggesting the subfactors of the g... series are conformational at the
molecular level. The amino acids comprising each of the subfactor epitopes were not
definable by sequence analysis. Results of epitope mapping, by two approaches,
support the view that the g... series phase-1 flagellin B-ceil epitopes are
conformational. Based upon amino acid comparison, the fliC gene of S. califomia, S.
monschaui and S. oranienberg may be the gene of choice for substitution(s) since it
may tolerate amino acid interchanges in region V better than the fliC gene of other
members of the g... series. Hydrophobicity and surface exposure plots suggest three
potential sites, two within region IV and one within region VI, for epitope substitution
within the flagellin of any g... series member Phylogenetic analysis and comparative
analysis shows S. califomia, S. monschaui and S. oranienberg to be the most diverse
g... series serovars. A combination of spontaneous mutation, insertion and deletion
most likely generated the antigenic polymorphism seen with salmonellar g... series
flagellins.
VI
LIST OF TABLES
2.1 Salmonella serovars used for genomic DNA preparations 10
2.2 Nucleotide sequences of the ^/Cg... series primers used 15
2.3 Absorption scheme used to prepare factor-specific antisera 31
3.1 Salmonella serovars used for genomic DNA preparations 48
3.2 Nucleotide sequences of the ^/Cg... series primers used 49
3.3 Differences in nucleotides between S. dublin fliC PCR products and recombinant clones, each containing a S. dublin fliC-PCR insert 50
3.4 Comparison of 17 Salmonella phase-1 g... series flagellins 51
3.5 Comparison of 17 members of the Salmonella fliC flagellin g... series on the basis of amino acid (aa) and nucleic acid (na) homologies with the g... series consensus sequence 55
3.6 Comparison of 17 members of the Salmonella fliC flagellin g... series with fliC flagellin a on the basis of amino acid (aa) and nucleic acid (na) homologies 57
3.7 Titration of unabsorbed antisera by a microagglutination test 67
3.8 Titration of factor-specific antisera by a microagglutination test 68
3.9 Titration of S. dublin antisemm before and after absorption to obtain p factor-specific antisemm 69
3.10 Titration of S. moscow antisemm before and after absorption to obtain q factor-specific antisemm 69
3.11 Titration of S. montevideo antisemm before and after absorption to obtain s factor-specific antisemm 70
3.12 Titration of S. rostock antisemm before and after absorption to obtain u factor-specific antisemm 70
3.13 Titration of S. enteritidis antisemm by a microagglutination test before and after absorptions used to obtain m1 factor-specific antisemm 71
3.14 Titration of S. enteritidis antisemm before and after absorption to obtain m1 factor-specific antisemm 72
3.15 Titration of S. oranienberg antisemm by a microagglutination test before and after absorptions used to obtain m2 factor-specific antisemm 73
vii
3.16 Titration of S. oranienberg antisemm before and after absorption to obtain m2 factor-specific antisemm 75
3.17 Titration of S. oranienberg antisemm before and after absorption to obtain t1 factor-specific antisemm 76
3.18 Titration of S. berta antisemm before and after absorption to obtain t2 factor-specific antisemm 76
3.19 Titration of S.senftenberg antisemm before and after absorption to obtain f3 factor-specific antisemm 77
3.20 Titration by a microagglutination test of trial absorptions to obtain f factor-specific antisemm 78
3.21 Titration of S. derby antisemm before and after absorption to obtain anti-
S. denbyf factor-specific semm 79
3.22 Confirmation of the flagellar antigens expressed by Salmonella serovars 80
3.23 Confirmation of the fiagellar antigens expressed by Lc2a-fiiC clones 81
3.24 Location, size and orientation of recombinant clones 85
VIM
y • ^
LIST OF FIGURES
2.1 Protein sequence used to generate sequences of progressive overiapping octameric peptides 33
3.1 Hydrophobicity and surface analysis of S. adeladie, S. berta, and S. budapest flageWins 58
3.2 Hydrophobicity and surface analysis of S. califomia, S. chaco, and S. daynsz flagellins 59
3.3 Hydrophobicity and surface analysis of S. derby, S. dublin, and S. entenf/d/s flagellins 60
3.4 Hydrophobicity and surface analysis of S. essen, S. jena, and S. monschaui f\age\\\ns 61
3.5 Hydrophobicity and surface analysis of S. montevideo, S. moscow, and S. oran/enbe/g flagellins 62
3.6 Hydrophobicity and surface analysis of S. rostock and S. senftenberg, flagellins 63
3.7 Evolutionary relatedness of Salmonella fiagelllns (phase-1 g... series, a, c, d, i, r and Salmonella phase-2 flagellin) and E. coli K-12, HI , H7 and H12 flagellins 65
3.8 Locations of S. moscow fliC inserts from positive and negative reacting recombinant clones screened with moscow antisemm 84
villi
CHAPTER I
INTRODUCTION
Bacteria of the genus Salmonella are Gram-negative, non-sporefonming,
facultatively anaerobic bacilli with simple growth requirements and are usually motile
with peritrichous flagella.
Salmonella are ubiquitous human and animal pathogens which produce
gastroenteritis, septicemia and enteric fever Pathogenic salmonella ingested in food
invade the mucosa in the small and large intestines and produce toxins. An acute
inflammatory response in the intestinal mucosa causes diantiea and may lead to
ulceration and destmction of mucosa. The bacteria can disseminate from the intestines
to cause systemic diseases. Salmonella can be grouped on the basis of host
preference: humans only (i.e., S. typhi associated with typhoid fever), hosts other than
humans, or a broad range of hosts. Because salmonellosis is a zoonosis and has an
enormous animal reservoir, the incidence of nontyphoidal salmonellosis has increased
with the growth of industrialized societies due to changes in animal husbandry, the
mechanism of food processing (particulariy eggs), and the mass distribution of food.
For example, 41 percent of turkeys examined in Califomia, 50 percent of chickens
examined in Massachusetts, and 21 percent of commercial frozen egg whites
examined in Spokane, Washington were contaminated with Salmonella and an
estimated 2 million Americans each year come down with salmonellosis (Giannela,
1991). From the records of the Ministry of Health and Public Health Laboratory
Services (Public Health Laboratory Service, 1989). 97% of the 12,791 isolates of food
poisonings in England and Wales between 1985 and 1987 were due to Salmonella. In
the developing countries, typhoid fever is still a major cause of disease, but in the
United States only about 500 isolates are reported annually.
The most common reservoirs for Salmonella are chickens, turi eys, pigs, and
cows. Other domestic and wild animals also harbor these organisms. Contaminated
food is the major mode of transmission of nontyphoidal Salmonella. Much effort has
been devoted to epidemiological methods which finely differentiate these organisms in
order to investigate outbreaks of salmonellosis and determine the source of infection
and main vehicle of transmission to correct the breakdown In food processing. To
identify a "species" of Salmonella, biochemical reactions are of little value because of
the similarity between individual "species." Serological analysis of surface antigens of
1
2
Salmonella using specific antisera offers clinical and epidemiologic advantages by
differentiating this group of organisms into a large number of "serotypes" (Edwards and
Ewing, 1972; Linberg, 1984).
The identification and labeling of the salmonellar antigens through the use of
absorbed semm was initiated by White in 1925 OA/hite, 1925) and 1926 (White, 1926).
This wori was confirmed and continued by Kauffmann (1941) who modified,
systematized, and extended it to form the "Kauffmann-White" Serological Classification
Schema of the genus Salmonella (KauffmanSalmonella (Kauffmann, 1964).
Three kinds of surface antigens (O, H, and K or Vi) determine an organism's
reaction with specific antisera. The designation O (Ger ohne, without), first applied to
nonswarming (i.e., nonflagellated) forms, is now used as a generic term for the
polysaccharide components of the lipopolysaccharide (LPS) somatic antigen. The O
antigen forms the outer portion of the outer membrane and is important for the
vimlence of salmonella by inhibiting activation of the alternative complement pathway
and decreasing the susceptibility to phagocytosis. The designation H (Ger. hauch,
breath) was first used to describe the growth of Proteus bacilli on the surfaces of moist
agar plates; the film produced by the swarming of this highly motile organism resembles
the light mist caused by breathing on glass. The H, or flagellar antigen is associated
with the filamentous portion of the fiagella and appears to be an important vimlence
factor. Mutants that have lost fiagella seem to have a decreased capacity for survival
and growth in macrophages OA/einstein et al., 1984). Capsular antigens are called K
antigen (Ger. Kapsel), and a specific capsular antigen of S. typhi is called Vi because
of its role in vimlence. The polysaccharide Vi antigen is not suitable for classification
since in certain species it is usually too thin to be seen as a capsule and is rapidly lost
in cultures. Only the former two antigens, O and H, are used in the "Kauffmann-White"
classification and to date over 2,324 serotypes of Salmonella have been identified
(Popoffetal., 1992).
Determination of H antigens is complicated by "Phase Variation," a phenomena
described by Andrews (1922, 1925). Andrews found that if two serotypes of
Salmonella with related H antigens were plated, certain colonies were agglutinated by
antisera derived from both types, whereas other colonies were agglutinated only by
antisemm derived from the homologous type. Furthermore, subsequent inocula
derived from the colonies had a mari ed tendency to retain these properties but
eventually reverted to the mixed characters of the initial culture. He explained that an
individual wild-type population of Salmonella species will contain cells which alternate
3
between two distinct fiagellar serotypes, identical in appearance and physical
character Organisms agglutinated only by homologous antisemm were called the
"Specific Phase" while colonies agglutinated by both homologous and heterologous
antisera were designated as the "Nonspecific Phase." As further Salmonella types
were studied it became apparent that the specific phases of some serotypes often
occurred in other serotypes in combinations with different O antigens or different
nonspecific H antigens. For this reason the Salmonella Subcommittee (Salmonella
Committee, 1940) substituted the designation "Phase-1" and "Phase-2," respectively,
for the specific phase and nonspecific phase.
From genetic studies, Lederberg and lino (1956) showed that the 2 flagellar
phases were specified by 2 independent genes, distant on the chromosome. These
genes were named "H-1" and "H-2" for Phase-1 and Phase-2 genes, respectively, and
recently have been renamed fliC (phase-1) and fIjB (phase-2) (lino et al, 1988). Other
transduction experiments showed that donor FliC (Phase-1) and FIjB (Phase-2)
antigens usually replaced antigens in the corresponding phase of the recipient and that
control of the expression of the different flagellin genes was associated somehow with
the fIjB (H-2) locus (Edwards et al., 1955; Lederberg and lino, 1956). The expression
of fIjB is now known to be regulated by a recombination event involving a speciflc,
invertible segment of DNA (995 base pairs long) located upstream of fIjB and
containing the promoter region of the fIjB operon (Zieg et al., 1977; Silverman et al.,
1979; Zieg and Simon, 1980) Inversion of this segment aligns a promoter and fIjB,
allowing synthesis of the FIjB flagellin and of FIjA (previously named rhi), which
represses fliC transcription. This inversion is mediated by the product hin (previously
named vh2) (Silverman and Simon, 1980; Scott and Simon, 1982), a recombinase,
which is on this segment (Zieg and Simon, 1980). Re-inversion of this region turns off
transcription of fIjB and fIjA genes, allowing the cell again to synthesize FliC flagellin.
To date, over 40 flagellar genes have been identifled which code for
components of the flagellar system (flagellation, motility and chemotactic
responsiveness) with at least 13, known on biochemical grounds and another six
suspected on genetic grounds, encoding stmctural genes (Macnab and Parkinson,
1991). Several other genes are known or suspected to control gene expression,
processes of flagellin export, and processes of flagellum assembly (Macnab and
Pari<inson, 1991). Flagellar system genes are clustered in four regions of the
chromosome (Kutsukake et al, 1988; Sanderson and Roth, 1988) with region I, II, III,
4
and IV containing 3, 2, at least 4, and 1 operons, respectively. Region III contains the
fliC gene and region IV includes the fIjAB operon and the hin gene.
In addition to the serological importance mentioned above, flagella are
especially interesting as they are simple motor organelles whose filaments have
defined geometrical properties imposing stmctural and functional constraints, yet are
able to adopt any one of several discrete helical shapes under appropriate conditions.
DePamphilis and Adier (1971) defined three regions of the flagellar organelle. The
complex basal body, which is embedded in the cell wall, curved hook and helical
filament function as a rotary motor, universal joint and propeller, respectively (Macnab,
1987). The Salmonella flagella fliament is a rigid helical tube, typically 5-10 ^m long
and with an apparent outer diameter of about 20 ^m (Macnab and Aizawa, 1984). The
filament is a self-assembling polymer of thousands of molecules (Namba et al., 1989)
of a single protein, termed "flagellin" (Astbury et al., 1955) specifled by either the fliC
(phase-1) gene or the fIjB (phase-2) gene (lino et al, 1988). The flagellin monomers
are an^anged in 11 vertical rows ("flbrils") parallel to the fliament axis forming a hollow
channel with a diameter of 6 nm (Namba et al., 1989; Jones and Aizawa, 1991). Using
pulse-labeling, Emerson et al. (1970) demonstrated that the filament grows In a polar
fashion, i.e., the subunits are added to the distal end of the filament A hypothesis for
this growth is that the monomers are synthesized inside the cell and then transported
down the length of the filament through the hollow central channel (Silverman and
Simon, 1977). Filament assembly requires the presence of the HAP2 protein (hook-
associated protein 2) which enables fiagellin monomers synthesized within the cell to
assemble onto the filament (Ikeda et al., 1987), while preventing addition of
exogenously added monomers (Homma et al., 1986).
In the resting condition, wild-type filaments nomnally assume a left-handed helix
(called normal-type filament) (Macnab and Koshland, 1974) with a pitch of about 2.5^
m. Counter-clockwise rotation of this left-handed helix exerts force on the cell body,
moving it in some direction, and allowing hydrodynamic forces to align the filaments
into a bundle which serves to propel the bacterium smoothly in one direction (Macnab
and Koshland, 1974). The flagellar bundle appears as a tail behind the swimming cell.
When the basal body changes its direction of rotation from counter-clockwise to
clockwise, the flagellum assumes an alternative right-handed helix (called curiy-type
filament) of smaller pitch whose rotation causes disaggregation of the bundle and
cessation of directional movement so that the bacterium "tumbles" chaotically (Macnab
and Koshland, 1974).
dtt^Mtaaiom
5
Polymorphic transition has been explained by regarding this phenomenon as an
allosteric transition. In models, a fliament is composed of 11 vertical rows of flagellin
subunits and the subunits in each row are assumed to be exclusively in one of two
conformational states, R or L (Asakura, 1970; Calladine, 1978). Co-operative
transitions along one or more rows results in changes in the overall filament shape.
Different combinations of R-rows and L-rows are thought to produce various discrete
helical types (straight, coiled, semi-coiled, curiy (I) and curiy (II)) (Calladine, 1978) with
two distinct straight polymorphs consisting solely of flagellin in one conformation of the
other The coexistence of flbrils in different states in the same fliament produces stress
which is relieved by deformation of the straight fliament into a superhelix (Calladine,
1978).
Polymorphoic forms of filaments may also result from changes in the
environment (i.e., pH, temperature, ionic strength, organic solvent, or viscous flow), or
in the flagellin stmcture. For example, at low pH, filaments undergo a transition from
the normal helical form to the curiy form (Shimada et al., 1975). Changes in flagellin
stmcture, either by mutation (O'Brien et al., 1972; lino and Mitani, 1967; lino and
Oguchi, 1974; Hyman and Trachtenberg, 1991) or the use of amino-acid analogs (lino,
1969) can result in the fliament having one of the polymorphic shapes under nonnal
conditions. When Kanto et al. (1991) analyzed Salmonella typhimurium wild-type
flagellin and flagellins from 17 flagellar-shape mutants, they found by amino acid
sequencing, that the mutational sites were localized to the temninal regions of flagellin.
Mutations which affect the ability of flagellin to polymerize into filaments have also been
localized to the terminal regions of the molecule (Horiguchi et al., 1975; lino, 1977;
Yamaguchi et al., 1984; Homma et al., 1987).
With strict functional constraints placed upon the filament, one would anticipate
only limited possible variations in fiagellin stmcture. However, flagellins differing widely
in molecular weight have been isolated from different bacteria (McDonough and Smith,
1976) and as depicted in the Kauffman-White Scheme, there is great variability In
flagellar antigens. By chemical and immunological analysis of f,g antigen flagellin.
Parish, Wistar and Ada (1969) demonstrated that all the antigenic specificities reside in
the central region of the flagellin polypeptide and from a purely genetic approach, lino
(1977) provided evidence that the central area of the molecule contained the
antigenically variable regions. Previous reports have shown that Salmonella flagellins
with antigens a, c, and d (Wei and Joys, 1985), / (Joys, 1985) and r (Wei and Joys,
1986) consist of extremely conserved temriinal regions with a variable center section.
6
The central area embodies a hypervariable segment, Region IV, of ca. 120 amino acids
(Wei and Joys, 1985). Wei and Joys (1985) found that the predicted amino acid
sequence of Region IV showed no greater than 30% homology for any pairwise
comparison between alleles and suggested this region was not involved in flagella
localization or function, but contributed to antigenic specificity.
The region of an antigen that is recognized by the binding site of an
immunoglobulin is called an antigenic determinant or epitope. Epitopes are usually
classified as either continuous or discontinuous. A continuous epitope is a short, linear
peptide fragment of the antigen that is able to bind to antibodies raised against the
intact protein. A discontinuous epitope consists of a cluster of residues brought
together by the folding of the polypeptide chain. The vast majority of B-cell epitopes
are discontinuous and are usually recognized by antibodies only if the protein molecule
is intact and its confonnation preserved. However, it is possible that about 10% of
antibodies directed to discontinuous epitopes are able to react with linear peptide
fragments of the protein (Amon and Van Regenmortel, 1992). The method most
commonly used for localizing protein epitopes consist of identifying which peptide
fragments of the molecule are able to cross-react with antibodies raised against the
intact protein (Amon and Van Regenmortel, 1992). Synthetic peptides attached to a
solid phase as antigenic probes allows the detection of continuous epitopes (3-8
residues) crossing reacting with antibody to cognate protein. Bacterial cloning library
containing clones, each expressing a peptide derived from the protein under study,
allows the detection of continuous and, depending on the peptide size, discontinuous
epitopes.
As mentioned above, the N- and C-terminal ends of flagellin molecules are
highly conserved whereas the central region, in which antigenic reactivities are
determined, is more variable. A previously identified epitope of flagellar antigen / (Joys
and Martin, 1973) and two major epitopes of antigen d (Joys and Schodel, 1991) were
located within segment IV. These properties led others (Newton et al., 1989; Wu et al.,
1989; Newton et al., 1990) to suggest that flagella might be useful in vaccine
development if part of the hypervariable region could be substituted with a medically
important epitope-specifying oligonucleotide, in correct orientation and reading frame,
resulting in exposure of the epitope at the surface of the flagella. To date, such
substitutions have been limited to a unique restriction site in the stmctural gene for the /
antigen flagellin of Salmonella typhimurium (Joys,T.M. unpublished report in Newton et
al., 1990) and to a 48 base pair segment in region IV (Wei and Joys, 1985) of the
7
stmctural gene for the d flagellar antigen of Salmonella muenchen (Majarian et al.,
1989; Newton et al., 1989; Wu et al.. 1989; Newton et al., 1990; Stocker, 1990; Brey et
al., 1991; Newton et al., 1991; McEwen et al., 1992). which is readily excised by the
restriction enzyme, EcoRV. Results have been only partially successful. The primary
problem seems to be that substitutions in the flagellin cause inability of the molecules
to polymerize into flagellar filaments. In retrospect, the EcoRV site may not be the
most desired insertion site because the EcoR\/-EcoR\/ segment does not overiap any
of the reported (Joys and Schodel, 1991) cf flagellar antigen B-cell epitopes. It occurred
to Joys (1991) that an examination of other flagellins may reveal more suitable
molecules for interchange with known epitopes. The d and / flagellins investigated to
date are both defined (Kauffmann, 1964; Edwards and Ewing, 1972; Le Minor and
Popoff, 1987) by their production of a single major flagellar antigen. Other flagellar
antigens have been divided into sub-factors (Kauffmann, 1964; Edwards and Ewing,
1972; Le Minor and Popoff, 1987), and it would seem a pnon that the existence of such
natural variation might reveal a site at which manipulation could be attempted with less
effect on the molecule's properties. In the Kauffmann-White scheme (Kauffman,
1964), eight major factors (f, g, m, p, q, s, t and u) of the Salmonella phase-1 g antigen
were described and it has been shown that the factor g itself is a complex composed of
two or more of at least five factors, g1 to g5 (Yamaguchi and lino, 1969). Based upon
amino acid composition, McDonough (1965) determined that members of the g... series
(selected serovars with flagellar antigens g,m; g,p and g,s,f) showed greater variability
than single factor flagellins. For this reason, we selected flagellins from the g... series
of Salmonella flagellar antigens in the expectation that the sub-factors which separate
the different serovars would be located in a common region that would make an
efficient site for directed substitutions.
The Salmonella g... series flagellin serotypes selected for analyses are as
follows: S. adelaide (Atkinson, 1943), S. berta (Hormaeche et al., 1938), S. budapest
(Rauss, 1939). S. califomia (Edwards et al., 1950). S. chaco (Savino and Menendez,
1935), S. danysz (Bahr, 1928), S. derby (Peckham. 1923), S. dublin (Jensen, 1892-
1893), S. enteritidis (Gartner, 1888), S. essen (Hohn and Henmann. 1936). S. jena
(Kauffmann. 1930a). S. monschaui (Cariquist and Coates, 1947). S. montevideo
(Honnaeche and Peluffo. 1936), S. moscow (Hicks, 1929), S. oranienberg (Kauffmann,
1930a), S. Aosfoc/c (Kauffmann, 1930b), and S. senftenberg (Kauffmann, 1929).
This dissertation reports the comparison of the amino acid sequences and
phylogenetic analyses for 17 fliC genes that specify the FliC antigens: f,g (S. adelaide),
8
f,g,t (S. berta); g,t (S. budapest); g,m,t (S. califomia); g,m (S. chaco); g,m (S. danysz);
f,g (S. derby);, g,p (S. dublin); g,m (S. enteritidis); g,m (S. essen); g,m (S. jena); m,t (S.
monschaui); g,m,s (S. montevideo); g,q (S. moscow); m,t (S. oranienberg): g,p,u (S.
rostock); and g,s,t (S. senftenberg). A polyethylene pin-based epitope mapping study
of the central portion of the g... series flagellins from S. berta, S. budapest, S. derby, S.
dublin, S. enteritidis, S. montevideo, S. moscow, S. oranienberg, S. rostock and S.
senftenberg, is presented. In addition, an epitope mapping study based on a library of
bacterial clones, each of which expresses a small peptide derived from the flagellin of
S. moscow is reported.
CHAPTER II
MATERIALS AND METHODS
General
Bacterial Strains
Salmonella adelaide ATCC 10718 {fliC '9), S. berta ATCC 8392 (fliC ^'9>^, S.
califomia ATCC 23201 {fliC 9'"^'^, S. chaco ATCC 49214 {fliC 9,fri), S. danysz ATCC
{fliC 9.rn)^ s. derby ATCC 6960 {fliC f>9\ S. dublin ATCC 15480 (fliC 9>P), S. enteritidis
ATCC 13076 {fliC 9,^)^ § e^sen ATCC 49219 {fliC 9.^)^ s jena ATCC 49221 {fliC
9,m), S. montevideo ATCC 8387 [fliC 9.m,S), S. oranienberg ATCC 9239 {fliC '^ and
S. senftenberg ATCC 8400 {fliC 9,s,t) were obtained from the American Type Culture
Collection (Rockville, MD.). S. budapest CDC 23 (fliC 9.t)^ s. moscow CDC 67(fl'/C 9,9)
and S. rostock CDC 66 {fliC 9,P>U) v^ere obtained from the Centers for Disease Control
(Atlanta. GA.). S. monschaui TDH BE-04 {fliC '^•^ was a generous gift from Susan
Gibson (Texas State Department of Health, Austin. TX.). Salmonellar serovars are
listed in Table 2.1. Escherichia coli DH5a (F", end>A/, hsdR17, supE44, lambda-, thl-1,
gryA96, relA1) (Gibco BRL Life Technologies Inc., Grand Island, NY) was used as the
initial host for recombinant plasmids in transformation experiments involving
salmonellar flagellin gene cloning. E. coli (C600 mK" rK", hag ") (Zeig et al., 1977),
called LC2a in our laboratory, was a generous gift from Dr M. Simon (California
Institute of Technology, Pasadena, CA) and was used to detect flagellin production by
recombinant plasmids. NovaBlue (DE3) (Novagen, Inc., Madison, Wl) and NovaBlue
(Novagen, Inc.) cells were used in epitope mapping of the flagellin of S. moscow by the
NovaTope System (Novagen, Inc.). NovaBlue (DE3) competent cells were used as a
host for pTOPE T-vector (Novagen, Inc.) recombinant plasmids in the creation of a
library of bacterial clones, each of which expresses a small peptide derived from the
flagellin of S. moscow, and ultimately examined by immunoscreening. NovaBlue
competent cells, were used as a host for pTOPE T-vector recombinant plasmids in the
creation of a library of bacterial clones, each of which contains a small fragment of DNA
derived from the fliC gene of S. moscow, and ultimately screened by colony
hybridization. NovaBlue (DE3) is an expression host for the T7 expression vector,
pTOPE T since these cells contains a chromosomal copy of the gene for T7 RNA
polymerase. NovaBlue is a cloning host for pTOPE T since these cells do not contain
T7 RNA polymerase.
Table 2.1. Salmonella serovars used for genomic DNA preparations. 10
Serovars
S. adelaide S. berta S. budapest S. califomia S. chaco S. danysz S. derby S. dublin S. enteritidis S. essen S. jena S. monschaui S. montevideo S. moscow S. oranienberg S. rostock S. senftenberg
Source
ATCC 10718 ATCC 8392 CDC 23 ATCC 23201 ATCC 49214 ATCC 49216 ATCC 6960 ATCC 15480 ATCC 13076 ATCC 49219 ATCC 49221 TDH BE-04 ATCC 8387 CDC 67 ATCC 9239 CDC 66 ATCC 8400
Antigenic Formula''
35:f,g:-19.12:f,g,^
1.4.12,27:g,^-4.12:g,m,f 9,12:g,m 9,12:g,m
l,4,[51.12:f,g:[1,2] 19,12,[Vil:g,p:-19.12:g,m:[1,71
l,9,12:g,n7 9,12:g,m 35.m,t
6.7:g,m,s:-9,12:g,g:-6,7:m,^-
19,12:g,p,a:-1.3,19:g,[s],^-
1 Brackets [ ] indicate that the antigen may be absent. An underscore indicates that the bacterial strain has been lysogenized.
11
Plasmid Vectors
Plasmid pAMP 1 (Gibco BRL Life Technologies Inc.) was used as the vector in
transformation experiments involving salmonellar flagellin gene cloning. Plasmid pAMP
1 is a linearized plasmid pSPORT 1 DNA. Plasmid pTOPE T-Vector (Novagen, Inc.)
was the vector used to create a library of recombinant plasmids used in epitope
mapping of the flagellin of S. moscow by the NovaTope System (Novagen, Inc.).
Media
Media were sterilized by autoclaving unless othenvise indicated. Salmonella
serovars were cultured in 2YT broth (1.6% [wt/vol] Bacto-tryptone (Difco Laboratories,
Detroit, Ml), 1.0% [wt/vol] Bacto-yeast extract (Difco Laboratories) and 1.0% sodium
chloride (NaCI) (Sigma Chemical Co.). Cells (DH5a or LC2a) from the transformation
experiments were plated on 2YT plates (1.5% [wt/vol] Bacto-agar (Difco Laboratories)
in 2YT broth) containing 100 ng of ampicillin (Sigma Chemical Co.) per ml. LC2a cells
transformed with pAMP 1 vector-annealed polymerase chain reaction (PCR) product
were selected on the basis of motility in semi-solid medium (0.2% [wt/vol] Bacto-agar
and 100 ^g of ampicillin per ml in 2YT broth). The serological identities of the fliC
antigens were confirmed with semisolid medium (0.4% [wt/vol] Bacto-agar in 2YT broth)
containing factor-specific antisera (for Salmonella serovars) or factor-specific antisera
and 100 ^g of ampicillin per ml (for clones). NovaBlue (DE3) and NovaBlue cells
transformed with pTOPE T-Vector recombinant plasmids were plated on LB agar (1.0%
[wt/vol] Bacto-tryptone, 0.5% [wt/vol] Bacto-yeast extract, 1.0% NaCI and 1.5% [wt/vol]
Bacto-agar) plates containing 50 ^g of ampicillin per ml plus 15 ^g of tetracycline
(Sigma Chemical Co.) per ml. NovaBlue (DE3) and NovaBlue cells transfomied with
pTOPE T-Vector recombinant plasmids were cultured in LB broth (1.0% [wt/vol] Bacto-
tryptone. 0.5% [wt/vol] Bacto-yeast extract, and 1.0% NaCI) containing 50 \x.g of
ampicillin per ml plus 15 ^g of tetracycline (Sigma Chemical Co.) per ml.
Ethanol Precipitation of DNA
One tenth volume cold (4°C) 3M sodium acetate (Fisher Scientific Co., Fairiawn,
NJ), pH 5.2 and 2.5 volumes of 95% cold (-20°C) ethyl alcohol (ethanol) (Aaper Alcohol
and Chemical Co., Shelbyville, KY), were added to the DNA and mixed by vortexing
(S/P Vortex Mixer; American Scientific Products, McGraw Pari<, IL) for 20 seconds. The
mixture was left at -70''C (Ultracold Freezer; Kelvinator, Manitowoc, Wi.) for 30 minutes.
DNA was collected by centrifugation (14000 X g) (Eppendorf Microcentrifuge Model
12
5415; Brinkmann Instmment Co., Westbury, NY) at 4 X for 20 minutes. After
centrifugation, liquid was removed with a fine-tip pasteur pipette and the DNA was dried
under vacuum (Savant Speed Vac Concentrator) for 5 minutes. The dried DNA was
redissolved in an appropriate volume of liquid.
Agarose Gel Electrophoresis
By microwave heating, 0.75% Type I: Low EEO agarose (Sigma Chemical Co.)
in 1 X KADO (Kado and Liu, 1981) electrophoresis buffer was boiled 1 minute. KADO
E buffer, prepared as a 20 X stock solution, contains 0.8 M
trihydroxymethylaminomethane (Tris) (Research Plus Laboratories, Inc., Denville, NJ)
and 40 mM ethylenediamine tetra-acetic acid (EDTA) disodium salt, (BDH Chemicals
Ltd., Poole, England), pH to 7.9 with glacial acetic acid (Malinckrodt Specialty
Chemicals Co., Paris, KY). Volume lost through evaporation was replaced with water
The gel mixture was reheated just to boiling and poured into an apparatus assembled
for gel casting. Two different gel electrophoresis systems were used, depending upon
the number of samples that needed to be mn. The Gibco BRL Horizon 58 Gel
Electrophoresis System was used with a Buchler Instmments Voltage and Current
Regulated D.C. Power Supply Model 1471. The Jordan Scientific Co. Submarine Gel
Electrophoresis System was used with a Bio Rad Model 1410 Power Supply. The
loading buffer for agarose gel electrophoresis contains 30% glycerol (Intemational
Biotechnologies, Inc., New Haven, CT), 0.2% (wt/vol) tetra brom phenol sulfonphthalein
(bromphenol blue) indicator (Allied Chemical, Monistown, NJ) in 1 X KADO E buffer.
DNA samples to be examined and quantified are mn along with lambda DNA Hind III
fragments and pUC18 Hind lll/BAP (Phamriacia LKB Biotechnology, Piscataway, NJ)
standards. Electrophoresis was earned out at 100 mA with 1 X KADO E buffer
containing 0.01^1 2,7-diamino-10-ethyl-9-phenyl-phenanthridinium bromide (ethidium
bromide) (Sigma Chemical Co.) per ml. Gels were laid on a Fotodyne long (304 nm)
wave ultra-violet transilluminator (Fotodyne Inc., New Beriin, Wl) and photographed
using a Polaroid MP4 land camera (Polaroid, Cambridge, MA) with appropriate filters
and Polaroid 667 high speed coateriess instant film (Polaroid Corp.. Cambridge. MA).
Chromosomal Amplification by PCR
Genomic DNA Isolation
The BIO 101 G NOME DNA Kit (BIO 101 Inc.. La Jolla, CA) and protocol were
used with slight modifications. A single colony of bacteria was inoculated in a 15 ml
.saoW-y y
13
plastic tube containing 5 ml 2YT growth media and grown ovemight in a ZTC water
bath shaker (Gyrotory Water Bath Shaker; New Bmnswick Scientific. Edison. NJ) with
225 rpm shaking. Cells were pelleted by centrifugation (2550-3400 X g) (Model GLC
2b Centrifuge; DuPont Sorvall. Wilmington, DE) at room temperature for 5 minutes.
The supematant was removed and 2 ml of cell suspension solution was added and the
stock solution mixed until it appeared homogeneous. Fifty-five 1 of RNase mixx was
added, the stock solution thoroughly mixed, followed by the addition of 35 1 cell
lysis/denaturing solution, after which the stock solution was once again mixed well. If a
precipitate fomried in the stock solution, the stock solution tube was placed in a 60°C
water bath for 10 to 20 minutes. The stock solution was then incubated in a 55°C water
bath for 30 minutes, at which time 1 ml "salt-out" mixture was added and the
suspension gently yet thoroughly mixed. The stock solution was divided into 1.5 ml
centrifuge tubes and centrifuged (14000 X g) for 2 minutes. The supematant was
carefully collected with a sterile plastic transfer pipette (Bio-Rad Laboratories,
Richmond, CA) and transfen-ed to a clean 15 ml plastic tube. The pellet was discarded.
If a precipitate remained in the supematant, the collected supematant was centrifuged
until clear and then transfen-ed. To the collected supematant, 50 1 spooling salts and
6 ml of 100% (-20°C) ethanol were added. The ethanol phase was gently stirred into
the DNA/aqueous phase with a clean glass rod. The genomic DNA collected on the
rod was gently blotted on a Kimwipe to remove any ethanol present and dissolved in
200 \3\ sterile water in a 1.5 ml microcentrifuge tube. The collected genomic DNA
dissolved in the water within 24 hours and was stored at -20°C (Scientific Freezer;
VWR, San Francisco. Ca.).
Phenol:Chloroform Extraction of Genomic DNA
Two hundred il of TES buffer (50 mM Tris/HCL pH 8.0. 5 mM EDTA and 50 mM
NaCI) and 600 1 of phenol:chloroform premlxed with isoamyl alcohol (Amresco. Solon.
OH) were added to 100 il isolated genomic DNA in a 1.5 ml microcentrifuge tube and
mixed by vortexing for 1 minute. The mixture was centrifuged (14000 X g) at room
temperature for 5 minutes. The upper layer was collected with a sterile plastic transfer
pipette and transferred to a clean tube. One hundred 1 of TES buffer was added to
the lower layer and mixed for 20 seconds. The mixture was centrifuged (14000 X g) at
room temperature for 5 minutes. The upper layer was transferred to the tube
containing the previously collected upper layer and the lower layer was discarded. To
the collected solution. 800 1 of a 24:1 chloroform (HPLC grade) (Aldrich Chemical Co..
14
Inc.. Milwaukee. WI):isoamyl alcohol (Sigma Chemical Co.) was added followed by a 20
second vortex. The upper layer was again collected into a new tube and concentrated
by ethanol precipitation.
PCR Amplification
For amplification of the fliC gene for sequencing, the GeneAmp PCR Reagent
Kit (Peri<in-Elmer Cetus, Norwalk. CT) was used along with 20-mer primers (Table 2.2).
synthesized by Midland Certified Reagent Co. (Midland. TX). derived from the upstream
and downstream regions of the fliC gene: sense primer PCR1S, -241 bp upstream
(Szekely and Simon, 1983), and antisense primer PCR1AS, -84 bp downstream (Wei
and Joys, unpubl.). PCR amplification was done in a final volume of 100 il containing
100 ng of chromosomal DNA and overiaid with 100 1 of mineral oil (Sigma Chemical
Co.). When using ISS thermal cycler tubes (ISS, Natick, MA), no oil was added. The
amplification buffer contained 10 1 of [10X] reaction buffer, 200 nM dATP, 200 iM
dCTP, 200 iM dGTP and 200 ^M dTTP. Primers were added to a final concentration
of 1 iM. AmpliTaq DNA polymerase was added at a final concentration of 2.5 Units per
100 111. After an initial denaturation at 95''C for 3 minutes, amplification was perfomned
for 29 cycles on a DNA thermal cycler (Tempcycler Model 50/60; Coy Laboratory
Products, Ann Arbor, Ml). The cycle consisted of a denaturation at 94°C for 1 minute, a
primer annealing step at 55°C for 1 minute, and an extension step at 72°C for 2
minutes. After the last cycle, the sample was mn through a modified cycle in which the
polymerization step was extended to 5 minutes. The sample was then held at 4°C. To
visualize the PCR product, after amplification 5 |il of sample was electrophoresis in a
0.75% agarose gel along with lambda DNA Hind III fragments (Gibco BRL Life
Technologies, Inc.) sample.
Purification of PCR Products
PCR amplified DNA was purified using the Promega Magic PCR Preps DNA
Purification System (Promega, Madison, Wl) and protocol for direct purification from
PCR reactions. To a 12 X 75 mm polypropylene tube, 100 1 of direct purification
buffer and 30 - 300 il of PCR reaction was added and mixed by vortexing for 5
seconds. One ml of Magic PCR Preps resin was added to the tube and briefly mixed 3
times over a 1 minute period. The Magic PCR Preps DNA purification resin containing
the bound DNA was transfen-ed using a pipette to a plastic 3 cc syringe (Becton,
Dickinson and Co., Rutherford, NJ) ban-el connected to a Promega mini-column. The
15
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16
plastic syringe plunger was inserted and the slurry was gently pushed into the mini-
column. The syringe ban-el with plunger was disconnected from the mini-column and
the ban-el reconnected without the plunger. The mini-column was washed with 2 ml of
Magic PCR Preps column wash solution by gently pushing the column wash through
the mini-column with the syringe plunger The syringe barrel was removed, the mini-
column transfen-ed to a 1.5 ml microcentrifuge tube and centrifuged (14000 X g) at
room temperature for 20 seconds. The mini-column was transfen-ed to a clean 1.5 ml
microcentrifuge tube and the bound DNA fragment was eluted by adding 50 nl of sterile
water for 1 minute followed by centrifugation (14,000 X g) for 20 seconds. The purified
DNA was examined and quantified by electrophoresis in a 0.75% agarose gel with
lambda DNA Hind III fragments and pUC18 Hind lll/BAP (Phannacia LKB
Biotechnology, Piscataway, NJ) standards. The purified DNA was stored at -20°C.
Seouencino in General
Cleanino Seouencino Plates
Since the sequencing gels are covalently linked to and dried on to the small
glass plate, both the large (41.9 cm x 33.3 cm) and small (39.4 cm x 33.3 cm) glass
sequencing plates must be thoroughly cleaned before use. After removing the previous
gel from the small plate, both the large and small plates were wiped free of grease with
95% ethanol and scmbbed with an S.O.S. extra thick steel wool soap pad (Miles Inc.,
Chicago. IL). The plates were rinsed with distilled water, wiped on both sides with a 5%
wt/vol potassium hydroxide (KOH) (Matheson, Coleman & Bell Manufacturing Chemists,
NoHA ood, OH)/methyl alcohol (Aldrich Chemical Company, Inc.) solution to remove the
silicone, then rinsed once again with distilled water After placing one plate, with the
side that will contact the gel facing upwards, into a 2N sodium hydroxide (NaOH)
(Fisher Scientific Co.) bath, 50 - 75 g NaOH pellets were evenly distributed onto the
plate along with two glass rod separators. The next plate is loaded the same way as
the first. After soaking ovemight, both plates are thoroughly rinsed with distilled water
and either blot dried or air dried.
Treatment of Cleaned Sequencing Plates
The small plate, treated with Bind-Silane (Pharmacia LKB Biotechnology), is the
one to which the gel will be covalently bonded, while the large plate, treated with
Sigmacote (Sigma Chemical Co.), is the one which must repel the gel. The small plates
were treated with Bind-Sllane according to manufacturer's instmctions. Eight ml
17
Bind-Silane was added to 2000 ml distilled water, adjusted to pH 3.5 with acetic acid,
and mixed with a magnetic stin-er until the solution was clear. Each small plate was
immersed, with the side that will contact the gel facing upwards, in the Bind-Silane
solution for 60 minutes. Treated plates were removed, rinsed with distilled water and
either blot dried or air dried. The large plates were treated with Sigmacote according to
manufacturer's instmctions. The large plate was placed on a lab countertop with the
side that will contact the gel facing upwards. The plate was wiped with a Kimwipe
soaked with ethanol and Kimwipe dried. Two to three ml of Sigmacote was pipetted
down the center of the plate and immediately evenly wiped over the surface. After
drying, the applied Sigmacote was buffed with a Kimwipe and wiped with 95% ethanol
and buffed dry.
Sandwichino of Treated Sequencing Plates
A large and small sequencing plate were placed, treated side up, on a bench
towel. Using a syringe filled with high vacuum grease (Dow Coming Corp., Midland,
Ml), a line of grease was deposited 0.2 - 0.3 cm along the sides and bottom of the large
plate. A dean 0.4 mm end spacer (Gibco BRL Life Technologies Inc.) and two clean
0.4 mm side spacers (Intemational Biotechnologies, Inc.) were placed over the grease
lines with a 1/8 inch gap between the side spacers and the end spacer A line of
grease was deposited on the spacers with a daub of grease filling the 1/8 inch gap.
The small plate, treated side down, was placed on the spacers. Three large binder
clips (Charies Leonard Inc., Chicago, IL) were evenly applied to the positioned end
spacer The side spacers were adjusted so that the side and end spacers met with the
grease block joining them. Three small binder clips (Charies Leonard Inc.) were evenly
applied to the positioned side spacers. The plate sandwich was placed at a slightly
tilted 45° angle for gel pouring.
Preparation of Seouencing Gel-Fonmino Solutions
Hydrolink Long Ranger (AT Biochem, Inc., Malvem. PA) gel-fomning solution
was used to prepare a 5% and 8% gel following the 'MAX Readability 1.2 X T gel
instmctions. Ten X TBE was prepared by reconstituting one package of UltraPure Gel-
Mix Running Mate TBE buffer (Gibco BRL Life Technologies Inc.) In 99 ml of distilled
water Gel solution was prepared in a final volume of 100 ml using a 250 ml glass
beaker containing 42 g urea (ammonium-free, absolute grade) (Research Plus
Laboratories. Inc.). 12 ml of 10 X TBE buffer, and 10 ml or 16 ml of Hydrolink Long
18
Ranger for a 5% or 8% gel. respectively. The gel solution was mixed with a magnetic
stin-er until dissolved and filtered with a 0.2 ^m Nalgene disposable sterilization filter
(Nalge Co.. Rochester. NY).
Pourinq Sequencinq Gels
Five hundred 1 of 10% ultra pure ammonium persulfate (electrophoresis grade)
(Gibco BRL Life Technologies Inc.) and 50 1 of ultra pure N'.N'.N', N'-
tetramethylethylenediamine (TEMED) (electrophoresis grade) (Gibco BRL Life
Technologies, Inc.) were added to the filter gel solution and mixed with a magnetic
stirrer for 30 seconds. The gel solution was transfen-ed to a 60 cc syringe connected to
a canula tip. The tip was positioned and the gel solution slowly added along one side
of the plate sandwich which was placed at a slightly tilted 45° angle. Any bubbles
formed were removed by tapping the plate sandwich with pliers. After filling, the plate
sandwich was laid at a 15° angle and 0.4mm shari<stooth sequencing combs (Gibco
BRL Life Technologies, Inc.) were inserted and clamped in place. Gels were allowed to
polymerize at least 60 minutes before use.
Electrophoresis of Sequencing Gels
Ten X TBE buffer was prepared by reconstituting one package of UltraPure Gel-
Mix Running Mate TBE Buffer In 99 ml of distilled water. A mnning buffer of 0.6 X TBE
was used. The gel-plate sandwich was mounted on a Gibco BRL Model S2
Sequencing Apparatus according to manufacturer's instmctions. A DAN-KAR Corp.
(Reading, MA) Electrophoresis Power Supply was used to carry out the electrophoresis.
Before pre-mnning, each loading well was rinsed using a syringe filled with buffer
After a 10 minute pre-mn, the wells were rinsed with buffer, and a 3 1 sample was
loaded. An AT Biochem Hydrolink temperature strip was used to monitor gel
temperature. Sequencing gels were mn between 45-49°C. Once the electrophoresis
was completed, the power supply was disconnected and the glass sandwich removed
and placed on a bench towel. Using a spatula, the sandwich was separated and the
large plate and spacers removed. The small plate with the bound gel was placed, for
30 minutes, in a fixative bath containing 2 liters of 10% glacial acetic acid and 20%
methyl alcohol. After fixing, the plate was placed in a 4 inch-deep water bath for
4 minutes, gently dried with paper towels, and transferred to a Fisher Isotemp Oven
200 Series Model 230F preheated to 80°C. After drying for 2 hours, the plate was
removed from the oven and allowed to cool before exposing ovemight to X-Omat AR
19
film (Eastman Kodak Co., Rochester, NY). A Fisher Biotech Electrophoresis System
Autoradiograph Cassette FBAC 1417 or a Kodak X-ray exposure holder were used.
Developing Exposed Autoradiograms
Exposed autoradiograph film was placed in GBX developer and replenisher
(Eastman Kodak Co.) for 7 minutes, a 3.57% glacial acetic acid stop bath with constant
agitation for 30 seconds, and then into GBX fixer and replenisher (Eastman Kodak Co.)
for 2 minutes after a 5 second agitation. The developed autoradiogram was washed
with mnning water for 20 minutes before air drying.
Data Interpretation
The images (bands) on the autoradiogram resulted from radiolabeled-primed
DNA chains which were prematurely terminated due to the incorporation of a 2',3'
dideoxynucleotide In place of the nomfial deoxynucleotide. The bands appear as
horizontal lines in each of the four dideoxynucleotide lanes, each lane representing a
separate enzymatic reaction. The absence of a 3'-hydroxyl residue prevents the
formation of a phosphodiester bond with the succeeding deoxynucleotide, so each
band represents the 3'-terminal nucleotide of the dideoxynucleotide used.
Data Handling
The sequences, sense and antisense, obtained from the autoradiograms were
entered and stored in spreadsheet form in a BI-LInk 386 25mHz computer system (Bl-
Link Computer, Inc., Whittler, CA) using Microsoft Excel (Microsoft Corporation,
Redmond, WA). Each serovar's sense and antisense strands were saved in text
fonnat, allowing other programs to access the data. The Mount and Conrad (Mount
and Conrad, 1984) program was used to make a DNA complementary strand from the
antisense strand data and make a translation product from the sense strand data.
Using the Pearson HMATCH executable file which came with the Mount and Conrad
program, the complementary strand obtained from the antisense data was compared to
the sense strand data to confirm agreement Using the sense strand data, nucleic acid
alignments were accomplished using the Pearson HMATCH executable file. Amino
acid alignments were accomplished using the Lipman and Pearson FASTP (Lipman
and Pearson, 1985) program. An MTRANS (Masten, 1992) program was used to
transfer files, sense strand data or translation products, from a Mount and Conrad
format to an Excel spreadsheet for nucleotide or amino acid comparisons based on
20
obtained alignment data. Using the PHYLIP (Phylogeny Inference Package) Version
3.4 PROTPARS (Protein Sequence Parsimony Method) (Felsenstein, 1991) program,
an unrooted phylogeny tree from protein sequences was obtained. Protyize (Scientific
& Educational Software, State Line, PA), a protein stmcture prediction program was
used to prepare hydrophobicity and surface exposure plots. When necessary, file
editing was accomplished using Microsoft Windows version 3.1 Notepad Editor
(Microsoft Corp.). Micrografx Window Draw (Micrografx, Inc., Richardson, TX) and
Microsoft Word for Windows (Microsoft Corp.) was used for graph preparation. The
AUTHORIN (Intelligenetics/GenBank, Los Alamos, NM) program was used to submit
the nucleotide sequences of the fliC flagellar proteins to GenBank (Los Alamos, NM).
Their ultimate deposition was in the EMBL Data Library (Heidelberg, Gennany) with the
following accession numbers: S. berta, Z15064 ('BERTA'); S. budapest, Z15065
('BUDA'); S. derby, Z15066 ('DERBY); S. dublin, Z15067 ('DUB'); S. enteritidis, Z15068
('ENTER'); S. montevideo, Z15069 ('MONT'); S. oranienberg, Z15070 ('ORAN'); S.
rostock, Z15071 ('ROS'); S. senftenberg, Z15072 ('SENF'); and S. moscow, Z15086
CMOS'.
Seouencing of Purified PCR Products
Purified PCR products were directly sequenced using the fmol DNA Sequencing
System (Promega) and protocol with walking primers (Table 2.2) end-labeled with [y^2.
P]ATP (New England Nuclear, Boston, MA). The fmol DNA Sequencing System is
based upon the Sanger dideoxy-mediated chain termination method (Sanger et al.,
1977) of DNA sequencing. The Sanger sequencing method depends upon the use of
2',3'-dideoxynucleoside triphosphates (ddNTPs) which can be incorporated by DNA
polymerase into a growing DNA chain through their 5' triphosphate group, but due to an
absence of a 3'-hydroxyl residue, prevent fonnation of a phosphodiester bond with the
succeeding deoxynudeoside triphosphate (dNTP). A small amount of one ddNTP is
included with the four conventional dNTPs in a reaction mixture for DNA synthesis.
There is competition between extension of the DNA chain and infrequent, but specific,
termination, yielding products which are a series of oligonucleotide chains whose
lengths are detemined by the distance between the terminus of the primer used to
initiate DNA synthesis and the sites of premature termination. By using the four
different ddNTPs in four separate enzymatic reactions, populations of oligonucleotides
are generated that teminate at positions occupied by every A, C, G, or T in the
template strand. Newly synthesized chains are separated on a sequencing gel. The
^
21
nudeotide sequence of each PCR product was detennined in both orientations.
Starting primers (Midland Certified Reagent Co.) were derived from the upstream and
downstream regions of the fliC gene: sense primer BM1S, -61 bp upstream (Szekely
and Simon, 1983), and antisense primer BM1AS, -41 bp downstream (Wei and Joys,
unpubl.). Progressive walking primers (Midland Certified Reagent Co.), BM2S - BM8S
and BM2AS - BM8AS, were derived as sequences were determined.
Primer Radiolabeling
Primers were labeled using the fmol DNA Sequencing System (Promega) and
protocol. Using a 0.6 ml microcentrifuge tube stored on ice, primer end-labeling was
accomplished in a final volume of 10 1 containing 10 pmoles primer, 10 pmoles of
adenosine-5' triphosphate tetra (triethylammonium) salt [y^2p] . (6000 curies per
milimole) in 0.01 M tricine (N-tris [hydroxymethyl] methylglydne) (New England Nudear,
Boston, MA), 1 il T4 polynucleotide kinase 10X buffer and 5 Units of T4 polynudeotide
kinase. This protocol was designed to label enough primer for 6 sets of double-
stranded sequencing reactions and can be scaled proportionately according to the
number of reactions to be performed. After mixing gently and a brief spin (14000 X g),
the labeling reaction was incubated in a dry heat block at 37°C for 30 minutes followed
by a kinase inactivation step in a dry heat block at 90°C for 2 minutes. The labeling
reaction was placed on ice for 5 minutes, then centrifuged (14000 X g) briefly to collect
condensation. The end-labeled primers were either used immediately or stored at -
20°C for up to 2 weeks, depending on the age of the y22p
Extension/Tennination Reactions
For each of the sequencing reactions, four 0.6 ml microcentrifuge tubes (A, C,
G, T) were labeled, placed on ice, and the appropriate diluted d/ddNTP was added.
Using a 0.6 ml tube stored on ice, the primer/template mix was prepared in a final
volume of 16 .1 containing 40 fmoles of purified PCR product, 4.25 1 fmol sequencing
5X buffer (Promega), and 1.5 jil (1.5 pmoles) labeled primer One 1 of sequencing
grade Taq DNA polymerase (5U/ il) was added to the primer/template solution and
mixed briefly by pipetting up and down. The reaction tubes were centrifuged (14000 X
g) briefly. Four 1 of the enzyme/primer/template mix was added to the inside wall of
each tube containing appropriately diluted d/ddNTP and gently mixed by tapping. The
reaction tubes were centrifuged briefly (14,000 X g). The reaction tubes were placed in
a programmable oven (Integrated Separation Systems, Hyde Parte, MA) that had been
_ ^
22
preheated to 95°C. After the reaction tubes were held at 95°C for 2 minutes,
amplification and extension was performed for 30 cydes. The cyde consisted of a
denaturation at 95°C for 30 seconds, a primer annealing step at 42°C for 30 seconds,
and an extension step at 70°C for 1 minute. After the last cycle, the reaction tubes
were held at room temperature. The reaction tubes were removed from the oven and
centrifuged (14000 X g) briefly to collect condensation and placed on ice. Three (J of
fmol sequencing stop solution was added to the inside wall of each tube followed by a
brief spin to temriinate the reaction. Before loading 3 il of each reaction on a
sequencing gel, the reactions were heated in a dry heat block at 70°C for 2 minutes.
Cloning PCR-Generated Products
Unless indicated othenvise, all recombinant DNA techniques used were
described by Sambrook et al. (1989). The fIC gene was originally cloned using the
CloneAmp System (Gibco BRL Life Technologies Inc., Grand Isaind, NY) with
Subdoning Efficiency DH5a competent cells (Gibco BRL Life Technologies Inc.) and
according to the manufacturer's instmctions. The CloneAmp System utilizes uracil DNA
glycosylase (UDG) to facilitate directional cloning of PCR products. With this system,
DNA to be cloned is amplified by PCR using primers having a specified 12-base 5'
sequence that contains dUMP residues in place of dTMP. The PCR products then
contain the dUMP residues at each 5' terminus. Treatment with UDG renders dUMP
residues abasic. dismpting base-pairing which results in 3' protmding termini. Plasmid
pAMP 1 vector, which contains 3* ends that are complementary to the 3' protmding
termini of the UDG-treated amplification product, and UDG are both added to the
amplification product. UDG action and annealing of the PCR produ<::t to the vector
occur simultaneously, producing recombinant molecules ready for transformation.
Plasmid DNA containing the insert were ultimately transformed into an E. coli strain,
LC2a.
Amplification of fliC bv PCR
For amplification of the fliC gene for doning, primers PCR2S and PCR2AS,
which contain the same sequence as primer PCR1S and primer PCR1AS, respectively,
with the addition of CUACUACUACUA to their 5' ends were used. Primers were
prepared by the Midland Certified Reagent Company. Conditions for PCR amplification
and PCR product purification followed the same protocol as "Chromosomal
Amplification by PCR" and "Purification of PCR Products" mentioned above.
23
Annealing and Transformation
Using a 0.6 ml microcentrifuge tube stored on ice, annealing was performed in a
final volume of 19 il containing 50 ng purified PCR product, 50 ng pAMP 1 vector DNA
(Gibco BRL Life Technologies Inc.). 1 X (final concentration) GeneAmp buffer (Peri<in
Elmer Cetus) and 1 Unit of uradi DNA glycosylase (Gibco BRL Life Technologies Inc.).
The annealing reaction was mixed by tapping, briefly centrifuged (14000 X g) and
incubated In a water bath at 37°C for 30 minutes. Four 1 of annealing mix was added
to 100 1 of competent DH5a cells in a 15 ml plastic tube on ice by moving the pipette
through the cells while dispensing. After incubating the transfomriation mixture on ice
for 30 minutes, the cells were heat shocked in a 37°C water bath for 45 seconds, then
placed on ice for 2 minutes. Nine hundred and fifty il of room temperature UltraPure
S.O.C medium (Gibco BRL Life Technologies Inc.) was added and the transformation
reaction incubated for 1 hour in a 37°C water bath with 225 rpm shaking. After
expression the cells were mixed briefly and 100 J of the transformation reaction was
spread on 2YT plates containing 100 ig of ampicillin per ml. The remaining
transformation reaction was pelleted by centrifugation (2550-3400 X g), resuspended in
10 nl of 2YT broth containing 100 ^g of ampicillin per ml, and spread on 2YT plates
containing 100 |ig of ampicillin per ml. The inoculated plates were incubated ovemight
in a 37°C incubator (Model 1510E Incubator; VWR, San Francisco, Ca.).
Screening for Plasmid-lnsert-Containing Colonies
Colonies were selected and analyzed for plasmid insert size by rapid plasmid
preparation, Miniprep Kit Plus (Pharmacia LKB Biotechnology, Piscataway, NJ),
followed by a double restriction enzyme digestion. Procedure 'A' of the Alkaline Lysis
Miniprep protocol was followed with slight modification. A single colony of bacteria
inoculated in a 15 ml plastic tube containing 5 ml of 2YT growth media with 100 ig of
ampicillin per ml was grown ovemight in a 37°C water bath with 225 rpm shaking. One
and four tenths ml of the ovemight culture was transfen-ed to a 1.5 ml microcentrifuge
tube and centrifuged (14000 X g) for 1 minute. The supematant was removed with a
sterile plastic transfer pipette and 100 1 of cold solution I was added to the pellet. The
pellet was resuspended by vigorous vortexing and incubated on ice for 5 minutes. Two
hundred \i I of solution 11 was added, mixed by inversion and incubated on ice for
5 minutes. One hundred and fifty nl of solution III was added, mixed by inversion,
incubated on Ice for 5 minutes, and centrifuged (14000 X g) at room temperature for 5
minutes. The supematant was transfen-ed, with a sterile plastic transfer pipette, to a
24
dean 1.5 ml microcentrifuge tube or the pelleted predpitate was removed carefully with
a sterile toothpick. Four hundred and fifty 1 of room temperature anhydrous
isopropanol (Sigma Chemical Co., St Louis, MO) was added to the supematant, mixed
by inversion, and incubated at room temperature for 10 minutes. The DNA was
pelleted by centrifugation (14000 X g) at room temperature for 5 minutes, the
supernatant was removed with a transfer pipette, and 1 ml of 70% cold (-20°C) ethanol
was gently added and removed. The DNA pellet was dried under vacuum for 5
minutes. The dried DNA was redissolved in 20 \i\ of TE buffer (50 mM Tris/HCL pH 8.0
and 5 mM EDTA). Using a 0.6 ml microcentrifuge tube stored on ice, the first restriction
enzyme digestion was performed in a final volume of 10 nl containing 5 nJ Miniprep
plasmid DNA, 1 1 One-Phor-AII Buffer Plus 10X (Phamriada LKB Biotechnology), 1 ng
acetylated nudease-free BSA (Promega) and 10 units of Sma I (Pharmacia LKB
Biotechnology). The digestion reaction was performed in duplicate. The digestion
reaction was mixed by tapping, briefly centrifuged (14000 X g). then incubated for 1
hour in a 30°C water bath. After inactivating the Sma I enzyme by incubating the
digestion reaction at 65°C for 20 minutes. 10 units of Bam HI (Pharmacia LKB
Biotechnology) was added to only one digestion reaction, gently mixed by tapping,
centrifuged (14000 X g) briefly, and incubated for 1 hour in a 37°C water bath. 2 d of
0.1% bromphenol blue was added to each digestion mixture and 10 il samples were
analyzed on a agarose gel. The Sma I only digestion and the Sma I followed by Bam
HI digestion were analyzed side-by-side on the agarose gel.
Transformation of LC2a
Plasmid DNA containing the insert was isolated using the Phamnada Miniprep
Kit Plus as described above and transformed to a non-flagellated strain of E. coli,
termed LC2a. The transfonnation buffer and MnCl2 solution were prepared the day of
transformation. The transformation buffer was prepared with 2.72 g sodium acetate
trihydrate (Sigma Chemical Co.), 1.67 g caldum chloride anhydrous (J.T. Baker
Chemical Co., Phillipsburg, NJ) and 500 ml distilled water and the buffer adjusted to pH
5.6 with glacial acetic acid (Malinckrodt Specialty Chemicals Co.). The buffer was
stored on ice. Using a cold (-20°C) graduated cylinder, 100 ml of cold (on ice) buffer
was added to a 150 ml glass beaker containing 1.39 g of manganese chloride (Sigma
Chemical Co.). The MnCl2 solution was mixed with a magnetic stin-er until dissolved
and filtered with a cold (-20°C) 0.2 micron Nalgene disposable sterilization filter (Nalge
Co.) and placed on ice until needed. A single colony of LC2a inoculated in a 15 ml
25
plastic tube containing 5 ml of 2YT growth media was grown ovemight in a 37°C water
bath with 225 rpm shaking. One ml of the ovemight culture was added to a 500 ml
baffled flask (Belico Biotechnology, Vineland, NJ) containing 100 ml of 2YT broth and
incubated in a 37°C water bath with 225 rpm shaking until an optical density (OD) of
0.145 at 590 nm was attained. The growth was monitored using a Spectronic 20
spectrometer (Bausch and Lomb, Rochester, NY) and 6 ml samples. When the OD590
reached 0.145, 25 ml of cell culture each was transfen-ed to two SS-34 tubes (DuPont
Sorvall, Wilmington, DE) and centrifuged (RC-5 Superspeed Refrigerated Centrifuge;
DuPont Sorvall, Wilmington, DE) at 6785 X g for 10 minutes at 4°C. The supematant
was removed and the pellet was resuspended in 2.5 ml cold (on ice) MnCl2 solution,
pooled Into one tube and incubated on ice, with occasional swiriing, for 20 minutes.
The competent cells were centrifuged at 6785 X g for 10 minutes, the supematant
removed, and the pellet resuspended in 1.7 ml cold (on ice) MnCl2 solution. Two
hundred p,i of competent cells were dispensed into cold (on ice) sterile 15 ml plastic
tubes and 5 1 of Miniprep DNA, sterilized with a Spin-X low binding 0.45 ^m cellulose
acetate centrifuge fliter unit (Costar, Cambridge MA), was added by moving the pipette
through the cells while dispensing. After the transformation mixture was incubated on
ice for 1 hour, cells were heat shocked in a 37°C water bath with swiriing for 2 minutes.
One ml of 2YT broth was added, the cells mixed by vortexing, and incubated for 1 hour
in a 37°C water bath with 225 rpm shaking. After expression the cells were vortex
briefly and 100 pi of a 1:10 dilution of the transformation reaction was spread on 2YT
plates containing 100 ng of ampicillin per ml. The remaining transformation reaction
was pelleted by centrifugation (2550-3400 X g), resuspended in 50 \x\ of 2YT broth
containing 100 ig of ampicillin per ml, and 20 1 streaked in semi-solid 2YT plates
containing 2 mg of agar per ml and 100 ^g per ml of ampicillin. The inoculated plates
were incubated ovemight, or if necessary longer, in a 37°C incubator E coli containing
the vector-annealed PCR product were selected based on motility in the semi-solid
medium. Motile LC2a clones were replated in semi-solid 2YT plates containing
ampicillin and incubated at 37°C until motile clones swarmed approximately 5 cm.
Seouencing Cloned Plasmid DNA in LC2a
Cloned plasmid DNA for sequencing was isolated from transformed LC2a using
the QIAGEN Plasmid Kit (Qiagen, Chatsworth, CA) following the Plasmid Midi Protocol.
Clones were sequenced using the dsDNA Cyde Sequendng System (Gibco BRL Life
Technologies Inc.) with end-labeled primers (Table 2.2) following the manufacturer's
26
instmctions. The nucleotide sequence of each clone DNA was determined in both
orientations. Starting primers and progressive walking primers were the same as used
for sequencing purified PCR products.
Isolation of Clone Plasmid DNA
The QIAGEN Plasmid Kit and Plasmid Midi Protocol was used to isolate done
plasmid DNA A single colony was inoculated in a 250 ml baffled flask (Belico
Biotechnology) containing 200 ml 2YT broth and 100 ^g of ampidllin per ml and was
grown ovemight in a 37°C water bath with 225 rpm shaking. The ovemight culture was
transfenred to a GSA tube (DuPont Sorvall, Wilmington, DE) and centrifuged (28000 X
g) at 4°C for 30 minutes. After removing the supematant, the pellet was resuspended
in 4 ml of buffer PI by vortexing and transferred to a SS-34 tube (DuPont Son/all).
Four ml of buffer P2 was added, mixed gently, and incubated at room temperature for 5
minutes. Four ml of buffer P3 was added, mixed immediately but gently, and
centrifuged (42000 X g) at 4°C for 30 minutes. The supematant was carefully removed
and transfen-ed to a clean SS-34 tube and centrifuged (42000 X g) at 4°C for 10
minutes. The particle-free clear lysate was applied to a QIAGEN-tip 100 column,
previously equilibrated with 3 ml of buffer QBT, and the lysate was allowed to enter by
gravity flow. The column was washed with 10 ml of buffer QC and the DNA was eluted
into a clean SS-34 tube with 5 ml of buffer QF. Using 3.5 ml of room temperature
anhydrous isopropanol, the DNA was precipitated and centrifuged (42000 X g) at 4°C
for 30 minutes. After removing the supematant, 10 ml of 70% cold (-20°C) ethanol was
added and centrifuged (42000 X g) at 4°C for 5 minutes. The ethanol was removed
and the DNA was dried under vacuum for 5 minutes. The dried DNA was redissolved in
100 il of sterile water Five l of plasmid/LC2a DNA was electrophoresed on a 0.75%
agarose gel along with a lambda DNA Hind III fragment sample and pUC 18 Hind
lll/BAP standards (Phamada LKB Biotechnology).
Primer Radiolabeling Using a 0.6 ml microcentrifuge tube stored on ice, primer end-labeling was
accomplished in a flnal volume of 5 1 containing 1 pmoles primer, 1 \i\ of adenosine-5'
triphosphate tetra (triethylammonium) salt [g^2p]. (sooo curies per milimole) in 0.01 M
tridne (N-tris [hydroxymethyl] methylglydne) (New England Nudear). 1 1 5X kinase
buffer (Gibco BRL Life Technologies Inc.) and 1 unit of T4 polynudeotide kinase (1 Unit
per il) (Gibco BRL Life Technologies Inc.). When the same primer was used for
27
multiple templates, the above labeling protocol was scaled lineariy as required in a
single tube. After mixing gently and a brief spin (14000 X g), the labeling reaction was
incubated in a dry heat block at 37°C for 30 minutes followed by a kinase inactivation
step in a 55°C dry heat block for 2 minutes. The labeling reaction was placed on ice for
5 minutes, then centrifuged (14000 X g) briefly to collect condensation. The end-
labeled primers were either used immediately or stored at -20°C.
Seouencing Reactions
For each of the sequencing reactions, four 0.6 ml microcentrifuge tubes (A, C,
G, T) were labeled, placed on ice, and 2 1 of the appropriate termination mix
(con-esponding d/ddNTP) was added. Using a 0.6 ml tube stored on ice, the
prereaction mixture mix was prepared in a final volume of 36 il containing 50 fmoles of
plasmid/LC2a DNA, 4.5 il 10X Taq sequencing buffer (Gibco BRL Life Technologies
Inc.), and 5 pi of labeled primer One-half 1 of Taq DNA polymerase (5U/ il) (Gibco
BRL Life Technologies Inc.) was added to the prereaction mixture and mixed briefly by
pipetting up and down. The reaction tubes were centrifuged (14000 X g) briefly. Eight
[i\ of the enzyme/prereaction mixture was added to the inside wall of each tube
containing the appropriate termination mix and gently mixed by tapping. The reaction
tubes were centrifuged briefly (14,000 X g). The reaction tubes were placed in a ISS
Programmable Oven that had been preheated to 95°C. After the reaction tubes were
held at 95°C for 3 minutes, an initial ampliflcation and extension was performed for 20
cycles followed by a second extension for 10 cycles. The flrst cycle set consisted of a
primer annealing step at 55°C for 30 seconds, and an extension step at 70°C for 1
minute, and a denaturation at 95°C for 30 seconds. The second cycle set consisted of
a extension step at 70°C for 1 minute followed by denaturation at 95°C for 30 seconds.
After the last cycle, the reaction tubes were held at room temperature. The reaction
tubes were removed from the oven and centrifuged (14000 X g) briefly to collect
condensation and placed on ice. Five il of stop solution (Gibco BRL Life Technologies
Inc.) was added to the inside wall of each tube followed by a brief spin to tenninate the
reaction. Before loading 3 il of each reaction on a sequendng gel, the reactions were
heated in a dry heat block at 90°C for 5 minutes, followed by a brief spin (14000 X g).
28
Seroloov
The identity of each flagellar antigen was confirmed by the Motility Inhibition
Test (Gard, 1938), in which bacterial motility is inhibited in semi-solid medium by
incorporation of factor-specific antisemm. Antisera was raised (Berieeley Antibody Co.,
Richmond, CA) in rabbits with formalinized broth cultures of flagellated strains using
standard techniques (Ewing, 1986) and titrated with a microagglutination test (Joys
andStocker, 1969). To obtain factor-speciflc antisera, antisera was absortsed following
established methods (Ewing, 1986) and titrated.
Preparation of Agglutinable H Suspension
Strains of bacteria used for preparing agglutinable H suspensions were flrst
passed through semi-solid 2YT medium, to ensure good development of flagella, then
plated on 2YT medium. A loopful of bacteria was mixed with 25 il of 1:100 dilution, in
sterile 0.85% NaCI, of Salmonella H antisemm g complex (Difco Laboratories) on a
slide and examined by naked eye for agglutination to confirm fiagellar antigens. A
loopful of flagellated bacteria was inoculated into 100 ml of 2YT broth and incubated in
a 37°C water bath until an optical density (OD) of approximately 0.7 at 590 nm was
reached. A loopful of the cell culture was analyzed by low-power darie-fleld microscopy
(Olympus BH) for motility and spontaneous agglutination. One hundred ml of a sterile
0.85% (wt/vol) NaCI/ 0.3% (vol/vol) formaldehyde (Matheson, Coleman & Bell
Manufacturing Chemists) solution was added, gently mixed, and incubated ovemight in
a 37°C incubator After incubation, 25 1 of cell culture was analyzed by low-power
dark-field microscopy for spontaneous agglutination, while another 25 .1 of cell culture
was mixed with 25 1 of 1:100 dilution, in sterile 0.85% NaCI. of Salmonella H antisemm
g complex on a slide and examined by naked eye or microscopy for agglutination.
Each cell suspension was labeled with the strain of bacteria and the title. "H
Suspension." As a viability check, 1 ml of H Suspension was added to 100 ml 2YT
medium and incubated for 48 hours, then checked for growth. H suspensions were
stored at 4°C.
Preparation of Antisera
Strains of bacteria used for preparing antisera were first passed three times
through semi-solid 2YT medium, to ensure good development of flagella. A loopful of
bacteria was inoculated into 5 ml 2YT broth. After incubating unshaken at 37°C for 7
hours, 5 ml of sterile 0.85% NaCI and 834 pi of 36% formaldehyde were added, gently
29
mixed, and incubated ovemight at 37°C. After incubation, 25 1 of cell culture was
analyzed by low-power dari<-fleld microscopy for spontaneous agglutination, while
another 25 l of cell culture was mixed with 25 il of 1:100 dilution, in sterile 0.85%
NaCI, of Salmonella H antisemm g complex on a slide and examined by naked eye or
microscopy for agglutination. Each cell suspension was labeled with the strain of
bacteria and the title, "Injecting Suspension." As a viability check, 1 ml of Injecting
Suspension was added to 100 ml 2YT medium and incubated for 48 hours, then
checked for growth. Serovar -specific antisera was prepared by the Beri<eley Antibody
Company using each nonviable Injecting Suspension as antigen. Five intravenous
injections were accomplished 5 to 6 days apart, followed by a single bleed 8 to 10 days
after the last injection. The first, second, third, fourth and fifth injection contained 0.25
ml, 0.5 ml, 1.0 ml, 1.0 ml and 1.0 ml Injecting Suspension, respectively.
Sera Titration Using H Suspensions
Sera were titrated by twofold steps, starting at 1:500 and ending at 1:64000, by
a microagglutination test (Joys and Stocker, 1969). Sera were diluted using sterile
0.85% (wt/vol) NaCI. In the micromethod, 25 p.1 of H Suspension and 25 .1 of semm
were mixed on a glass plate and incubated In a damp chamber (14 cm petri dish
containing a damp paper towel and a bent glass rod for plate support). After incubating
2 hours at 37°C and 1 hour at room temperature, the semm/cell suspension was
examined for agglutination by naked eye or low-power darie-field microscopy. As a
negative control, H Suspension was mixed with sterile 0.85% (wt/vol) Nad. To avoid
non-specific clumping, the plates were cleaned with a sulfuric acid (Fisher Scientific
Co.)/sodium dichromate (Fisher Scientific Co.) solution (sodium chromate was added to
750 ml of sulfuric acid in a glass beaker until the solution bec:ame saturated), rinsed in
distilled water, immersed in a 95% ethanol bath, dried with a Kimwipe and flamed
before use. The titers were recorded as the highest dilution of semm showing
agglutination.
Preparation of Absorbing Suspension
Strains of bacteria used for preparing absortsing suspensions were first passed
through semi-solid 2YT medium, to ensure good development of flagella, then plated
on 2YT medium. A loopful of bacteria was inoculated in 30 ml of 2YT broth and
incubated in a 37°C water bath until turt)id (approximately 5-6 hours). Ten ml of the
turbid cell culture was spread on sterile agar containing Bacto tryptone (1.6% wt/vol).
30
Bacto yeast extract (1.0% wt/vol), sodium chloride (1.0% wt/vol), Bacto agar (1.5%
wt/vol), sodium lactate (Matheson, Coleman & Bell Manufacturing Chemists) (0.5%
vol/vol) and sodium glycerophosphate (Matheson, Coleman & Bell Manufacturing
Chemists) (1.0% wt/vol), in a 41cm (L) x 25.5cm (\N) x 6.5cm (H) enamel tray. After
36-48 hours at 37°C the growth from a tray was harvested by adding 10 - 20 ml sterile
0.85% NaCI to the surface of the culture and gently transfening the growth into a sterile
50 ml centrifuge tube (Coming Inc., Coming, NY).
Preparation of Absorbed H Antisera
The protocol of Edwards and Ewing (Ewing, 1986) for 'Preparation of Absorbed
H Antisera' was followed in preparing p, q, s, t (labeled t1 in our lab) and u factor-
speciflc antisera. Modiflcation of the protocol, allowed factor "m" to be subdivided into
m^ and m2. The protocol published by Rauss (1939) for preparing f, t2 and t3 factor-
speciflc antisera was followed, with a slight modification allowing factor "f antisemm to
be anti-S. derby f-specific. The absorption scheme used is shown in Table 2.3. In a
SS-34 tube, one volume of undiluted antisemm was mixed with 3 volumes (except for
the preparation of m2 in which S. oranienberg antisemm was mixed with 4 volumes)
total of absorbing suspensions. After incubating 2 hours in a 37°C water bath and 1
hour at room temperature, the absorption mixture was centrifuged (30877 X g) at 4°C
for 30 minutes, the supematant transfen-ed to a clean SS-34 tube, and centrifuged
(30877 X g) at 4°C for 15 minutes. The cleared supematant was filter sterilized using a
0.45 pm low protein binding sterile Acrodisc (Gelman Sciences, Ann ArtDor, Ml).
AbsortDed sera was titrated as described above and stored at -20°C.
Motility Inhibition Test
The identity of each fiagellar antigen was confirmed by a Motility Inhibition Test
(Gard, 1938) In which bacterial motility is inhibited in semi-solid medium by
incorporation of factor-specific antisemm. Each strain to be tested was inoculated, by
stabbing, on to 4 ml per well of semi-solid (0.4% agar) 2YT medium containing 10 Units
per ml of different antisera per well in 12-well (well diameter 22 mm) fiat bottom plates
(Coming Inc., Coming, NY). One Unit of antisemm is defined as titer detennined. For
a control experiment, plates without the addition of antisera were inoculated. Plates
were incubated for approximately 5 hours and the distance of motility measured.
31 Table 2.3. Absorption scheme used to prepare factor-specific antisera. Antisera was
raised (Berteeley Antibody Co.) in rabbits with fonnalinized broth cultures of flagellated strains by using standard techniques (Ewing, 1986) and titrated with a microagglutination test (Joys and Stocker, 1969). To obtain factor-specific antisera, antisera was absort)ed with highly motile organisms of an absortjing strain by using established methods (Ewing, 1986- Rauss 1939).
AbsortDlng Salmonella cultures Factor Antiserum^ (flagellar antigen) (flagellar antigen)
S. de/tjy (gf)
m1 S. enteritidis (gm)
m2 S. oranienberg (mt)^
p S. dublin (gp)
q S. moscow (gq)
s S. montevideo (gms)
t1 S. oranienberg (mt)
t2 S. berta (gft)
S. budapest (gt), S. dublin (gp), S. enteritidis (gm), S. msocow (gms), S. rostock (gpu) and S. senftenberg (gsf)
S. berta (gft), S. budapest (gt), S. dublin (gp), S. moscow (gq) and S. senftenberg (9St)
S. berta (gft), S. budapest (gt),S. dublin (gp),S. moscow (gq), S. senftenberg (gst) and S. montevideo (gms)
S. enteritidis (gm) and S. senftenberg (gst)
S. enteritidis (gm)
S. enteritidis (gm) and S. oranienberg (mt)
S. montevideo (gms)
S. derby (gf), S. dublin (gp),S. enteritidis (gm), S. montevideo (gms), S. moscow
t3
u
S. senftenberg (gst)
S. rostock (gpu)
(gq) and S. rostock (gpu)
S. derby (fg), S. dublin (gp), S. enteritidis (gm), S. montevideo (gms), S. moscow (gq) and S. rostock (gpu)
S. dublin (gp)
''One volume of undiluted antisemm was mixed with three volumes total of absort ing suspension.
20ne volume of undiluted antisemm was mixed with four volumes total of absorbing suspension.
32
Epitope Mapping on Polvethvlene Pins
On the basis of the detennined amino add sequences for the central portion of
the g... series flagellins from S. berta, S. budapest, S. derby, S. dublin, S. enteritidis, S.
montevideo, S. moscow, S. oranienberg , S. nDstock and S. senftenberg, progressive
overiapping octamers were synthesized on polyethylene pins, using materials and
protocols purchased as a Pin Technology Epitope Scanning Kit (Cambridge Research
Biochemicals Ltd., Cheshire, UK). The protocols of this kit are based on the
procedures described by Geysen et al. (1987) which allows the synthesis and ELISA
screening of large numbers of peptides. Peptides were synthesized on solid
polyethylene pins that have been radiation grafted with acrylic acid, coupled with mono-
t-butyloxycarbonyl-1-6-diaminohexane (HMD) and capped with a 9-
fluorenylmethyloxycartjonyl (Fmoc)-protected 8-alanine spacer group. All amino adds
have their a-amino group protected with Fmoc and their side chains protected with
specific chemicals. Synthesis of peptides was accomplished by repetitive cyding of
Fmoc-deprotection, washing and coupling. After removal of the final Fmoc group, the
tennlnal amino group was capped by acetylation, side chains deprotected and, finally,
the peptides sonicated before use in an ELISA assay. All reagents and solvents used
were of the highest possible purity.
Generating Schedules for Synthesis of Peptides
The protein sequence (Figure 2.1) derived from the selected Salmonella g...
series flagellins, encompassing all amino acid differences from amino add position 181
to 345, was entered into a computer using the Utilities software provided with the
Epitope Scanning Kit. The General Net Synthesis (GNET) option from the Sequence
Maintenance Main menu was used to generate sequences of progressive overiapping
octameric peptides and a schedule for their synthesis.
33
571
1 51
101 151 201 251 301 351 401 451 501 551
VTGYDTYAVG ENNTAVDLFK GNDGNGKVST FTFDDKTKNE SGAWTDAAA AWTDDAAPD ATSIKGGKVG GASATDVNSA KSTAGTDEAK GNDGNGTVST TDVNAATLQD ATLQSSLTVA
ANKYRVDVNS TTKSTAGTAE TINGEKVTLT STGYDTYAAG PDKVYVNAAG KVYVNAANC3Q DTFDYKGVSF KIQSSKDVYT AIAGLFKTTK IDTKTGDDCa^ lATGATNVNA DIGIGAADVN
GAWTDTTAP AKAIAGAIKG VADITAGAAN ADKYRVDINS AWTDAVAPN LTTADAQNNT TIDTKAGDDG SWSC3QFTFA SAAGTDEAKD GKVSIDTKTG ATLQSSLTVA A
TVPDKVYVNA GKEGDTFDYK VDAATLQSSK GAWTDTYAA KVYVNAAAVA AVDLFKSTKS NGTVSTTING DKTKNESAEN EAKAIASAIK DGGNGKVSTT DITGGAANVD
ANGQLTTDDA GVTFTIDTKT NVYTSWNC^ GANKYRVDIN PDKVYVNASG AAGTDDAKAI EKVTLTISDI NTAVNLFKTT GGKETIDTKA LTVADIATGA ATAGAANVND
Figure 2.1. Protein sequence used to generate sequences of progressive overiapping octameric peptides and and a schedule for their synthesis.
Purification of N.N-Dimethvlfonmamide with Molecular Sieves
N,N-dimethylformamide (DMF) (Aldrich Chemical Co., Milwaukee, Wl), a solvent used
in Fmoc chemistry, must be pure and free of amines. Amines were removed by
standing DMF over activated 4A molecular sieves (Aldrich Chemical Co.) for a minimum
of one week. Four-A molecular sieves (1/8" beads, 4-8 mesh) were activated by
heating ovemight in a 300°C oven (Baxter Diagnostics Inc., Grand Prairie, TX), and left
to cool In a vacuum desiccator Activated molecular sieves (200 g) were added to 2.5
liters of DMF, left in the dark for 3 days standing, the bottle shaken, and then left for a
further 4 days further 4 days to stand. Prior to use, the DMF was filtered through
Whatman GF/A (VWR, San Frandsco, CA) glass fiber filter paper.
Svnthesis of Peptides
On the top surface of each block of pins, a letter identifying the block was
initially etched using a scriber Synthesis requires deprotection, washing, coupling and
washing
Fmoc-Deprotection and Washing. The Fmoc protecting group is extremely base
labile and is readily removed by treatment with piperidine in DMF. All washes were
perfonned at room temperature with agitation. Each block of pins was placed in a bath
which covered the pins to half height and contained 20% (vol/vol) piperidine (Aldrich
Chemical Co.) in DMF for 30 minutes at room temperature. After removing the blocks
34
from the bath, excess liquid was shaken off and the pins washed in a half-height DMF
bath for 5 minutes at room temperature. The blocks were transfen-ed, after shaking off
excess liquid, to a bath of methanol which completely covered the blocks for 2 minutes
then through three successive fresh half-height methanol baths 5 minutes each. Upon
removing the blocks and allowing them to air dry for a minimum of 10 minutes, the
prepared amino acid solutions were pipetted into appropriate wells of 96-well round
bottom microtiter plates (Baxter Scientific). Before positioning the pins in the amino
acid solutions, the blocks were washed in a fresh bath of DMF for 5 minutes.
Addition of Fmoc-Amino Acid Active Esters. According to the synthesis
schedule, amino acid derivatives for the relevant day were weighed out and prepared.
Into a clean flask, the amount of 1-hydroxybenzotriazole (HOBt) required for the day's
synthesis, as shown on the daily schedule, were weighed out and dissolved in the
required volume of purified DMF. Fmoc-amino acid active esters (weighed out for that
day) were dissolved in the required volume of HOBt solution and dispensed, while the
deprotected and washed pins were drying, until all the designed wells of each plate
were filled. Each tray containing dispensed amino acids was placed in the bottom of a
plastic container, each block of pins positioned into their appropriate wells, and the
container sealed with a lid. After 18 hours at room temperature, each block of pins was
washed in a half-height DMF bath for 2 minutes, covering methanol bath for 2 minutes,
and three successive fresh half-height methanol baths for 2 minutes each. Finally the
blocks were air dried for 10 minutes. At this point if another amino add is to be added,
the peptide-pins are deprotected as described above. The deprotection, washing,
coupling and washing steps are repeated until all amino acids have been coupled.
Once all the required peptides have been synthesized on all pins, the N-temninus of
each peptide must be acetylated and side-chains deprotected.
Acetvlation of Terminal Amino Groups. The N-temninus of each peptide is
acetylated once synthesis is complete in order to remove the charge associated with a
free terminal amino group. The washed and air dried blocks of pins were deprotected,
washed and then placed into a tray containing 150 il of DMF:acetic anhydride (Aldrich
Chemical Co.):triethylamine (Aldrich Chemical Co.) 50:5:1 (vol/vol/vol) per well. The
acetylation reaction was allowed to proceed for 90 minutes at room temperature in
sealed plastic containers. After this period the blocks of pins were removed and
washed as usual, i.e., in a DMF bath ( 1 X 2 minutes) and methanol baths ( 4 X 2
minutes), followed by air drying for at least 10 minutes. The peptides are now ready for
side-chain deprotection.
35
Side Chain Deprotection and Neutralization. All protecting groups used to
protect side-chain functionalities during synthesis must be removed from the
synthesized peptides prior to use. All amino acids supplied with the pin technology kit
have the following side chain protecting groups: f-butyl ether (-Bu') for serine,
threonine and tyrosine; f-butyl ester (-OBu') for aspartic add and glutamic add; /-
butyloxycartjonyl (Boc-) for lysine and histidine; 4-methoxy-2,3,6-
trimethylbenzenesulphonyl (Mtr-) for arginine; and trityl (Trt-) for cysteine). The
acetylated blocks of pins were placed into a close container half-height bath containing
trifluoroacetic acid (Aldrich Chemical Co.):anisole (Aldrich Chemical Co.):ethanedithiol
(Aldrich Chemical Co.) 95:2.5:2.5 (vol/vol/vol) for 4 hours at room temperature. On
removal from the deprotection bath, the pins were washed in two successive
dichloromethane (DCM) (Aldrich Chemical Co.) baths for 2 minutes each, two
successive baths of 5% diisopropylethylamine (Aldrich Chemical Co.) in DCM for 2
minutes each, a DCM bath for 2 minutes, followed by air drying for a minimum of 10
minutes. After air drying, without agitation, the pins were washed in a full height water
bath for 2 minutes followed by an ovemight full height methanol bath. The pins were
allowed to air dry a minimum of 10 minutes before use.
ELISA Testing
The Pin Technology Epitope Scanning Kit is based on the synthesis of
overiapping progressive octameric peptides, derived from the protein under study,
which are screened using a prepared antisera as the primary antibody, a second
antibody horseradish peroxidase conjugate, and o-phenylenediamine-hydrogen
peroxide color development substrate. When hydrogen peroxidase is reacted with a o-
phenylenediamine-hydrogen peroxide color development substrate, a yellow color
appears. Upon addition of hydrochloric acid, a brown color appears which is read
spectrometrically. Using this system, functional domains can be localized to 8 amino
acids. Before ELISA testing, the pins must be dismpted and sonicated.
Dismption and Sonication Procedures. Pins are dismpted before their first and
subsequent use. The following operations were carried out in a fume hood. Blocks of
pins to be cleaned were placed, tips down, into a 65°C pre-heated sonication bath
(Branson 2000 Ultrasonic Cleaner, Branson Ultrasonics Corp., Danbury, CT) containing
34.5 g sodium phosphate monobasic monohydrate (Sigma Chemical Co.) , 25 g SDS
(Serva), 2.5 ml 2-mercaptoethanol (Sigma Chemical Co.), 2.5 liters distilled water, pH to
7.2 with 50% NaOH. Pins were sonicated at 65°C for 30 minutes, washed
36
( 3 X 5 minutes) with hot (55-60°C) water covering the blocks and agitation, placed in a
boiling methanol bath (Water bath 182. Predsion Sdentific Inc.. Chicago, IL) for 5
minutes, and allowed to air dry for a minimum of 5 minutes. The pins were either used
directly for ELISA testing or stored at room temperature in a desiccator containing silica
gel.
ELISA Testing Procedure. The ELISA testing procedures of Joys and Schodel
(1991) were followed. The following incubations and washes were performed for each
block of pins at room temperature with agitation. The washes covered the pins and
between each wash, excess liquid was shaken off. Non-specific protein binding sites
were blocked by covering the pins in H-PBS buffer for 1 hour H-PBS buffer contains
5% Tween-20 (Serva) in 1 X E-PBS buffer E-PBS buffer, prepared as a 10 X stock
solution, contains 1.39% (wt/vol) sodium phosphate dibasic anhydrous (Sigma
Chemical Co.), 0.24% (wt/vol) potassium phosphate dibasic anhydrous (Fisher
Scientific Co.), 9.76% (wt/vol), pH to 7.4 with 50% NaOH. Antisera was diluted to a titer
of 1:100 in H-PBS and dispensed in 200 pi volumes in round bottom 96-well microtiter
plates (Coming). Each tray containing dispensed antisera was placed in the bottom of
a plastic container, each block of pins positioned into their appropriate wells, and the
container sealed with a lid. Following a minimum 2 hour incubation with the primary
antibody, pins were washed three times with H-PBS buffer for 5 minutes each. Goat
anti-rabbit IgG (whole molecule) horseradish peroxidase conjugate (Sigma Chemical
Co.) diluted 1:5,000 in H-PBS was dispensed in 200 pi volumes in round bottom 96-well
microtiter plates. Each tray containing dispensed antisera was placed in the bottom of
a plastic container, each block of pins positioned into their appropriate wells, and the
container sealed with a lid. Following a minimum 1 hour incubation with the secondary
antibody, pins were washed in H-PBS ( 3 X 5 minutes) followed by a 5 minute wash with
E-PBS. Pins were incubated in flat bottom 96-well microtiter plates (Coming) with 150
pi of Substrate Solution at room temperature in the darie with agitation for 30-40
minutes. Substrate Solution contains 51.5% (vol/vol) 0.2 M sodium phosphate dibasic
anhydrous, 48.5 % (vol/vol) 0.1 M citric acid (Fisher Scientific Co.), 0.0005% (wt/vol) o-
phenylenediamine (Sigma Chemical Co.) and 0.0005% (vol/vol) of 30% hydrogen
peroxide (Sigma Chemical Co.). To neutralize the substrate solution, 150 pi of 1 N
hydrochloric acid (Sigma Chemical Co.) was added after removing the pins. The plates
were read immediately with a Dynatech MR300 spectrophotometic microplate reader
(Dynatech Laboratories Inc., Chantilly, VA.) at a wavelength of 490 nm. Pins were
stored in a 1 X E-PBS bath until dismpted and sonicated.
37
Epitope Mapping bv the Novatope Svstem
The flagellin of S. moscow was analyzed for epitopes using the NovaTope
System (Novagen, Inc., Madison, Wl) according to the manufacturer's instmctions. The
NovaTope System is based on the creation of a library of bacterial clones, each of
which expresses a small peptide derived from the protein under study. The library is
screened by standard colony lift methods using an antibody as the probe. Positive
clones are directly analyzed by DNA sequencing to determine the precise amino acid
sequence of the target epitope. Using this system, functional domains can be localized
to 10-20 amino acids. In practice, the library was constmcted using DNase I, in the
presence of manganese, to randomly cleave the starting gene into fragments averaging
50 to 150bp in size. The DNA fragments were either (a) fractionated by
electrophoresis in a low-melting point agarose gel, then recovered and purified by
phenol:chloroform extraction or (b) visualized by electrophoresis in an agarose gel and
selected samples purified by phenol:chloroform extraction. The selected DNA
fragments were treated successively with T4 DNA polymerase and Tth DNA
polymerase, which repairs and then adds a single dA residue to each 3' end. The
prepared DNA fragments were ligated into the pTOPE T-vector plasmid which contains
single dT overhangs complementary to the inserted fragments. The pTOPE T-vector is
designed for the expression of small peptides since they are produced as part of a
larger fusion protein (the major capsid from T7), which prevents breakdown by cellular
proteases. Expression is controlled by a T7 promoter For protein screening, ligation
reactions were transformed into NovaBlue (DE3) host cells carrying the gene for T7
RNA polymerase, which results in the accumulation of the fusion protein in the cell.
Colonies were transfen-ed to nitrocellulose filters, lysed and screened with antisera as
the probe. Plasmid DNA from positive clones was prepared and the epitope DNA
sequence determined by standard DNA sequencing methods. For initial screening by
colony hybridization, ligation reactions containing fragments with nucleotides from 1-
540, 524e-870, or 842-1479 were transformed into NovaBlue host cells which do not
contain T7 RNA polymerase. Plasmid DNA from positive dones was prepared
sequenced by standard DNA sequencing methods. Recombinant plasmids containing
the appropriate insert in the right orientation were transfomied to NovaBlue (DE3) and
screened by protein expression.
y
38
PCR Amplification
For amplification of the S. moscow fliC gene for initial epitope mapping, primers
PCR1S and PCR1AS were used. When epitope mapping defined areas, the following
PCR primers were used to amplify selected fragments of the S. moscow fliC gene:
primers PCR3S and PCR3AS for a fragment encompassing the sequence from
nudeotide 1 to 540; primers PCR4S and PCR4AS for a fragment encompassing the
sequence from nudeotide 524e to 870; and primers PCR5S and PCR5AS for a
fragment encompassing the sequence from nucleotide 842 to 1479. Conditions for
PCR amplification and PCR product purification followed the same protocol as
"Chromosomal Amplification by PCR" and "Purification of PCR products" mentioned
eariier All purified PCR products were concentrated by ethanol precipitation. PCR
products amplified with primers PCR3S/PCR3AS and PCR5S/PCR5AS were directly
ligated to the pTOPE-T vector PCR products amplified with primers PCR4S/PCR4AS
were both directly ligated to the pTOPE T-vector, or after DNase I digestion, dA tailed
and ligated to the pTOPE T-vector PCR products amplified with primers
PCR1S/PCR1AS were DNase I digested, dA tailed and ligated to the pTOPE T-vector
DNase Shotgun Cleavage
The NovaTope System DNase Shotgun Cleavage Kit (Novagen, Inc.) and
protocol were used to generate overiapping DNA fragments of varying sizes. The
method is based on the observation that bovine pancreatic DNase I causes random
double strand scission of DNA in the presence of manganese and cleavage can be
controlled by varying the enzyme concentration, temperature and/or incubation time.
The protocol uses constant temperature and a fixed amount of DNA with increasing
dilutions of DNase I to find conditions that produce fragments in the desired size range.
Immediately before use, DNase I was diluted with IX buffer containing manganese
chloride to 1:133, 1:200, 1:300 and 1:450 dilutions. For PCR products amplified with
primers PCR4S/PCR4AS, the DNase I enzyme was further diluted to 1:675, 1:1013 and
1:1519 dilutions. Using 0.6 ml microcentrifuge tubes stored at room temperature, each
DNase I digestion was performed in a separate tube in a final volume of 10 pi
containing 0.9 pi of 10X DNase I buffer, 0.9 pi of 10X manganese chloride, 4 pg
ethanol precipitated PCR product, diluted DNase I, and sterile water After adding the
enzyme and mixing gently, the tubes were incubated at room temperature for exactly
10 minutes. Immediately, 2 pi (0.2 volume) 6X stop buffer, containing lOOmM EDTA,
30% glycerol, and tracking dyes, was added to stop the reaction. One pi samples were
39
analyzed by electrophoresis In a 2% agarose gel beside 5 pi PCR mariners (50, 150,
300, 500, 750 and lOOObp). Cleavage reactions containing fragments in the 50 to
400bp size range were either (a) pooled and electrophoresed at 4°C and 50V in a 2%
SeaPlaque (FMC, Rockland, ME) low melting temperature agarose gel containing 0.5 p
g of ethidium bromide per ml, and then recovered and purified following techniques
described by Sambrook et al. (1989) or (b) pooled and directly purified by
phenol:chloroform extraction. DNA was concentrated by ethanol precipitation to a
minimum concentration of 0.1 pg per pi in water and electrophoresed in a 2% agarose
gel along with PCR mariners and pUC18 Hind lll/BAP standards. The purified DNA was
stored at -20°C.
Single dA Tailing
The NovaTope System Single dA Tailing Kit (Novagen, Inc.) and protocol were
used to prepare DNase I digested fragments having various types of ends for T-
cloning. Following treatment with T4 DNA polymerase in the presence of all four
dNTPs to ensure flush ends, a single 3' dA residue is added using Tth DNA
polymerase. The Tth DNA polymerase preferentially adds a dA residue even though all
four dNTPs are present. Using a 0.6 ml microcentrifuge tube stored on ice, the flushing
reaction was performed in a final volume of 25 pi containing DNA (30 pmol ends), 2.5 pi
10X flush buffer, 2.5 pi 10X dNTP mix (1 mM each dCTP, dGTP, dTTP, 10 mM dATP).
1.25 pi 20X DTT (100 mM), 0.5 pi T4 DNA polymerase (1-2 units), and sterile water
After adding the enzyme and mixing gently with the pipette tip, the tube was incubated
at 11°C for 20 minutes. The reaction was stopped by inactivating the enzyme at 75°C
for 10 minutes. Single dA tailing was performed by adding to the flushing reaction, 8.5
pi 10X dA tailing buffer, 0.5 pi Tth DNA polymerase (1.25 units) and sterile water to a
final volume of 85 pi . After adding the enzyme and mixing gently with the pipette tip,
the tube was incubated at 70°C for 15 minutes. One volume of 24:1
chloroform:isoamyl alcohol was added to the reaction, vortex vigorously for 60 seconds,
and centrifuged at 14,000 X g for 1 minute. The aqueous phase containing the DNA
was transfen-ed to a fresh tube and stored at -20°C. To monitor the perfonnance of the
test DNA, the positive control 50 mer supplied with the kit was used in a parallel
reaction.
40
Ligation to oTQPE T-Vector
The NovaTope System DNA Ligation Kit (Novagen, Inc.) and pTOPE T-vector
Kit (Novagen, Inc.) and protocol were used to directly ligate single dA tailed fragments
with the pTOPE T-vecAor The pTOPE T-vector is designed for expression of inserts as
stable fusion proteins (a fusion containing the first 260 amino adds of the T7 gene 10
protein) controlled by T7 DNA polymerase. It contains a high copy number origin of
replication for superior plasmid yields and has single 3' dT residues at each end so that
inserts having single 3' dA overtiangs can be ligated directly into the vector Using a
1.5 ml microcentrifuge tube stored on ice, ligation was performed in a final volume of 10
pi containing 1 pi 10X llgase buffer, 0.5 pi 100 mM DTT, 0.5 pi 10 mM ATP, 1 pi of 50
ng per pi pTOPE T-vector (0.03 pmol), 0.5 pi T4 DNA llgase (2-3 Weiss units), 0.2 pmol
target DNA, and sterile water After adding the enzyme, the tube was incubated at
16°C ovemight in a refrigerated circulating bath (Model 1420, Bio-Rad). To test the
efficiency of ligation, 5 ng of the positive control T-vector insert provided with the kit
was used in place of the target DNA.
Transfomriation Using NovaBlue (DE3) Cells
NovaBlue (DE3) (Novagen, Inc.) is an expression host for the T7 expression
vector, pTOPE T-vector since these cells contains a chromosomal copy of the gene for
T7 RNA polymerase. Although the T7 RNA polymerase is under lacUVS control,
substantial amounts of protein are produced even in the absence of IPTG induction.
Under most conditions proteins accumulate as insoluble inclusion bodies which provide
the advantage of sequestering expressed proteins so that they are less susceptible to
proteolytic breakdown, and so that potentially harmful products do not affect cell
viability. One pi of ligation mix (or 1 pi [1-30 ng] of Magic Minipreps purified plasmid
DNA) was added to 20 pi of competent NovaBlue (DE3) cells in a 15 ml plastic tube on
ice by moving the pipette through the cells while dispensing. After incubating the
transformation mixture on ice for 30 minutes, the cells were heat shocked in a 42°C
water bath for 40 seconds, then placed on ice for 2 minutes. Eighty pi of room
temperature S.O.C (Novagen, Inc.) medium was added and the transformation reaction
incubated for 1 hour in a 37°C water bath with 225 rpm shaking. After expression the
cells were vortex briefiy and 50 pi of the transfonnation reaction was spread on LB
plates containing 100 pg of ampicillin per ml and 15 pg of tetracycline per ml. The
inoculated plates were incubated ovemight, or if necessary longer, in a 37°C incubator.
41
Colony Screening bv Protein Expression
The NovaTope System Immunoscreening protocol (Novagen, Inc.) was used to
process nitrocellulose filters by successive incubations with prepared antisera as the
primary antibody, a second antibody alkaline phosphatase conjugate, and
bromochloroindolyl phosphate - nitro blue tetrazolium as color development substrate.
When alkaline phosphatase is reacted with a bromochloroindolyl phosphate - nitro blue
tetrazolium substrate, a darie purple precipitate appears. The number of clones
required to achieve a given probability that a given sequence will be present in a gene
library is N = ln(1-P)/ln(1-1/n), where N = the number of clones required, P = the
probability desired, and 1/n = the fractional proportion of the total sequence
represented by target sequence (Sambrook et al., 1989). In a pTOPE library screened
by expression of target DNA, this number is multiplied by a factor of 6 to account for 3
possible reading frames and 2 possible orientations. Plates containing transformants
were chilled at 4°C for one-half hour prior to making colony lifts so that the agar would
not stick to the nitrocellulose. The plates were overiayed with BA85 87 mm
nitrocellulose filters (Schleicher and Schuell, Keene, NH) for one minute and marieed by
poking a pipette tip into 3 asymmetric places into the filter and plate. The plates were
retumed to a 37°C incubator to regenerate colonies. Bacterial colonies were lysed by
placing the colony lifts into a chloroform chamber In a fume hood, colony lifts were
placed on dampened paper towels in a large glass dish, along with a small beaker
containing Kimwipes soaked with chlorofonn (HPLC grade) (Aldrich Chemical Co., Inc.,
Milwaukee, Wl). The dish was covered with aluminum foil and left at room temperature
for 15 minutes. Bacterial colonies were denatured by placing the lifts, colony side up,
on a piece of Whatman 3MM paper (VWR, San Frandsco, CA) saturated with colony
denaturing solution (20 mM Tris (Research Plus Laboratories, Inc., Denville, NJ), pH to
7.9 with hydrochloric acid (Fisher Scientific Co., Fairiawn, NJ); 6 M urea (Research Plus
Laboratories, Inc.) and 0.5 M NaCI (Sigma Chemical Co., St. Louis, MO)) for 15 minutes
at room temperature. The following incubations and washes were performed at room
temperature with agitation. Non-specific protein binding sites were blocked by
immersing the filters in TBST buffer plus 1% gelatin (Bio Rad Laboratories, Richmond,
CA) for 30 minutes. TBST buffer contains 10 mM Tris, pH to 8.0 with hydrochloric add;
0.5% Tween-20 (Aldrich Chemical Co., Inc., Milwaukee, WL), 150 mM Nad and 0.02%
sodium azide (Fisher Scientific Co.). Filters were washed with TBST three times for 15
minutes each, followed by a 30 minute incubation with antisera diluted to a titer of
1:100 in TBST. Following incubation with the primary antibody, the filters were washed
42
three times with 15-20 ml of TBST per filter for 10 minutes each, then incubated for 30
minutes with goat anti-rabbit IgG (whole molecule) alkaline phosphatase conjugate
(Sigma Chemical Co.) diluted 1:10,000 in TBST. After washing the filters three times
with 15-20 ml TBST for 10 minutes each, the filters were placed on paper towels to
absortD excess liquid, each transfen-ed to a petri dish lid, and incubated with 6 ml of
color development solution until a strong signal developed (within 1-10 minutes). Color
development solution contains IX alkaline phosphatase buffer and 4 pi per ml of the
NBT and BCIP color development substrates. Alkaline phosphatase buffer, prepared
as a 20X stock, contains 2 M Tris, pH to 9.5 with hydrochloric acid, 2 M NaCI and 100
mM magnesium chloride (BDH Chemicals Ltd., Poole, England). NBT color
development substrate contains 83 mg of 2,2' Di-p-nitrophenyl-5,5'-diphenyl-3,3'-(3,3'-
dimethoxy-4,4'-diphenylene)ditetrazolium chloride (Nitro blue tetrazolium; NBT) (Sigma
Chemical Co., St Louis, MO) per ml of 70% dimethyl formamide (Sigma Chemical Co.).
BCIP color development substrate contains 42 mg of 5-bromo-4-chloro-3-indolyl
phosphate (BCIP), p-toluidine salt (Sigma Chemical Co.) per ml of 100% dimethyl
formamide. To stop color development, the filters were rinsed several times in water
and allowed to dry. Positive colony areas were restreaked to a fresh plate and
rescreened the following day using the above protocol. To monitor performance of
reagents, S. typhimurium {fliC 0 and Salmonella H antisemm / (Difco Laboratories) were
used as a positive control. The second antibody only was also used to detemriine non-
spedfic binding.
Transformation Usino NovaBlue Cells
NovaBlue is a cloning host for pTOPE T since these cells do not contain T7
RNA polymerase. One pi of ligation mix was added to 20 pi of competent NovaBlue
cells in a 15 ml plastic tube on ice by moving the pipette through the cells while
dispensing. After incubating the transfomriation mixture on ice for 30 minutes, the cells
were heat shocked in a 42°C water bath for 40 seconds, then placed on ice for 2
minutes. Eighty pi of room temperature S.O.C medium was added and the
transfonnation reaction incubated for 1 hour in a 37°C water bath with 225 rpm
shaking. After expression the cells were vortex briefiy and 50 pi of the transformation
reaction was spread on LB plates containing 100 pg of ampicillin per ml and 15 pg of
tetracydine per ml. The inoculated plates were incubated ovemight, or if necessary
longer, in a 37°C incubator
43
Colony Screening bv Hybridization
Colony hybridization was used to identify NovaBlue cells that contain
recombinant plasmids.
Colony Lvsis. Plates containing transformants were overiayed with BA85 87
mm nitrocellulose filters (Schleicher and Schuell) for one minute and marieed by poking
a pipette tip into 3 asymmetric places into the filter and plate. The plates were retumed
to a 37°C incubator to regenerate colonies. Bacterial colonies were lysed by pladng
the colony lifts successively in four glass petri dishes containing Whatman 3M paper
saturated with (1) 0.5 N sodium hydroxide for 7 minutes, (2) 1 M Tris-HCL, pH 7.4
buffer for 2 minutes, (3) 1 M Tris-HCL, pH 7.4 buffer for 2 minutes, and (4) 0.5 M Tris-
HCL, pH 7.4, 1.5 M NaCI buffer for 4 minutes. Each filter was rinsed using a vacuum
filtration device (Schleicher and Schuell) with 100 ml of 0.5 M Tris-HCL, pH 7.4, 1.5 M
NaCI buffer and three 10 ml volumes of chloroform. Filters were laid, colony side up,
on Whatman 3M paper to dry for 30 minutes at room temperature, sandwiched
between two pieces of Whatman 3M paper, baked in an 80°C vacuum oven for 2
hours, and stored in a room temperature desiccator under vacuum.
Oligonudeotide Probe Labeling. Using a 0.6 ml microcentrifuge tube stored on
ice, probe end-labeling was accomplished in a final volume of 50 pi containing 15
pmoles probe, 50 pmoles of adenosine-5' triphosphate tetra (triethylammonium) salt [y
32p] - (6000 curies per milimole) in 0.01 M tricine (N-tris [hydroxymethyl] methylglydne)
(New England Nuclear), 5 pi T4 polynucleotide kinase 10X buffer (Promega, Madison,
Wl), 20 Units of T4 polynucleotide kinase (Promega, Madison, Wl) and sterile water.
After mixing gently and a brief spin (14000 X g), the labeling reaction was incubated in
a dry heat block at 37°C for 30 minutes followed by a kinase inactivation step in a dry
heat block at 90°C for 2 minutes. The labeling reaction was placed on ice for 5
minutes, then centrifuged (14000 X g) briefiy to collect condensation. The end-labeled
probe was either used immediately or stored at -20°C. Immediately before use, the
radioactive probe was diluted with 6 ml of 6X SSC and filtered using a 0.45 pm low
protein binding sterile Acrodisc (Gelman Sciences, Ann ArtDor, Ml). The filter was
rinsed with 2 ml of 6X SSC, yielding 8 ml of diluted probe. SSC, prepared as a 20X
stock and autodaved to sterilize, contains 17.53% (wt/vol)NaCI and 8.82% (wt/vol)
sodium citrate. pH to 7.0. This protocol was designed to label enough probe for 2 filter
hybridizations.
Hybridization. The following protocol is designed for 2 circular nitrocellulose
filters, 87 mm In diameter. Prehybridlzation solution contains 5 ml 50X Denhart's
44
solution (Sigma Chemical Co.), 7.5 ml 20X SSC, 12.5 ml sterile water and 50 mg
dodecylsulfate-Na-salt (SDS) (Serva, Heidelberg, Gennany). While two 1 liter glass
beakers, containing 12.5 ml of prehybridization solution each, were incubating for 10
minutes in a 67°C water bath, two filters were wet by submersion in 30 ml of 6X SSC
for 5 minutes. Filters were transfenred to the heated prehybridization solutions,
incubated for 5 minutes with occasional agitation, washed in 200 ml of room
temperature 6X SSC. and placed colony side down onto 4 ml of heated probe (heated
for 5 minutes at 67°C) in a plastic petri dish lid. Hybridization occun-ed at room
temperature for 1 hour Filters were washed three times in 100 ml of fresh room
temperature 6X SSC for 1 minute each, followed by an incubation for 30 minutes in 100
ml of 6X SSC in a glass baking dish at 50°C (or 55°C when necessary for higher
stringency) with 30 rpm shaking. Filters were removed, rinsed with 6X SSC, dried in
the air at room temperature on Whatman 3M paper, an-anged on a sheet of dry
Whatman 3M paper, labeled with autoradiograph Radtape (Diversified Biotech, Newton
Centre, MA) and covered with Reynolds film 914 (Reynolds, Richmond, VA). A Kodak
X-ray exposure holder and intensifying screen were used to expose the filters to X-
Omat AR film (Eastman Kodak Co., Rochester, NY) for 30 minutes at room
temperature. Exposed autoradiograph film was developed as described under
Sequencing in General, Developing Exposed Autoradiograms. Positive colonies were
identified when the 3 asymmetric mari<s left by the autoradiograph tape on the
developed film was aligned with the corresponding 3 asymmetric mari<s on the agar
plate. When necessary, positive colony areas were restreaked to a fresh plate and
rescreened the following day using the above protocol, allowing a single, well-isolated
positive colony to be picked for further analysis.
Plasmid Preparation and Screening for Plasmid-lnsert-Containing Colonies
Colonies, positive by protein screening or colony hybridization, were selected
and analyzed for plasmid insert by rapid plasmid preparation, Insta-Prep Kit (5 Prime ->
3 Prime, Inc., Boulder, CO), followed by a restriction enzyme digestion with BST XI
(Promega). A single colony of bacteria inoculated in a 15 ml plastic tube containing 5
ml of LB growth media with 50 pg of ampicillin per ml and 15 mg of tetracycline per ml
was grown ovemight in a 37°C water bath with 225 rpm shaking. Cells were pelleted
by centrifugation (2550-3400 X g) (Model GLC Centrifuge; DuPont Sorvall, Wilmington,
DE) at room temperature for 5 minutes. The supematant was removed and the pellet
45
resuspended in 100 pi of sterile water Three hundred pi of PCI (50 phenol:49
chlorofonn: 1 isoamyl alcohol) solution was added and mixed by inversion. The entire
contents of the tube was transfen-ed, using a large bore pipette tip, to a pre-spun
(14,000 X g for 10 seconds) INSTA-PREP tube and centrifuged (14,000 X g) for 30
seconds. Three hundred pi of CI (49 chlorofonn: 1 isoamyl alcohol) solution was added,
the two upper liquid phases briefly mixed by inversion, and centrifuged (14,000 X g) for
30 seconds. The plasmid DNA was recovered by pipetting the topmost phase to a
fresh 0.6 ml microcentrifuge tube. Using a 0.6 ml microcentrifuge tube stored on ice,
the restriction enzyme digestion was performed in a flnal volume of 10 pi containing 3.5
pi INSTA-PREP DNA, 1 pi 10X buffer D (Promega), 4 units of BST XI (Promega). The
digestion reaction was mixed by tapping, briefly centrifuged (14000 X g), then
incubated for 1 hour in a 55°C water bath. After inactivating the BST XI enzyme by
Incubating the digestion reaction at 65°C for 20 minutes, 1 pi of 10X RNase Plus gel
loading buffer (5 Prime -> 3 Prime, Inc., Boulder, CO) was added to each digestion
mixture and 10 pi samples were analyzed on a 2% agarose gel beside PCR mariners
(Novagen). Undigested and BST XI digested INSTA-PREP were analyzed side-by-side
on the agarose gel. Plasmid DNA containing insert was ethanol precipitated prior to
sequencing.
The Magic Minipreps DNA Puriflcation System (Promega) was used to prepare
plasmid DNA for transformation to NovaBlue (DE3) cells. The "Plasmid Puriflcation
without a Vacuum Manifold" procedure was followed with slight modiflcation. A single
colony of bacteria inoculated in a 15 ml plastic tube containing 5 ml of LB growth media
with 50 pg of ampicillin per ml and 15 mg of tetracycline per ml was grown ovemight in
a 37°C water bath with 225 rpm shaking. Cells were pelleted by centrifugation (2550-
3400 X g) (Model GLC Centrifuge; DuPont Sorvall) at room temperature for 5 minutes.
The supematant was removed and the pellet resuspended in 200 pi of cell
resuspension solution and transferred to a 1.5 ml microcentrifuge tube. Two hundred p
I of cell lysis solution was added and mixed by inversion until the suspension cleared.
Two hundred pi of neutralization solution was added, mixed by inversion, and
centrifuged (14000 X g) at room temperature for 5 minutes. The supematant was
transfen-ed, with a sterile plastic transfer pipette, to a clean 12 X 75 polypropylene tube.
1 ml of Magic Minipreps DNA resin was added to the tube and mixed by inversion. The
Magic Minipreps DNA purification resin containing the bound DNA was transfen-ed
using a pipette to a plastic 3 cc syringe (Becton, Dickinson and Co., Rutherford, N.J.)
barrel connected to a Promega mini-column. The plastic syringe plunger was inserted
^
46
and the slurry was gently pushed into the mini-column. The syringe ban-el with plunger
was disconnected from the mini-column and the barrel reconnected without the
plunger The mini-column was washed with 2 ml of column wash solution by gently
pushing the column wash through the mini-column with the syringe plunger The
syringe ban-el was removed, the mini-column transfen-ed to a 1.5 ml microcentrifuge
tube and centrifuged (14000 X g) at room temperature for 20 seconds to dry the resin.
The mini-column was transferred to a clean 1.5 ml microcentrifuge tube and the bound
DNA fragment was eluted by adding 50 pi of preheated (65-70°C) sterile water for 1
minute followed by centrifugation (14,000 X g) for 20 seconds. The purified DNA was
electrophoresis in a 0.75% agarose gel along with a lambda DNA Hind III fragment
sample and pUC18 Hind lll/BAP (Phannada LKB Biotechnology, Piscataway, NJ)
standards. The purified DNA was stored at -20°C.
Seouencing Clone Plasmid DNA
Clones were sequenced using the fmol DNA Sequencing System (Promega)
and protocol (described eariier under the title, "SEQUENCING OF PURIFIED PCR
PRODUCTS") with vector-specific primers (primer T7 gene 10 and primer T7
terminator) provided with the NovaTope Epitope Mapping System (Novagen Inc.).
Clone plasmid DNA for sequencing was isolated from transfomied NovaBlue (DE3) or
NovaBlue cells using the Insta-Prep Kit (5 Prime -> 3 Prime, Inc.) followed by ethanol
precipitation. One hundred ng of plasmid DNA was used for each sequencing reaction.
CHAPTER III
RESULTS
PCR Amplification. Cloning and Seouendnq of fliC
Genomic DNA from the 17 g.. series members (Table 3.1) was used as a
substrate for PCR reactions with primers (Table 3.2) derived from the upstream
(Szekely and Simon, 1983) and downstream (Wei and Joys, Unpubl.) regions of the fliC
gene. Both PCR primers are spedfic for the fliC gene, do not amplify the fIjB gene, and
amplify a single PCR product Purified PCR products primed with PCR IS and
PCR IAS were directly sequenced (in both directions) using progressive walking
primers (Table 3.2) derived as sequences were determined. Purified PCR products
primed with PCR2S and PCR2AS were doned using the plasmid pAMPI as the vector
and DH5a cells as host Plasmid DNA from colonies containing the pAMPI with insert
was transformed to LC2a, a non-flagellated strain of E. coli which has most of its fliC
(previously temried hag) gene deleted (Zieg et al., 1977) so that recombination between
the fliC gene of the recipient and the fliC gene of the donor does not occur Clone DNA
was isolated from LC2a and sequenced (in both directions) using the sequencing
primers listed in Table 3.2. Cloned-derived DNA sequences agreed with PCR product
DNA sequences, except for S. dublin. As shown in Table 3.3, 14 nucleotide sites were
in contention between sequence data derived from two S. dublin fliC PCR products and
five clones, each containing a S. dublin fliC PCR-derived insert. Position numbers are
based on nucleotide alignment with fliC^ (HI-A) as shown in Appendix A and
equivalent to those previously reported (Wei and Joys, 1985) for the fliC^ flagellin
gene.
Comparative Analyses of Salmonella a... Series Serovars
An amino acid comparison based on the g... series sequencing data is
presented in Table 3.4. Position numbers are based on amino acid alignment with fliC^
(HI-A) as shown in Appendix B and equivalent to those previously reported (Wei and
Joys, 1985) for the fliC^ flagellin. Additional amino acids are indicated by subdivision
(i.e., a, b, c, ...). Amino acid comparisons and alignments were accomplished using the
Lipman and Pearson (1985) FASTP program, along with an MTRANS (Masten, 1992)
program and an Excel spreadsheet The FASTP program of Lipman and Pearson
(1985) permitted the alignment of two protein sequences with allowance for gaps and
insertions. The MTRANS program of Masten (1992) allowed the transfer of the Mount
47
MiiiiiiliMiiArili
48
Table 3.1. Salmonella serovars used for genomic DNA preparations.
Serovars
S. adelaide S. berta S. budapest S. califomia S. chaco S. danysz S. derby S. dublin S. enteritidis S. essen S. jena S. monschaui S. montevideo S. moscow S. oranienberg S. rostock S. senftenberg
Source
ATCC 10718 ATCC 8392 CDC 23 ATCC 23201 ATCC 49214 ATCC 49216 ATCC 6960 ATCC 15480 ATCC 13076 ATCC 49219 ATCC 49221 TDH BE-04 ATCC 8387 CDC 67 ATCC 9239 CDC 66 ATCC 8400
Antigenic Formula''
35:f,g:-l,9,^2:f,g,t.-
lA^2,27•.g,t-4,12:g,m,f 9,12:g,m 9,12:g,m
l,4,[5],12:/;g:[1,2] l,9,12,[Vi]:g,p:. 1.9,12:g,m:[1,7]
l,9,12:g,m 9,12:g,m 35:m,t
6,7.g,m,s:-9,12:g,g:-6,7:m,t-
l,B,^2•.g,p,u•.-l,3,^9•.g,[s],t-
'' Brackets [ ] indicate that the antigen may be absent An underscore indicates that the bacterial strain has been lysogenized.
•au^ iStaBUi ^
49
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m m m m m
M CO (/)(/>(/)(/) c < C O C O C O C O C O C O C O C O < < < < < D ^ < < < < < < < < C N c o ^ i n . W Q ^ T - r g c O ' ^ i n < D i ^ c o Q ^ a : f r r y ^ 0 5 5 5 2 5 5 5 5 0 0 0 0 c o C L Q Q O Q Q D C Q Q Q Q Q Q Q C Q C L ( L Q . ( L
Table 3.3. Differences in nucleotides between S. dublin fliC PCR products and recombinant clones, each containing a S. dublin fliC-PCR insert. Position numbers are based on amino acid alignment with fliC^ (HI-A) as shown in Appendix B and equivalent to those previously reported (Wei and Joys, 1985) for the fliC^ flagellin.
50
Nucleotide Position
636
717
783
844
913
916
917
921
928
934
1080
1114
1146
1164
PCR Products
PCR1
A/G
C/T
A/G
A/G
A/G
A/G
G/T
C/T
A/G
A/G
C/T
A/G
C/T
T
PCR2
A
C/T
A/G
A/G
A/G
A/G
G/T
C/T
A/G
A/G
C
G
nd
nd
DU8
G
T
A
G
G
A
T
C
G
A
T
A/G
C
A
Recombinant clones
DUA2-10 DUB1-3 DUB1-8
A
C
G
A
A
G
G
T
A
G
C
G
C
T
A
C
G
A
A
G
G
T
A
nd
C
G
C
T
A
C
G
nd""
A
G
G
T
A
nd
C
G
C
T
DUB1-13
A
C
G
A
A
G
G
T
A
nd
C
G
C
T
Consensus
A
C
G
A
A
G
G
T
A
G
C
G
C
T
1 Not done.
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54
and Conrad translation files to an Excel spreadsheet for sequence comparisons based
on the FASTP alignment data. Comparative analyses of the infen-ed covalent
structures of the 17 Salmonella g... series serovars showed that both ends of the
flagellin molecule (regions I, II, and III [amino acids 1 to 180] and region VIII [amino
acids 421 to 493]) were conserved in the amino acid sequences. Region Vi,
embodying amino acids 323 to 359, showed complete amino acid conservation for all
serovars except S. califomia, S. monschaui and S. oranienberg, which showed the
same differences at amino acid positions 327 and 333. Region VII, embodying amino
acids 360 to 420, showed a high degree of amino acid homology (except for S. daynsz)
with differences at amino acid positions 368 and 372. Due to a large deletion
encompassing 39 amino acids from amino acid positions 367 to 405, S. daynsz showed
a low degree of homology in region VII. Region IV (amino acids 181 to 299) and
region V (amino acids 300 to 322), which are located in the central portion of the
flagellin molecule, showed the greatest diversity among the 17 serovars with S.
califomia, S. monschaui and S. oranienberg being the most divergent. While the other
fourteen g... series serovars demonstrated 93 to 100% amino acid homology in region
IV and 78 to 100% amino acid homology in region V, S. califomia, S. monschaui and S.
oranienberg showed only 82 to 84% amino acid homology In region IV and 61% amino
acid homology in region V (Table 3.5). The infen-ed flagellin amino acid sequence of
13 of the 17 g... series serovars showed a 9 bp deletion at amino acid positions 213 to
215, located in region IV. The other four serovars, S. berta, S. califomia, S. monschaui
and S. oranienberg, showed threonine, valine and proline, respectively, at amino acid
positions 213 to 215. The five Salmonella g... series serovars expressing g,m flagellar
antigens exhibited, excluding the large deletion in S. daynsz, a high degree of
homology with differences detected at amino acid position 158 in S. daynsz and amino
acid position 282 in S. enteritidis. S. chaco, S. jena and S. essen , all expressing g,m
antigens showed complete homology.. Salmonella g... series serovars expressing f,g
flagellar antigens exhibited a high degree of homology with three differences detected
at amino acid positions 210, 306 and 368. S. monschaui and S. oranienberg
expressing antigens m,t, showed only one amino acid difference at position 252. Upon
comparing S. califomia {fliC9>'^'^ with S. monschaui {fliC^'^ and S. oranienberg
{fliC^'^, amino acid differences were detected at position 247 and at positions 247 and
252, respectively. Interestingly S. senftenberg {fliC9>s.t) and S. montevideo {fliCd.rr^.s)
express the s antigen and show an aspartic acid at amino acid position 209, while all
the other g... series serovars show a threonine or alanine. Aspartic acid at amino acid
55
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position 209 may contribute to the expression of the s antigen in S. Senftenberg and S.
montevideo. S. moscow {fliCd'Q) is the only serovar with a glycine present at amino
acid position 283. The other g... series serovars show an aspartic acid at this position,
suggesting in the fliC9,9 flagellin of S. moscow, glycline at amino acid position 283 may
contribute to the expression of the q antigen.
Comparative Analvses of Salmonella fliC a. a... Series, c. d. i. r. and E. coli fliC Flagellins
Based on nucleotide and amino acid homology with flagellin a. Salmonella
flagellin from the g... series were compared with the previously reported flagellins c, d, i,
and rand E. coli fliC (Table 3.6). Using the sense strand data, nucleic acid alignments
and comparisons were accomplished using the Pearson HMATCH executable file
included in the Mount and Conrad (1984) program, along with an MTRANS (Masten,
1992) program and an Excel spreadsheet. Amino acid alignments and comparisons
were accomplished as stated above. The g... series flagellins showed more diversity
than the single factor Salmonella flagellins and the E. co//flagellin. In regions I and VIII
where flagellins a, c, d, i, and rwere highly conserved, flagellins of the g... series were
less homologous with flagellin a. Although the central area (regions III, IV, V, VI and
VII) of flagellin is known to be variable between serovars and species, the g... series
flagellins demonstrated more variability than single factor flagellins (c, d, i, and r) and E.
coli flagellin when compared to flagellin a. The best example is seen in region V where
the g... series flagellin show little (4%) or no homology when compared to flagellin a.
Hvdrooathic Character of Flagellin
Hydrophobicity and surface exposure plots, based on the g... series flagellin
sequences and g... series numbering (Appendix C) are presented in Figure 3.1 to
Figure 3.6. Interpretations are converted to the fliC^ numbering system (Appendix B).
The polar-apolar nature of flagellin was analyzed using the Protyize Predictor version
3.01 program (Scientific & Educational Software). This program estimates
hydrophobicity and hydrophilicity from protein sequences using the method of Kyte and
Doolittle (1982) or of Hopp and White (1981). For both methods, values which fall on
the horizontal axis are neither hydrophilic nor hydrophobic. The distance from the axis
line is proportional to the degree of hydrophilicity or hydrophobicity. Kyte and Doolittle
(1982) represents a composite hydrophobicity scale derived from interpretation of free
57
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64
energy changes on a water-vapor phase transition and an analysis of buried side
chains. Each value is the average of the values of 5 adjacent residues and is plotted at
the middle residue. The range of values is approximately ± 4 relative units. Hopp and
Wood (1981) derived hydrophobicity values from a study of antigenicity and adjusted
the values to maximize the accuracy of predicting antigenic determinants. Each value
is the average of the values of 6 adjacent residues and is plotted at the middle point.
The range of values is approximately ± 3 relative units. Surface exposure analysis use
the date of Janin et al. (1978) which provides values representing the fraction of
residues of a given amino acid that have a surface area of greater than 20 angstroms
squared. High values therefore represent amino acids that are likely to be exposed on
the surface of the protein. Plotted values are the average of 6 residues and are plotted
at the middle point. The vertical axis is the proportion of surface exposure of amino
acids. The horizontal axis line represents the overall mean value for all amino acids.
Values which fall on the horizontal axis line are neither predicted to be exposed on the
surface of buried within the protein. Peak values which fall above the axis line are
predicted to be exposed on the surface of the protein, while those below the axis line
are not predicted to be exposed. Three locations showing hydrophilic character and
demonstrating surface exposure were identified. Two hydrophilic clusters were found
within region IV encompassing amino acids 262 to 268c (9 amino acids total) and 277
to 285 (9 amino acids total), respectively. The largest and third region likely to be
exposed to the surface of g... series flagellin embodies amino acids 331 to 341 (11
amino acids total) found in region VI.
Evolutionary Relatedness of Salmonella fliC a. c. d. i. r and a... Series and E. coli Flaoellin
An unrooted phylogenetic tree, based on the flagellin sequences is presented in
Figure 3.7. C. jejuni was chosen as the outgroup species. Evolutionary relatedness
was analyzed using the PROTPARS program of the PHYLIP version 3.4 (Felsenstein,
1991) analysis package. This program estimates phylogenies from protein sequences,
using the parsimony method, in a variant intermediate between the approaches of Eck
and Dayhoff (1966) and Fitch (1971). Eck and Dayhoff (1966) allowed any amino acid
to change to any other, and counted the number of such changes needed to evolve the
protein sequences on each given phylogeny. This approach allows replacements
which are not consistent with the genetic code to be counted equally with replacements
that are consistent. Fitch (1971), on the other hand, counted the minimum number of
65
I
-26 +
+-25 t -I I
C. jajtini.
E. aoli K-12 (fl±C)
S. H2aDchan (fli.cf')
S. cbolarma-muia (flicf)
S. parM typhi CfliC*)
+ 20
+-21 + -! +-24 ! ! ! +—S. typhimurxtm (fli.c'-) +-22 +-23
! +—S. xvhi.al*w (fli.(f)
-27
+ ~ 3
+-- l e
-S. abortaB-aqai. (fljB*''^''')
-S. QraiUanbarg (fliC^'^)
'• + ~ S . Monschaui (fl±C^'^) +-17
+—S. Cal i fomia f f l iC*'" ' ' ;
-16 -S. BartM (fli.C^''^'^)
-15
+—4
+-! !
+-13
+ 12
+--S. Dublin (fliC^'P) -5 +~S. Darby (fliC^'^}
*—S. Sanftmbarg (fltc^'''^) -14
+ ~ S . Budapest (fliC^'^)
+—S. DayoMz (fliC^'') -19
+—S. HoDtarxdao (flLC^'''')
S. RoatocJc (fliC^'P'") + 11 ! ! +—S. ttoaaatr (fli.C^''^) ! +-10
-7 +—S. ISitaritidia (fltC^'')
t—S. Jan* (fliC^'')
8 +--S. Essen ffliC^'"; ! + S. Chaco (fli.(^'')
+ S ide la ide (fliC^'^)
•E. ooli H7 CfliC; +--2
• *—E. coli HI ("flic; +—1
+ ~ I . oo l i H12 CfliC;
Figure 3.7. Evolutionary relatedness of Salmonella flagellins (phase-1 g... series, a (Wei and Joys, 1985), c (Wei and Joys, 1985), d (Wei and Joys, 1985), / (Joys, 1985), r (Wei and Joys, 1986), and Salmonella phase-2 flagellin (Okazaki et al., 1993)) and E. coli K-12 (Kuwajima et al., 1986), HI (Schoenhals and Whitfield , 1993), H7 (Schoenhals and Whitfield , 1993) and H12 (Schoenhals and Whitfield , 1993) flagellins. This is an unrooted tree, with Campylobacter jejuni (Khawaja et al., 1992) as the outgroup species. Flagellin antigens are shown in parentheses.
66
nucleotide substitutions that would be needed to achieve the given protein sequences.
This approach has the problem of counting silent changes equally with those that
change the amino acid. The PROTPARS approach insists that any changes of amino
acids be consistent with the genetic code and changes between two amino acids via a
third are allowed and counted as two changes if each of the two replacements is
individually allowed. Silent changes are not counted since this approach assumes that
synonymous changes are considerably faster and easier than ones that change the
amino acid. A total of 54 trees were found, with the one in Fig. 3.7 being
representative. Three major divisions, branching from C. jejuni are detected. One
section is formed by the Salmonella strains exhibiting single factor antigen flagellins, a
phase-2 flagellin and the E. coli K-12 flagellin. The second branch encompasses the
gf... series serovars which further divides into two major offshoots. S. oranienberg, S.
monschaui and S. califomia form one offshoot of the branch while the remaining ^A g...
series serovars form the other division. Within the offshoot encompassing the majority
of the g... series serovars, a subdivision segregates S. berta from the other the
serovars. The third major division encompasses the rest of the E. coli strains.
Preparation and Titration of Absorijed H Antisera
Antisera raised in rabbits using a standard protocol was titrated against 10
Salmonella gf... series members with a microagglutination test (Joys and Stocker,
1969). Summaries of the titers for unabsorbed antisera and factor-specific antisera are
shown in Table 3.7 and Table 3.8, respectively. To obtain p, q, s and u factor-specific
antisera, the Edwards and Ewing protocol was followed and titers before and after
absorption shown in Tables 3.9 to 3.12. m factor-specific antisemm prepared by the
Edwards and Ewing protocol (Ewing, 1986), and labeled mt in our lab, detected the m
in S. enteritidis and S. montevideo, but not in S. oranienberg (Table 3.13 and Table
3.14). To determine if the m in S. enteritidis and S. montevideo were the same, second
absorptions were performed using S. enteritidis or S. montevideo absorption cultures.
Both second absorbed antiseras lost reactivity with S. enteritidis and S. montevideo,
suggesting these two serovars share the same m flagellar antigen, m factor-specific
antisemm prepared with S. oranienberg antisemm in place of S. enteritidis
demonstrated reactivity with S. enteritidis, S. montevideo, and S. oranienberg (Table
3.15;. To determine if the m in S. oranienberg was different from the m in S. enteritidis
and S. montevideo, second absorptions were performed with S. enteritidis and S.
montevideo absorption cultures. Both second absortDed antiseras lost reactivity
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cow
; or,
S
dilu
tion
sho
wi
CD ^ 0 CO
0
o
0 CO • o 0
o CO
. 0 0 c
3
0
^
«
3
0
CO
iH C
O) .2^ i5 c U- 0
X (O
s i 0 CO c: c o 0
• 5 CO 0 3
CO <«
0
0 Q 0 0 0 0 0 0 0 o S 9 0 0 0 0 0 0 i o S V ? ' ^ ' ^ o t f ) i n i r ) * ' 0 O V V V - ^ V V
o o o g o o o o S S S Q O O O O « ? o o g o o o i o V CN T- «£ -^ CN CN
0 0 0 0 0 0 0 0 0 (3 0 0 0 0 0 0 i n IT) 0 0 10 ' r 00 V V T- T-
S 0 0 0 0 Q 0 0 0 S
2 2 0 0 0 2 Q § 0 0 u? u) 0 0 0 V V " ' T -
2 0 0 0 2 0 0 0 "? S 0 ^ V ^^ ^^
0 0 0 0 0 0 10 0 0 V TT
0 0 0 0 0 0 ' ^
0 0 0 CN
0 0 0 "^
TT
1600
0
0
^~
1600
0
0
"^
6400
0
0
' "
0 0 0 00
0
8000
0 0 0 - ^
0
V
6400
0
0
V
1600
0
<500
V
0 0 0 00
0
0 0 0 00
0 0 0 ^ •
r-) 0 0 10 0 V T -
2000
20
00
0 0 0
0 0 0 J:: 0 10 0 2
0 0 0 0 0 0 0 0 0 0 0 10 0 0 00 00 V ' J- -^
0 0 0 0 0 0 0 0 0 0 0 0 0 0 ir> 0 0 "^ CN CN V 00 T-
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 IT) 0 m 0 0 -^ CN V CN V "^ CN
0 0 0 0 0 0 0 o o o o o o o o O O O O O l O O O ^ O O O O C N " ^ V T - 0 0
o o o o o o o o 0 0 0 ^ 0 0 0 0 0 o o o S m o o i o o TT CN ^~ V ^ T— V ( [N
0 ^
0 3 0 ^ C O ^ T3 T3 0
c: o 5
o o CO
o
0 -Q
.0
c
o
o o
8
5> 0
0 CO
cocococococococococo
68
2 0
CO l _
0
CO a>
0 u o
CO "O c 0 CO >» o
4 ^ CO
B c c .2
0 .c • B l _D C3)
C3> ^
0 o> 2-1 .2 ^ 0 <«
0 ^ •^ E C D O CO
«•— • -
o '' 0) <i>
8-.2> O 0) o .c 0 —
• ^ CO •«— 0
°-o C 0
•2 "2 28
. t i 0 00 CO
.2 0
2 0 CO
c 0
.o o 0 QJ CO
?o
Sii
CO
c\
i ; ; CO
Ui .2^ i5 ^ Li. 0
CO c g 0 CO
fo
o
0 CO w>
c 0 Cl l CO
D
O O O Q O O O O O O
V V v ^ V V V V c o V
g g o o o o o o o g T - ^ " ^ V V V V V V $ ^
o o CN CN CO CO
CN O O O O O O r-> T - T - T - T - CO 2 V V V V T - V ° "
o o o o o o o o o «^ «^ V. C.J c^ c^ T7 TT T7 TT CM T^ vD CD CD V V V V V CN
0 0 0 o 0 0 0 0 r - . 0
V V V V CD V V CN CD
O O O O
V V V V
C J C ^ J C J O Q Q O O O O T ~ ^ ~ ^ ~ ^— * " ' ^ ^ CVI ^— ^— ^—
o o o o CN CO V V V
2 2 2 o o o o i i ^ i r i ^ o o o o r- -«- CN V V CO V
O Q O O O O O O O Q
CN V V V V V CD V
o o o o o o o o o o v v v v c o c o v v v V
o o § o O o o o o o T-CNjQCNy v V V V V
i§ill|ll|l
0
CO 0
c:
c: 0
o
o
o CJ CO
o 5
5* 0
•C3
c .0 c 2 o
15 o CO
8
5> 0 c
0 CO
cocococococococococo
69
Table 3.9. Titration of S. dublin antisemm before and after absorption to obtain p factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gft) budapest (gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption
<500 4000 1000
64000 4000 2000 2000 <500 8000 1000
dilution
1:4
1:4
after absorption''
<10 <10 <10 1280 20 10
<10 <10 320 <10
''The p factor-specific semm was prepared by absorbing S. dublin {gp) antisemm with S. enteritidis {gm) and S. senftenberg {gsf).
Table 3.10. Titration of S. moscow antisemm before and after absorption to obtain q factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gfC) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption
<500 16000 1000 4000 16000 8000
64000 <500 2000 2000
after dilution absorption'
<10 <10 <10 <10
1:4 20 10
320 <10 <10 <10
''The q factor-specific semm was prepared by absorbing S. moscow {gq) antisemm with S. enteritidis {gm).
^m H
70
Table 3.11 Titration of S. montevideo antisemm before and after absorption to obtain s factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption
<500 <500 500 1000 1000 4000 1000 <500 1000 16000
dilution
1:4
1:4
after absorption'
<10 <10 <10 10
<10 640 <10 <10 20 640
''The s factor-specific semm was prepared by absortDing S. montevideo {gms) antisemm with S. enteritidis {gm) and S. oranienberg {mf).
Table 3.12. Titration of S. rostock antisemm before and after absorption to obtain u factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption
after dilution absorption
<500 2000 1000 16000 4000 2000 2000 <500 8000 1000
1:4
1
<10 <10 <10 <10 <10 <10 <10 <10 320 <10
''The u factor-specific semm was prepared by absortDing S. rostock {gpu) antisemm with S. dublin {gp).
71
0 i _ O •^• 0 ia „«^, a> CD a ^~ i_r 0
j ^
o o •*->
(0 "D C 0 CO >% O
- 3
*^
tes
c g 0 c
'•t.^ D Ui C3> 0 O
icr
E 0 >»
.Q
E 3 i _
0 CO
^^rf
c 0 .<o :6 c: 0 H S
c: 0
CO l ^ -
o c g
'4.^
2 4 - >
1-c6 T—
CO 0 .Q 0
E 3 ^ 0 CO
'•«.^ c 0 g
«^ 0 0 a. CO
1 0 u 0
*»— T ~
5 c "0 JQ 0 0
T3 0 CO
CO C
g '•*-»
a. 0 CO
0 ^ 0
0 • D C 0
CO S 0 5 "^ 5 •PCN
o i O — CO t r I- o 0 CO ^ "9 0 0
o
c: o
CO
.<0
c:
0
CO
c g
•u
2 g • - & 5> o $ - CO 0 .Q
0
C
g
c 0) .2
% o
0
CO c 0
c .o 2 c
JS CO CO
S CO 0
0 «« i5 CO X i
^ ^ ^ ^ 0 0 ^ 0 ^ ^
c c c c V V c c
^ * - * - . . - o o ^ o ^ ^
CN CN
O O O O O O O O O O x - T - x - T - C N C N " * - - * - " * - " * " V V V V C O C O V V V V
"^ -^
in V
2 o o o o o o o o O Q O O O O O O O S o o o o o i n o o ^T^'^roooooo V " -
3 C3> 0
53«
CO
C3> 5 CO O) ^ 3 ^ >b. >S
3 o 3 "5 5
"§ c 2 T3 0 C
0
o o CO
o
. 0
c 2 o
0> 0
^ c O 0
o *; o ^ C CO cocococococococococo
CO S
^ a (0 %
<*s CO
0 -o •Q 0)
CO 0 CO
SI .Q
1 " > CO
"O ^ 0 H •e si 0 3
(gfm
) se
rum
was
d S
. se
nfte
nber
g
.CO 0
:6 ^ "S o-? ^ ^ ^
0 0 0
CO 8
CD S CO
o>3 c
L U ^
SI "O • * - <
> CO
c 3 "E « 0 g 0 S-0 CO 0 T3 3
T -
E 0 CO
c 0 u
it—
0 Q. CO
0 4 - <
0 0 .>
^ • 0 0 0 .Q 0
0
0
CO C 0
Uirf Q. 0 CO .Q 0 ^^ CO
0 • • ^
"O c 0 0 0 CO
0" >» E c 0 0
0 ^ .c c CO ^ - ^ 0 0
<D 0
1 e 1 CO
. "O c^ S 0
0 c CO CJ
1? 0 ^^
0 § CO §
c c
« ™ £ ? Jt= CO
0 =» C "D
E ® <1> n
f £ CN
72
Table 3.14. Titration of S. enteritidis antisemm before and after absorption to obtain m1 factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mt) rostock {gpu) senftenberg {gst)
before absort)tion
<500 16000 1000 4000 8000 8000 8000 <500 4000 4000
dilution
1:4 1:4
1:4
1:4
1:4
after absorption''
<10 <10 <10 <10 320 320 <10 <10 <10 <10
''The m1 factor-specific semm was prepared by absorbing S. enteritidis {gm) antisemm with S. berta {gff), S. budapest {gf), S. dublin {gp), S. moscow {gq) and S. senftenberg {fsf). This absortjed sera detects the 'm' in S. enteritidis and S. montevideo, but not in S. oranienberg.
y
0
0
"O c 0
0 k -
o »*-0 .Q ^.^ C3) CD (Ji T-
kJ" 0 . ^ o o •^-t
if) •o c 0
CO >» o 3 *-> Ui 0 *.» c g '*.» 0 c
"•«-«
3 C3> C3> 0 o c o E 0
>* .Q
E k_ 0 W
"c 0
5> 0
c: .<!> C
2 o
CO
o c g w 0 k—
P
•
E zs ^ 0 CO
c 0 g
i * -O 0
CO 1
k -O
t) 0
CN
5 c 0
s o o *-> • o 0 CO D CO
c g "S. k -
o CO
0
CO
.2
.Q 0
c o
o CO j a 0 • a c o o 0 CO k . 0 *-» «»-0
CO
51 0
c
c 0 CO
co 0
•Q
IN O
:§
o
CO
"c:
0
c g
'•u
11 S o C CO 0 Si
0
c g
•u
c
0 s o U
0
c c c c ^ ^ *^ s ••^ •^ CO
• - • - • - • - o o C C C C <^ ' ^
- 2 *- *-
73
C C C C ' ^ ' ^ C C N C C
CN CN CN CN
. ^ • - .5- * - o o C C C C CO CO
CD 4.^ 0 0 *•* ^^
" ^ - ^
o o o o o o o g O O O C D O O O g o o to to o o '^r 00 V V
10 CD
o o o o m o
C3)
3 CJ> 0
" ^ Q. >^
tr "o ^ 43 XJ T3
CO
E CO C3>
3 o
.c"g .2
"O 0 c
3 0 s 0
O CD O 0
i5 S "S: c: o 2 E o
CO 0 CO
cocococococococococo
CO CO o
CJ -Q: 5
0 S.2 o • 2 . C
c: o E
0 CO 3
CO 0
E 0 CO
c 0
E
:3 - TJ CO
CO .^
3 0 CO 0 ) 0
CO 3* - • >
3 0 0 0
•Q
c:
2 CO
0
CO >
E a 0 CO
c 0
g o 0 CL Vi
"D 0
€ o CO Si 0 CO 0
E 3
E 0 Ui '.^
c c c 0 .Q O
0
x> 0
•*->
2 4-< CO c O E 0 •o
E ^. 0 CO
4-< C 0 "O 0 € o CO n 0 0
So 0 ^
O o 2 2 CL
•Q C 0 C 2 o CO CD
00
0 •Q C
c 0 CO
CO • D C 0
0 •Q C .0
LU 0
C3);g
CJ ? ^ 0
3g o
o CJ CO
o E
CO T3 C 0
0 0
c o
o CO
. Q 0 "O c o u 0 CO
o"
c: o E
CO T3 C 0
0
CO c
0 s: ^^ * - I0 E E o 3 — o 0 !5 II "o o .<2 E B>co
il CO :o c c:
— Q E c 0 * S C O ; t .c
E ® 0 E 0 o •o t :
^ £ CN
C
o
3 CO 0 a •Q
CO C3> C CO
D • D 0
E i -o t: 0 Q. 0 k_
0
CO
c o O CO
.Q 0
• a c o o 0 CO
CO
3 CO 0
a « .
3-c: • 2 CO
. Q CO
c o l
p E CO a 0 0 £ CO * ^ • ^ 0 C > 0 0
C3) e> "Q c 0 3 Is o :o
CO 0 C3) C C Q
c 0 c 0 ex. E 0 0
0
berg
f ha
s ra
niem
0
CO
_c E 0
ifth
0 c u
jJd
0 T3 0
CO
>* c 0
3
c: 0 <«: c: 0
CO "D c 0
^ E SCO n -0 0 c >v 0
• 0 w 0 o>
ely
obta
ii ft
enbe
rg
ultim
at
S. s
en
. ^.^
E O" a3 s ^
cifc
ant
ii .
mos
co
0 CO Q. . CO ' r* iL CX
0 3 ^ E 0 ^ • ^ ^ CN ^
E ^ - ^
/
74
with S. enteritidis and S. montevideo, but retained activity with S. oranienberg,
suggesting the m in S. oranienberg is different than the m in S. enteritidis and S.
montevideo. To determine if the first-absorbed antisemm contained a t component,
second absorptions were perfonned with S. budapest (gt) and S. senftenberg (gst)
absorption cultures. No t component was detected. Based on trial absorptions, S.
oranienberg antisemm was ultimately absorbed with S. berta, S. budapest, S. dublin, S.
moscow, S. senftenberg and S. montevideo (Table 3.16) This absortjed antisemm
detects the m in S. oranienberg, but not in S. enteritidis or S. montevideo and was
labeled m2 factor-specific antisemm. The protocol of Edwards and Ewing for t factor-
specific semm was followed in preparing t1 factor-specific antisera. Titers before and
after S. oranienberg antisemm was absortsed with S. montevideo is shown in Table
3.17. The protocol of Rauss (1939) was followed in preparing t2 and tS factor-specific
antisera and corresponding absorption titers are shown in Table 3.18 and Table 3.19,
respectively. The f factor-specific antisemm was more difficult to obtain. The Edwards
and Ewing protocol (Ewing, 1986) involves absorbing S. cfenby antisemm with S. dublin,
S. montevideo and S. senftenberg cultures. However, as shown in Table 3.20, S.
derby antisemm absorbed under these conditions was still reactive with S. budapest, a
non-f antigen containing serovar. Several trial absorptions using S. derby, S. berta and
S. budapest antisemm were conducted (Table 3.20) to detenmine if the cross-reactivity
with S. budapest could be eliminated. The f-serum protocol of Rauss (1939) involves
absortDing S. cfenby with S. dublin, S. enteritidis, S. montevideo, S. moscow, S. mstock,
S. berta and S. Senftenberg. Since absorption with S. berta or S. budapest gave
similar titers and S. berta expresses an f antigen, S. budapest was substituted for S.
berta in the protocol of Rauss. A trial absorption of S. derby antisemm with S. dublin,
S. enteritidis, S. moscow, S. rostock, S. Senftenberg and S. berta showed a titer of 320
with S. derby H suspension, a titer of 20 with S. dublin, and no detectable reactivity with
S. berta. This absorption scheme was followed in preparing bulk anti-S. cfenby f factor-
specific serum and titers before and after absorption are shown in Table 3.21.
g^erolooical Identities of fliC Antigens
The serological identities of the fliC antigens of Salmonella serovars (Table
3.22) and recombinant clones (Table 3.23) expressing the fliC gene were confimied by
the motility inhibition test (Gard, 1938) using factor-specific antisera. Serovars and
clones expressing p, q, s or u flagellar antigens were inhibited in semi-solid media
^
Table 3.16. Titration of S. oranienberg antisemm before and after absorption to obtain m2 factor-specific antisemm.
75
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption
4000 8000 <500 <500 1000 1000 <500 16000 <500 1000
dilution
1:5 1:5
1:5
1:5 1:5
1:5
after absorption''
<10 20 <10 <10 <10 <10 <10 640 <10 40
''The m2 factor-specific semm was prepared by absortDing S. oranienberg {mf) antisemm with S. berta {gff), S. budapest {gf), S. dublin {gp), S. moscow {gq), S. senftenberg {gsf) and S. montevideo {gms). This absortDed sera detects the m in S. oranienberg, but not in S. enteritidis and S. montevideo.
76
Table 3.17. Titration of S. oranienberg antisemm before and after absorption to obtain t1 factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before absorption dilu
4000 8000 <500 <500 1000
after tion absorption''
640 640 <10 <10 <10
1000 1:4 <10 <500 16000 <500 1000
<10 1280 <10 160
''The t1 factor-specific semm was prepared by absorbing S. oranienberg {mf) antisemm with S. montevideo {gms).
Table 3.18. Titration of S. absorption antisemm.
berta antisemm before and after to obtain t2 factor-specific
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) rostock {gpu) senftenberg {gst)
before after absorption dilution absorption''
4000 4000 2000 1000 500
320 320 20 <10 <10
<500 1:4 <10 1000 1000 <500 2000
<10 160 <10 80
''The t2 factor-specific semm was prepared by absortDing S. berta (fgt) antisemm with S. derby (fg), S. enteritidis (g,m), S. montevideo (gms), S. moscow (gq) and S. mstock (gpu).
77
Table 3.19. Titration of S.senftenberg antisemm before and after absorption to obtain tS factor-specific antisemm.
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) mstock {gpu) senftenberg {gst)
before absorption
500 8000 <500 <500 <500 4000 <500 <500 <500 8000
after dilution absorption''
160 1280 10
<10 <10
1:4 <10 <10 160 <10 1280
''The tS factor-specific semm was prepared by absorbing S. senftenberg (gst) antisemm with S. denby (fg), S. enteritidis (g,m), S. montevideo (gms), S. moscow (gq) and S. rostock (gpu).
78
gco (0 0
"Ji? "P "o 0 C fli a. Vi
io"2 ^3 • 5 rtf O 0 •*..
C • 0 (
.o o o 4 ^
CO
c
ptio
o CO
0
"co •c 4.^
o oT CD Oi ^~
tock
er,
CO XJ c 0 CO
c 2 0
yi cf 0 . . c 3 ^
CO 2f "O
^
CO
«
o o 0 0 0 ^ CO
0 4 - ^
h-
5> 0 •
5> Jfc 0 CO
CO
Vi
•ic CJ
5 CO
lo e 0 , . Q.CO
3 43
CO 3
>»-Q
St (
Jo
0 • • - •
0 Xi
C CO g
iggl
utin
at
CO
o O E 0 >
C
atio
i
k .
Tit
ci CN CO 0 sn 0
•a
ui c .2 0 • >
0
<
E 3 0 CO
' • ^
C 0
5> 0
c 0
2 o
. CO v.-o
o o CO
o E CO d' E
5
o E
^ c g e-o CO n to Ui 0
4 - ' 0 o T3 C
c g
4-» 0
^ r f
3 C3> CJ) 0
O) c > 5 CO
c g _3
^
E 3 0 CO
4 - ' CO 0 ^ C3>
0 .c 4 .^
c g CO c 0 CM CO 3 CO
jO
"0 c: o
0 CO ^
«
3 •Q
0 •Q
E S 2 2 ? 2 m "
iS o
111 o o C CO 0 0
CO
o ^ ^ o ^ ^ o - c c - c c -
° c c c c c ^
C J 4.^ 4.^ 4 - ' 4.^ 4.^ 4.^
T: c cz c c c c
o ^ ^ .^ .^ .^ o T7 c c c c c T7
o ^ .^ o ^ ^ ^ V ^ c : - «= c c
en ^ ^ ^ ^ ^ o ^ c c c c c -
4-> O •*-' C 00 c
r o C CN
O O O O O O CN 2 S CN CN CD CN CO ° ° ® CO CO T- CO
O 2 2 O O " ^ ^ v v T T 00 v v
o o o o V V v
0 3 3 J3 43 ^
-, r: 1^ . 0 0) 0 0 CO CO CO CO - - VI •^i "*
Ui 3 3 J3 3 : x 2 -6 "O T3 -2 1^ R r R 0 0 0 0 . 0 TJ "O T3 T3 T3 CO CO CO CO CO
O O
V V
3 •Q
0 0 0* CO - Q CO
e 2 8 o" d^ 6 E E E
0 0 0
=»" 3" .2^ "O T3 T3 R r R 4U -S -S CO CO CO
• ' J .
^ ^ ^ ^ ^ CO 0 0 0 ^
c c: c 0 0 0
C C C C C. Cl
c c c c c c
c c c c c c
C d C C d c
c c c c c c
c c c c c c
ccLCiCiCic:
o o o g o g <X) CN ^ -^ ^ ^
s°i°§i o o 2 o o o g^ T- ^ CN 00 00
c c c
c c: c
c c c
d c c
c c c
c c c
c c c
o ? ? CN V V
O c-> O '^ 2 CD CD 00
o o o V v v
0 CO
o o 3 OT C
=5 ^ X5 "D
0 0" 0* . . CO CO CO
fc ••.r H-T " ^ a E E E 0 . « . CO 3 3 3 - ^ --. (O "D "O T3 ^ ^ 0 § R R R R R R 0 0 0 0 0 0 0 .Q -Q -Q -Q -Q -Q -Q
CO CO CO CO CO CO CO I .1 .1 . ' .J . .J . - i
'•^ ""J— • * 5 * ^ ' ^ ' ^ " ^
c c c c: c c c 0 0 0 0 0 0 0
E 3 0 CO
0 CO
0 0
CO CO
^ E E E CO 0 S"
t l 3 3 O 412
3 3 "D "O R R 3 3 <Q 43
CO CO CO CO I . 1 . 1 . j _
4 .^ '«.,< 4 w 4..I
c c c cr 0 0 0 0
•o 0 "co 0
Table 3.21. Titration of S. derby antisemm before and after absorption to obtain anti-S. denby f factor-specific semm.
79
Salmonella H suspension (flagellar antigens)
berta {gff) budapest {gf) derby {gf) dublin {gp) enteritidis {gm) montevideo {gms) moscow {gq) oranienberg {mf) mstock {gpu) senftenberg {gst)
before absorption
4000 4000 16000 4000 2000 <500 2000 <500 4000 2000
dilution
1:4
1:4
1:4
after absorption''
10 20 640 20 <10 <10 <10 <10 10
<10
''The f factor-specific semm was prepared by absortDing S. derby {gf) antisemm with S. budapest (gt), S. dublin (gp), S. enteritidis (gm), S. msocow (gms), S. mstock and S. senftenberg {gsf).
80
0 ) 0 )
o .£ 0 <2 o ^
tl :2 0
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82
containing p, q, s, or u factor-specific antisera, respectively. Based on motility
inhibition experiments, S. chaco, S. daynsz, S. enteritidis, S. essen, S. jena and S.
montevideo are identified as serovars expressing the m1 flagellar antigen, whereas, S.
califomia, S. monschaui and S. oranienberg were serovars expressing the m2 antigen.
Motility inhibition experiments showed the f in S. cfenby to be different from the f in S.
adelaide and S. berta. The flagella of S. berta, S. budapest, S. califomia, S.
monschaui and S. oranienberg were found to express the t1 and t2 flagellar antigen.
The only serovar whose motility was inhibited with t3 factor-specific semm was S.
senftenberg, a serovar which was found to also express the t1 antigen.
Eoitooe MaooinQ on Polvethvlene Pins
On the basis of the determined amino acid sequences for the central portion of
the g... series flagellins from S. berta, S. budapest, S. derby, S. dublin, S. enteritidis, S.
montevideo, S. moscow, S. oranienberg , S. mstock and S. senftenberg, progressive
overiapping octamers were synthesized on polyethylene pins and screened by ELISA,
using prepared antisera as primary antibody. Sequential overiapping octameric
peptides were non-reactive with all antisera tested. These results support the
conclusion drawn from the g... series amino acid comparison; the epitopes of the gf...
series may be conformational.
Eoitooe MaoDino bv the Novatope Svstem
The flagellin of S. moscow was analyzed using the NovaTope System
(Novagen, Inc.). Five different bacterial clone libraries were created with this system:
(a) a library derived from the DNase I fragmentation of the fliC gene, (b) a library
derived from a PCR product encompassing nucleotides 1 to 540 (amino acids 1 to 180)
of the fliC gene, (c) a library derived from a PCR product encompassing nucleotides
843 to 1479 (amino acids 281 to 493) of the fliC gene, (d) a library derived from a PCR
product encompassing nucleotides 524f to 870 174d to 290) of the fliC gene, and (e) a
library derived from DNase I fragmentation of the PCR product used in (d) above. Each
library contained bacterial clones which expressed a portion of the S. moscow flagellin.
The library was screened by standard colony lift methods using an antibody as the
probe.
The first library created contained bacterial clones with inserts derived from
DNase I fragmentation of the fliC gene. After screening approximately 2200 clones by
protein expression, five clones reactive with S. moscow antisemm were obtained and
83
DNA sequencing showed four of the five clones to be different. As shown in Figure
3.8, these four clones (N0VA1, N0VA2, N0VA3, and N0VA5) contained sequences
overiapping in the central portion of the flagellin. To further discriminate reactive
regions of the flagellin, three libraries were created, each containing either amino-
temninal (amino acid residues 1-180), cartDOxyl-temninal (amino acid residues 281-493)
or a central area (amino acid residues 174d-290) fragment of the flagellin. Colony
hybridization was used to enrich for bacterial clones containing the appropriate insert.
Positive hybridization clones were sequenced to determine orientation of the insert.
Clones containing insert in Ciorrect orientation were screened by protein expression. As
shown in Figure 3.8, clones expressing either the amino-terminal (NOVA33) or carboxyl
terminal fragment (NOVA35) were non-reactive with S. moscow antisemm. Clones
(i.e., NOVA22) expressing a fragment derived from the central region (amino acid
residues 174d to 281) of the flagellin were partially reactive, showing a hybrid pattern of
reactivity. The fifth library screened contained clones, each expressing a different
length fragment derived from DNase I digestion of the central region (amino acid
residues 174d to 281) PCR product. Approximately 11,000 clones from this library
were screened by protein expression with no reactive clones detected. Interestingly,
when 48 clones from this library were analyzed for presence of a plasmid with insert, 34
(70.8%) recombinant clones were found. As shown in Table 3.24, out of the 34 clones
containing inserts, 10 were sequenced and only 3 of the 10 contained inserts in the
correct orientation with only 1 (NOVA 36) in the con-ect reading frame. The smallest
and largest size insert sequenced was 79 bp (NOVA36) and 343 bp, respectively.
Interestingly, clone 33 was not only backwards in orientation, but also had an added
TATAT sequence at its 5' end. Clone 19 was backwards and had an extra 'A at its 3'
end.' N0VA1, N0VA2, N0VA3, N0VA5 and NOVA22 clones were nonreactive when
screened with all factor-specific antisera and all unabsortDed antisera.
84
N0VA1
NOVA22
^175to28r
(174dto290)
(231 to 379)
(232 to 379)
N0VA3
NOVA33 C (1 to 180)
(267 to 290) NOVA361 I
N0VA2
NOVAS
(281 to 493) NOVA35 L
Region IV iVi VII VII VIII
Amino acid 1 100 160 180 299 322 359 420 493
Salmonella moscow phase-1 flagellin
Figure 3.8. Locations of S. moscow fliC inserts from positive ( • ) and negative (D) reacting recombinant clones screened with S. moscow antisemm. Position numbers are based on amino acid alignment with fliC^ (HI-A) as shown in Appendix B and equivalent to those previously reported (Wei and Joys, 1985) for the fliC^ flagellin.
^
85
Table 3.24. Location, size and orientation of recombinant clones. Position numbers are based on amino acid alignment with fliC^ (HI-A) as shown in Appendix B and equivalent to those previously reported (Wei and Joys, 1985) forthe/7/C3 flagellin.
Clone no.
Clone 6
Clone 9
Clone 10
Clone 19
Clone 26
Clone 29
Clone 31
Clone 33
Clone 35
Clone 36 (NOVA 36)
Location of nucleotide fragemnt
554 to 723
554-677
713 to 814
'A' plus 5931 to 828
unknown to 846
524eto714
526 to 846
524k to 798 plus TATAT'
717 to 869
798 to 869
Size of fragment in base pairs
173
127
108
237
unknown
205
343
287
160
79
Insert orientation
backward
forward (not in frame)
fonvard (not in frame)
backward
backward
backward
backward
backward
backward
fonvard (in-frame)
CHAPTER IV
DISCUSSION
In this work, I have sequenced the Salmonella fliC genes, encoding the phase-1
flagellins from 17 members of the g... series. The polymerase chain reaction was used
to amplify the fliC gene and a single PCR product was obtained for sequencing and
cloning. Cloned PCR-derived DNA sequences agreed with PCR product-derived DNA
sequences, except for S. dublin, in which 14 nucleotide sites were in contention (Table
3.3). Taq DNA polymerase was the enzyme used for DNA sequencing and to amplify
the fliC gene in vitro by the polymerase chain reaction. Taq DNA polymerase is a
thermostable DNA-dependent DNA polymerase which lacks editing functions and
incorporates an incorrect nucleotide at a rate of 2 X 10"^ nucleotides per cycle in
polymerase chain reactions. This rate of misincorporation translates into an overall
error frequency of 0.25% in a 30-cycle amplification (Saiki et al., 1988). For some
reason, the fliC gene of S. dublin had a high rate of misincorporation so that an
individual DNA molecule cloned from the amplified pool was unreliable. As the main
concern of this work was in defining epitopes, this problem was not pursued. A
consensus sequence for S. dublin was obtained upon sequencing several clones.
A comparison of the flagellin amino acid sequences (Table 3.4 and Table 3.5)
showed complete homology in the N-terminal (regions I, II and III) and C-terminal
(regions VIII) segments of the proteins. Differences in amino acids were found
throughout (regions IV, V, VI, and VII) the central portion of the flagellins. No localized
area specifying subfactor epitopes could be identified, suggesting that the subfactors of
the g... series are conformational at the molecular level. The amino acids comprising
each of the subfactor epitopes were not definable by sequence analysis. However, at
amino acid position 209, an aspartic acid residue found in S. senftenberg (///C^'S.^ and
S. montevideo {fliCg'^'^ flagellins may contribute to the expression of the s flagellar
antigen. All other g... series serovars analyzed showed a threonine or alanine amino
acid at this position. Alanine and threonine are both hydrophobic amino acids, with
threonine being slightly larger in size and polar. An aspartic acid residue is similar in
size to threonine, but is charged and polar (Sambrook et al., 1989). At amino acid
position 283, S. moscow {fliC9'9), the only serovar analyzed whose flagellin expresses
the q antigen, shows a glycine residue while all other g... series serovars show an
aspartic acid. Glycine is an amino acid which is small and non-polar and due to its one
hydrogen side chain, allows unusual chain confomiations in proteins.
86
87
Our expectation was that the linear epitopes which comprise the subfactors of
the gf... series would be localized to one region, providing an efficient site for directed
substitution with medically important epitopes. Although our expectation was not bome
out, a potential site for epitope substitution was observed upon comparison (Table 3.6)
of the gf... series flagellins with flagellin a and flagellins c, d, i, and rand E. co//flagellin.
Only in region V with the g... series flagellins did we find extremely low (4%) or
absolutely no homology with flagellin a, suggesting that this region in the gf... series has
potential for site directed substitutions. Joys and Schodel (1991) located a major
epitope of antigen d within region V. Interestingly, in region V, S. oranienberg and S.
monschaui which have no gf antigen but are members of the gf... series due to their m
and t antigens, demonstrated only 6 1 % amino acid homology (Table 3.5) with other
members of the g... series. A third serovar showing only 61% amino acid homology in
region V was S. califomia whose flagellin expresses the m and t antigens along with gf.
Other members of the g... series exhibited 78 to 100% homology (Table 3.5) in region
V. This information suggests that the fliC gene of S. oranienberg, S. monschaui or S.
califomia may be the gene of choice for substitution(s) since it may tolerate amino acid
interchanges in region V better than the fliC gene of other members of the g... series.
Hydrophobicity and surface exposure plots (Figure 3.1 to Figure 3.6) based on
g... series flagellin sequences suggested three potential sites for epitope substitution
within the flagellin of any g... series member. The largest and most hydophilic site was
found in region VI and embodied 11 amino acids demonstrating exposure to the
surface. Two other smaller, and less hydrophilic sites were found within region IV and
at each location a 9 amino acid cluster demonstrated exposure to the surface.
Interestingly none of the potential sites for epitope substitution identified by
hydrophobicity and surface exposure analyses are located within region V, the region
showing a very low degree of amino acid homology.
Based upon phylogenetic analyses (Figure 3.7) and comparative analyses of
Salmonella fliC a, g... series, c, d, i, and r and E. coli flagellins (Table 3.6), S.
oranienberg, S. monschaui and S. califomia flagellins expressing antigens m2 in
combination with t1 and t2 show the greatest diversity from all salmonellar flagellins
analyzed to date. The S. berta flagellin shows some similarity to these three flagellins
and also differs from the other flagellins. S. oranienberg, S. monschaui, S. califomia
and S. berta flagellins share the presence of threonine, valine and proline, respectively,
at amino acid position 213 to 215 (Table 3.4). The 14 non-m2-containing g... series
flagellins fonn one group with S. berta alone branching away from the other 13. The
88
g... series flagellins branch from a common ancestor which also gave rise to a non-g...
series branch which segregates into two offshoots, one consisting of the E. coli K-12
flagellin and the other consisting of the single factor Salmonella phase-1 flagellins and
the phase-2 flagellin of S.abortus-equi. This division is interesting since all of the single
factor phase-1 flagellins and the phase-2 flagellin contain histidine, while the E. coli K-
12 flagellin and the g... series flagellins do not. Also, the majority of the g... series
members are monphasic (Table 3.1), while the single factor serovars are diphasic,
except for S. paratyphi. The ancestor to the Salmonella flagellins and the E. coli K-12
flagellin also gave rise to a branch containing E. coli HI , H7 and H12 serotypes.
Interestingly, the flagellin of E. coli K-12 is grouped with the Salmonella non-g... series
flagellins, although serological (Ewing, 1986) and molecular weight (McDonough and
Smith, 1976) analyses show E. co//flagellins to be even more diverse than Salmonella
flagellins. E. coli K-12 may not have been grouped with the other E. coli serotypes due
to undertying sequence differences which contribute to disparity in their flagellar
filament morphotypes (Lawn, 1977; Lawn et al., 1977). E. coli HI , H7 and H12 are
members of the E classification of flagellar filament morphotypes, while E. coli K-12 is a
member of the C morphotype group. Possibly the sequences which contribute to the C
morphotype fonn share more homology with the Salmonella single factor flagellins and
phase-2 flagellins than with the E morphotype-expressing E. coli serotypes.
The Edwards and Ewing (Ewing, 1986) protocol for 'Preparation of AbsortDed H
Antisera" was followed in preparing p,q,s,t (labeled t1 in our lab) and u factor-specific
sera, while the protocol by Rauss (1939) was following in preparing f, t2 and t3, with a
slight modification for f Neither protocol addressed possible subdivisions for m factor-
specific antisemm. To obtain m specific antisemm by the Edwards and Ewing (Ewing,
1986) protocol, a semm mixture of S. enteritidis and S. oraneinberg is absortDed with S.
berta, S. budapest, S. dublin, S. moscow and S. senftenberg. An older version of the
Edwards and Ewing protocol (1962) describes absortDing m,t semm by double
absorption with S. senftenberg and S. berta. To obtain m specific antisemm by Rauss.
S. oranienberg antisemm is absorbed with S. berta and S. senftenberg. Interestingly,
when I absortDed S. enteritidis antisemm with S. berta, S. budapest, S. dublin, S.
moscow and S. senftenberg (Table 3.13) and S. oranienberg antisemm with S. berta,
S. budapest, S. dublin, S. moscow, S. senftenberg and S. montevideo (Table 3.15),
different patterns of anti-m reactivity were observed in motility inhibition experiments
(Table 3.22 and Table 3.23) with these two antisera. The absortDed S. enteritidis
antisemm, labeled m1, inhibited swamiing by S. chaco, S. daynsz, S. enteritidis, S.
89
essen, S. jena and S. montevideo. The absortDed S. oranienberg antisemm, labeled
m2, inhibited swamning by S. califomia, S. monshcaui and S. oranienberg.
Phylogenetic analysis (Figure 3.7) also showed a differentiation of m by segregating
m1 and m2 expressing serovars into two separate groups. Atkinson (1943) found the
f,g of S. derby and S. berta to be different than the f,g of S. adelaide. Motility inhibition
experiments (Tables 3.22 and 3.23) with anti-S. derby f specific semm support
Atkinson's conclusions and further suggests the f,g in S. berta to be different from the
f,g in S. cfenby Trial semm absorptions to obtain f factor-specific semm (Table 3.20)
suggest an unidentifiable cross-reactive component between S. berta and S. budapest.
The attempt to map, on polyethylene pins, the g... series phase-1 flagellin B-cell
epitopes was unsuccessful. This may be due to at least two reasons. If the epitopes
are confonmational, as suggested by the amino acid comparison of the g... series
flagellins, then screening for continuous epitopes would result in nonreactivity.
Secondly, the noise:signal ratio was high giving a high background with all antisera
tested.
As an alternative approach, epitope mapping with bacterial clones, each
expressing a small peptide derived from the flagellin of S. moscow (fliC99) was
attempted with no success. In theory, this method allows for the detection of
confonmational and linear epitopes, depending on the size of the insert being screened
against. Based on the number of clones I screened and the probability I calculated of 1
in 10 clones being in the correct reading frame, I expected to find at least 1,400
reactive clones if the B-cell epitopes were linear. After screening approximately 14,000
clones with S. moscow antiserum, only five reactive clones were obtained. N0VA1,
N0VA2, N0VA3, N0VA5 and NOVA22 contained inserts expressing peptides 87, 134,
298, 135 and 94 amino acids in length, respectively and overiapping in the central
region of flagellin. However, when these clones were screened with q factor-specific
antisemm, no reactive clones were obtained. The same results were found when these
clones were screened with all unabsortDed antisera. Although N0VA1 and NOVA22
expressed the exact amino acid sequence found in S. berta, S. derby, S. dublin, S.
enteritidis S. montevideo, and S. mstock, these clones were nonreactive when
screened with all subfactor-specific antisera and all other unabsorbed sera. As
mentioned above, based on amino acid comparison, a glycine residue at amino acid
position 283 may contribute to the expression of the q antigen in the S. moscow
flagellin. However, N0VA2, N0VA5 and N0VA3 clones all express this residue but
were nonreactive when screened with q factor-specific antisemm. Epitope mapping
mm
90
using bacterial clones did not identify the g or g epitopes present in the S. moscow
flagellin.
Recently, Selander et al. (1992) published data showing the variations in the
sequences of fliC flagellin genes from isolates of S. enteritidis (En 1: fliC g'^ and S.
dublin (Du 1 and Du 3: fliC g-P; "Du 2": fliC g>P>rrt), The fliC sequence of S. dublin {fliC
g'P) reported by us differs from that reported by Selander et al. (1992) in having 9
synonymous (amino acid positions 52, 54, 120, 239, 261, 307, 360, 382, and 388) and
6 nonsynonymous (amino acid positions 282, 305, 306, 310, 312, and 372)
substitutions when compared to Du 1 and Du 3, and in having 11 synonymous (amino
acid positions 52, 54, 59, 120. 137, 239, 261, 307. 360, 382, and 388) and 7
nonsynonymous (amino acid positions 210. 282. 305, 306, 310. 312, and 372)
substitutions when compared to "Du 2". Based on their fliC comparison, Selander et al.
(1992) deduced that the presence of alanine at amino acid position 309 is responsible
for the expression of the phase-1 flagellin p subfactor, while alanine at amino acid
position 210 and threonine at amino acid position 306 contributes to the m subfactor.
Interestingly, our phase-1 flagellin g... series comparison (Table 3.4) showed that at
amino acid position 309, alanine is present not only in S. dublin {fliC gP), but also in six
other g... series serovars, of which only one, S. mstock, manifests the p subfactor. At
amino acid position 210, alanine is present in S. chaco {fliC9'^, S. daynsz {fliC9>'^)' S.
enteritidis {fliC S''^, S. essen {fliC S^'^. S. jena {fliC ^ ' ^ and S. montevideo {fliC
9,m.S) as well as in four other non-n? subfactor serovars. Also, alanine is not present at
amino acid position 210 in S. califomia, S. monschaui and S. oranienberg, although
these flagellins have m and t antigens. At amino acid position 309. threonine is present
in all n7-expressing serovars. but it is also present in S. moscow {fliC 9'9), a non-m
subfactor serovar. Selander et al. (1992) also stated that the fliC sequence of S.
mstock (fliC g'P'U) differed from that of Du 1 {fliC g>P) only at amino acid position 305,
where glycine was present in S. mstock. Selander et al. (1992) suggested that this
glycine was responsible for the expression of the u subfactor in the S. mstock phase-1
flagellin. However, we found glycine at amino acid position 305 in S. oranienberg {fliC
^'^ as well. In addition, the comparison of our S. dublin {fliC gP) and S. mstock (fliC
g>P'U) fiiC sequences showed six nonsynonymous differences between the two
serovars. Thus, Selander's explanations do not form a basis for the serology results
when the data base is expanded.
Flagella are powerful immunogens, and the large number of Salmonella
flagellar antigens described by the Kauffmann-White Scheme (Kauffmann. 1964)
^ /
91
attests to their critical role in diagnosis and epidemiology of infection by Salmonella.
Based upon the data here and other genetic studies (Parish et al.. 1969; lino, 1977;
Wei and Joys. 1985; Joys. 1985). it seems likely that most, if not all, fliC flagellins of
Salmonella have been derived from an ancestral type which with time has tolerated
changes in the antigenic domains of the flagellin, as long as the changes were
compatible with the constraints imposed on flagellin in terms of con ect assembly,
stmcture and function. Both the accumulation of spontaneous mutations (Wei and
Joys. 1985) and zenologous (Gray and Fitch. 1983) replication (Selander and Smith,
1990, Smith, 1991, Smith et al., 1990) has been suggested to explain serotypic
variation in flagellin. Based upon the data presented here, it appears that a
combination of both mechanisms is likely to have occun^ed.
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Arnon. R. and M.H.V. Van Regenmortel. 1992. Stmctural basis of antigenic specificity and design of new vaccines. FASEB. 6:3265-3274.
Asakura. S. 1970. Polymerization of flagellin and polymorphism of flagella. Advan. Biophys. 1:99-105.
Astbury, W.T., E. Beighton and C. Weibull. 1955. The stmcture of bacterial flagella. Symp. Soc. Exp. Biol. 9:282-305.
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Brey, R.N., G.S. Bixlerand J.P. Fulginiti. 1991. Oral delivery of antigens in live baciterial vectors. Adv. Exp. Med. Biol. 303:169-184.
Calladine, C.R. 1978. Change of waveform in bacterial flagella: The role of mechanics at the mdecular level. J. Md. Biol. 118:457-479.
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APPENDIX A
NUCLEOTIDE ALIGNMENT OF 17 MEMBERS OF THE SALMONELLA
FliC FLAGELLIN g... SERIES AND c, d, i, AND r AND THE
E. COLI K-12 FliC FLAGELLIN WITH FliC FLAGELLIN a
Abbreviations: H1-A, S. paratyphi {y\le\ and Joys, 1985); AD, S. adelaide; BE, S.
berta; BU, S. budapest, CA, S. califomia; CH, S. chaco; DA, S.danysz; DE, S. derby;
DU, S. dublin; EN, S. enteritidis; ES, S. essen; MS, S. monschaui; MT, S. montevideo;
MO, S. moscow; OR, S. oranienberg; RO, S. mstock, SE, S. senftenberg; H1-C, S.
cholerae-suis (Wei and Joys. 1985); H1-D, S. muenchen (Wei and Joys, 1985); H1-I, S.
typhimurium (Joys, 1985); H1-R, S. mbislaw (Wei and Joys, 1986); E. coli, E. coli K-12
(Kuwajima et al., 1986).
1 2 5 5 0 + + +
HI - A GCACAAGTCATTAATACaUVACAOCXrrGTCXSCrrGTTGACCCAGAATAACCrrGAACAAATCCCAGTCCGCTC AD (SCACAAGTCATTAATACAAACy^CSCCTaTCCKTrGTTGACXXAGAATAACCrrCSAACAAATCrrCAGTCCTCAC BE GCACAAOTCATTAATACAAACAOCCrrGTC(3<?rGTTGACCCAGAATAACCrraAACAAATCrrCAGTCCrrCAC BU (KyVCaUUSTCATTAATACAAACy^GCCrrGTCCSCTGTTGACCCAGAATAACCTaAACa^AATCTrCAOTCCTCAC CA (3CACAAOTCATTAATAC:AAACAOCCrrGTCOCTGTTaACX:CACSAATAACCrraAACAAATC7rCAGTCC7rCAC ca GCACAAOTCATTAATACAAACAGCCTGTCCXrrGTTGACCCAaAATAACCrrGAACAAATCrrCAGTCCTCAC DA (3CACAAOTCATTAATACAAACAGC(rrGTCGCTGTTaACCCAaAATAACCrrQAACAAATCTCAGTCCrrc:AC DE CXZACAAGTCATTAATACAAACAGCCrrGTCGCTGTTGACCCAGAATAACCrrGAACAAATCTrCAGTCCrrcy^C DU CKIACAAGTCJ^TTAATACAAACAGCCrTGTCCXrrGTTGACCCAGAATAACCn'GAACAAATCrrCAGTCCTrCAC EH GCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCrrGAACAAATCTCZAGTCCTCAC ES GCACAAGTCIATTAATACAAACAGCCTGTCCXrrGTTGACCCAGAATAACCrrCSAACAAATCrrCAGTCCrTCAC JE GCACa^AGTCATTAATACAAACAGCCrrGTCGCrTGTTGACCCAGAATAACCrrGAACAAATCTCAGTCCrrCAC MS (Xa^CAAGTCrATTAATACy^AACAGCCrrGTCCSCTGTTGACCCAGAATAACrCTCjAACAAATCrrCAGTCCrTCAC MT GCACAAGTCATTAATACAAACAOCCTGTCCJCTGTTCjACCCy^GAATAACCrrCSAACAAATCTCAGTCCTCAC MO GCy^CAAGTCATTAATACAAACAGCCTGTCGCTGTTC3ACCCAGAATAACCrrGAACyU^TCTC:AGTCCTCAC OR GCACAAGT<yiTTAATACy^AACyVGCCTGTCC5CrrGTTGACCCAGAATAACCTGAACAAATCTCyVGTC<7rCAC RO GCACAAGTCATTAATACAAACAC3CCrrGTCCXrrGTTC3ACCCAGAATAACCTGAACAAATCrrCAGTC(rrCAC SE GCZACAAGTCATTAATACAAACy^GCCrTGTCMCTGTTQACCCAGAATAACCTGAACAAATCTCAGTCCTCAC HI - C GCACZAAGTCATTAATACZAAACAGCCrrGTCCXrrOTTaACCCy^GAATAACCrraAACAAATCCCAGTCCCSCTC HI - D GCAC:AAGTCATTAATAC:AAACAOCCTGTCCXrrGTTaACCCAaAATAACCTaAACAAATCCCAOTCCGCTC HI - 1 GCACAAGTCyVTTAATACAAACA£3CCrrGTCC3CTGTTGACCCAGAATAACCrraAACAAATCCCAOTCCC3CTC HI - R GCACAAGTCyVTTAATAC:AAACAaCCTOTCGCTGTTaACCCy^C5AATAACCrrC3AACAAATCCCAGTCCCXrrC E COLI GCACAAOTC:ATTAATACCAACAGCCrrCTCGCTaATCACrrCAAAATAATATCAACAAGAACCAGTCrrcx:c»C
102
103
HI AD BE BU CA CB
7 5 1 0 0 1 2 5 + + -- . + .
TGGGC^CCGCTATCGAGCGTCTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACQATGCGGCAGGTCA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCQAAAGACGATGCGGCAGGTCA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACC3ATGCGGCAGGCCA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACCSATGCGGCAGGCCA
DA TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGAC(3ATGCGGCAGGCCA
DE TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACaATGCGGCAGGCXA DU TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCXSTATCAACAGCGCGAAAGACGATGCGGCIAGGCCA EN TGAGTTCCGCTATTGAGCGTCTGTCCTCTGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGCCyv ES TGAGTTCCGCTATTGAGCX5TCrrGTCCTCTGGTCrrGCGTATCAACAGCGC<SAAAaACC3ATGCGGCAGGCCA JE TGAGTTCCGCTATTGAGCGTCTGTCCrrcrrGGTCTGCXSTATCAACAGCGCGAAAaACGATGCGGCAGGCCA MS TGAGTTCCG<rrATTGAGCGTC7rGTCCTCTGGTCTGCGTATCAACAGCGCaAAAaACGATGCG<X:AGGCCA MT TC5AGTTCCGCrrATTGAGCGTCrrGTCrrTCCGGTCrrGCGTATCAACAGCGCGAAAaACC3ATGCGGCAGGCCA MO TGAGTTCCCKTTATTGAGCGTCrPGTCCrrcrrGGTCTGCGTATCAACAGCGCGAAAGACCSATGCGCSCAGGCCA OR TGAGTTCCCKTTATTGAGCGTCrrGTCCrrCTCSGTCrrGCGTATCAACAGasaSAAAGACQATGCGCSCyUSGCXA RO TGAGTTC<:(3CTATTGAGCGTCrrGTCCrrc:T(KWCrr(KX3TATCAACAGCGCaWU^GACGATGCXX3CJU3GCCA SE TGAGTTCCGCn'ATTGAGCGTCn'GTCXrrCTGGTCTGCGTATCAACAGCGCGAAAGACXSATGCGGCy^GGCCA HI - C TG<3C3CACCG<?rATCQAGCX5TCrrGTCrrTCC(K5TCrrGCGTATCaUVCAGCGCGAAAGAC:GATGCX3GCAGGTCA HI - D T(3<3C3<::ACC(3<:TATCMAGCGTC?rGTCrrTCCMGTC7rGCGTATCAACAGCXXX3AAAGACaATGCG<3CAGGTCy^ HI - 1 TCKKK:ACC<XrrATCGAGCGTCrTGTCrrTCC(3K3TCn'CX:GTATCAACAGC(3CX5AAAGACGATC3C(30CyU3GTCA HI - R TG<3C3<::ACCCKrrATC(3AGCGTCrrGTCrrTCrrC3GTC7rCX:GTATCAACAGC(S<X3AAAGACC3ATGCG<3CAGGTCA E COLI TGTCX5AGTTC7rATCGAGCGTCrrGTCTTC?TG<3CTTC3CGTATTAACAGC(3C<3AAG<SATGACCK»GCCKXSTCA
1 5 0 1 7 5 2 0 0 + + +
HI - A GGCAATT(XrrAACCGTTTCACC(X:GAACATCAAAC3GTCrrGACTCAGG<7rTCCCGTAACX3CTAACXSM:CX3T AD (KX:GATTGCTAACC:(X7rTCACn'TC7rAATATCAAAG<3CCT(5AC7rCAG(3CTTCCX;GTAACCXrrAACC3ACaGT BE (3<3C(jATTGCTAACC(X7rTCAC7rTCrrAATATCAAAGCK:CTGACTCAOC3CTTCXXX3TAACX3CTAACXSACX3GT BU G<X:GATT(X7rAACCGCTTCACrrTCTAATATCAAAG<3TCrrGACTCAGGCTTCCX:GTAACGCTAAC»AC<;GC CA G<X:GATT(K7rAACCCXrrTCyvCrrTCTAATATCAAAGGTCTGACrrCAG<XrrTCC»:»TAAC<3<rrAACGACCK3C CH GGCGATTGCTAACCGCTTCy^CTrTCrrAATATCAAAGGTCrrGACrrCACKjCTTCCCXSTAACCXTrAACGACCKSC DA (KX:GATTGCTAACCG<7rTCACrrTCTAATATCAAAGGTCT(5ACrrcyU3<3<7rTCXX:GTAACG<::TAAC:GAC:(X3C DE G<3CGATT<XrTAACCGTTT<a^CGTCTAATATC»AAGGTCn'(3AC?rCAG<3C?rTCCXX}TAAC<3<rTAACaAC(3<3C DU GCX;(3ATT(3CrrAACCGTTTCACGTCTAATATCAAAGGT<7T(3ACTCAGGCTTC<X:GTAAC<3C:TAACC3AC(3C3C EN G<XX3ATTGCTAACCC3CTTCACrrTCrrAATATCa^AAGGTCrrGACTCAGC3<rrTCXXX3TAACXSC7rAAC<SACCXSC ES GGC(3ATT(3CTAACCC3CTTCACTTCTAATATCAAAOGTCrrGACTCawQC3<:TTCXXX»TAACCX7TAACCJAC:C3GC JE G<3C(3ATT(3CrrAACCCX7rTCACTTC:TAATATCAAAG6TCr(3ACrCAG<3CrrTCCX:GTAA£XJCTAACC3ACC3<3C MS G<3C(5ATTGCTAACC6TTTCACGTCTAATATCAAAC3GTC7TC3ACTCAC3<3CTTCX:CX5TAAC»3CTAACGACGGC MT G<X:C3ATTGCrAACCCK7rTCACTTC7rAATATCAAAGGTCTGACTC»GC3CrrTCCXX3TAAC(3CTAAC<3AC<3<3C MO GGCGATT(3CTAACCC3CTTCACTTCrrAATATCAAAGGTCn'GACrrC»GC3CTTCXX»TAACX3CTAACX3AC<3<X: OR G<3CGATT(KrrAACCGCTTCACrrTCn'AATATCAAAGGTCrrGACrrCAGGCTTCCCGTAACXX7rAACX3ACGGC RO GGCGATTGCTAACC:(3CTTCA<7rTC?rAATATCAAACX3TCrrC5ACTCAGGCTTCCXa3TAACX3CTAACX3ACGGC SE G<3C(3ATT(3CTAACCCXrrTCywCTTCrrAATATCAAAGGTC:TCSACTCAG<3CTTCX:CGTAAC<3CTAAC(SACG<3C HI - C G<3CGATTCSCrrAACCGTTTCACCCX:GAAC:yiTCAAAGGTC7rGACrrCAGC3CrrTCCCGTAACGCTAACaACXSGT HI - D GGCGATTG<rrAACCGTTTCACCCX:GAACATCAAAGGTCTGACrrC:AG<X7rTCCCGTAAC<S<rrAACGACGGT HI - 1 (3<3CGATTGCTAACCGTTTTACC(3CGAACATCAAAGGTCrTGAC7rCAG<XrrTCCCGTAAC(3CTAACC3ACGGT HI - R GGCGATTCXrrAACCGTTTCACXGCGAACATCAAAGGTCTGACTCAGGCTTCXXrGTAACGCTAACaAaSGT E COLI GGCGATTGCTAACCGTTTCACCTCrrAACATTAAAGGCCTGACTCAGGCGCXXrCXSTAACGCCAACGACGGT
104
2 2 5 2 5 0 2 7 5 + + +
HI - A ATCrrCC:ATTCX;CX:AfiACCACrrGAAGC3<:XK:<XrrGAACGAAATCAACAACAACXrrGCAG<:X3TGTGCGTGAAC AD ATCTCXATTCX:<3CAC3ACCACrr<5AAGC3C(3CGTTGAATGAAATTAACAACAACC7rCX»GCGTGTGCGTGAGT BE ATCn"CCATTGCX3CAGACCa^CrrC3AAGGCGCGTTGAATGAAATTAACAACAACXrr<;CyU3CGTGTGCGTGAGT BU ATTTCrrATTGCC3CAGACCACT(3AAGGTGC(3CTGAATGAAATCAACAACAACCn'GCAGCGTGTGCGTGAGT CA ATTTCTTATTCXXSCAGACXACrrGAAGGTCSCCSCTGAATCSAAATTAACAACAACCrrGCAGCGTGTGCXSTGAGT CH ATTTCn'ATTGC(XyU3ACCaVCn?(3AAGGTCK:<K?TGAATCSAAATCAACAACAAC<rrGGAGCGTGTGCGTGAGT DA ATTTCTTATTCKJCKAGACCJ^CTCaAAGGTCjCCSCTGAATGAAATCAACAACAACCrrGCAGCGTGTGCXjTGAGT DE ATTTCTATT(X:GCAGACCACTC3AAGGT(3CGCTC3AATC5AAATCAACAACAACCTG<:y^GCGTGTGCGTC3AGT DU ATTTCTATTCXXXaUjACCACTGAAGGTCSC(3CTGAATCaAAAT(:AACAACAACCn'GCAGCGTGTGCGTGAGT EN ATTTC:TATTCX;GCAGACC:ACn'(aAAGGTCX:CXrrC3AATC5AAATCAACAAC:AACC7rC3GAGCGTGTGCGT(5AGT ES ATTTCrrATTGCC3CyU3ACCy^CTGAA(3GTCX:C3CTC3AATQAAATCAACAACAACCn'(3CAGCGTGTGCGTGAGT JE ATTTCTATTCXXSCACaACCIACTCaAACKSTCXXKrrGAATCjAAATCIAACAACAACCTCKJVGCGTGTCKrGTGAGT MS ATTTC7rATT(K:(K:AGACCAC7T(3AAGGT(3C(3CTGAAT(aAAATTAACAACAAC(7r(3CAGCGTGT(3<:>3TaA6T MT ATTT(7rATT(3C(X:AGACXACTGAAGGC(3CG<rrGAATGAAATCAACAACAACC7r(X:AGCGTGTGCGTaAGT MO ATTTC7rATT(K:(3C:AGACCJU7rGAACX3TCX:(Xn^GAAT(SAAATC:AACAACAACC7r(X:AGCGTGT(3CGTGAGT OR ATTTCrrATTGCG<::AGA(XACT(3AAGGC(K:GCTGAATGAAATCAACAACAACCTGCAGCGTGTGCX3TaAOT RO ATTTCn'ATT(3C(K:AGACCACT(aAAGGTCKXKrrGAATGAAATCAACAACAACX7rGCJ^CX:GTGTGCGTaAST SE ATTTCrrATT(»^GCAGACC:AC7rGAAGGTGCGCTGAATGAAATTAACAACAACCTGCAGCGTGTCKX}TaAST H l - C ATTTCTATT(X:(X:AGACCACTGAACXX:(3C(X7rGAACGAAATCAACAACAACCrrCK:AGCGTGT(3CGTGAAC H l - D ATC7rCCATTGC(X:AGACCAC7r(3AAG<3CGCGCTC3AACGAAATCAACAACAACC7rCKJUK:(3TGTGCGTaAAC H I - I ATC7rCC:ATTGCGCAGACCACTGAAG<3C(K:(X7r(SAAC(3AAATCAACAACAACXTGCA6CGTGTGCGTaAAC H l - R ATCTCX:ATTGC(K:AGACCACn^(SAAG(3CGC:(3CTGAAC(3AAATCAACAACAACCrrGCAO<:GTGTGCGTGAAC E COLI ATCrrCX:GTTGC(3CAGACCACCGAAGC3C(3C(X7rGTCXX3AAATCAACAACAAC:TTACAGCGTGTGCGTaAAC
3 0 0 3 2 5 3 5 0 + + +
HI - A TCXX:CjGTTCAGTCrrCSCTAACAGCACCAACrrCX:CAGTCrrGAC<rrC(3ACn'(X:ATCCAOC3CTGAAATCACX:CA AD TGTC7rGT(3CAG<5CCACrrAAC<K5QACn'AAC?rC7rGATTCCX5ATCrrGAAATC:TATCX:ACK3ATGAAATCC:AGCA BE TGTCrrGTGCAG<3CCA<rrAACC3k3C3AC?rAACTCrr(3ATTCCGATCrrGAAATC7rATCCAG<3ATC5AAATCCAOCA BU TGTC7rGTTC»G<X:CACTAACG<3GACTAAC:TCrrGATTCCGATCrrGAAATCrrATCCAGGATGAAATTCAGCA CA TOTCrGTIGAGGCCACTAACGGCSACTAACTCrrGATTCCXSATCrrGAAATCrTATCCAGCSATGAAATTCAGGA CH TGTCTTGTTCAGCXXACrrAACGGGACn'AACTCTGATTCXXSATCTGAAATCTrATCCAGGATGAAATTCAGCA DA TGTCTGTTCAGGCCAC:TAACGGGAC7rAACTCTGATTCCGATC7rGAAATCTATCX:AG<3ATQAAATTCAGC:A DE TGTCTGTTCAG<3CCACTAACGGGACTAACrrCTGATTCCGATCrTGAAATCTATCCAGGATGAAATTCau3CA DU TGTCTTGTTCAGGCCACTAACGCKSACTAACTCTCSATTCCCSATCrrGAAATCTATCXZAGGATGAAATTCAGCA EN TGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATCSAAATTCAGCA ES TGTC?rGTTCAGGCCaiCrrAACG<3GACTAACTCrrGATTCCGATC7rGAAAT(n?ATCCAGGATaAAATTC»GC:A JE TGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTCSAAATCTATCCAOaATaAAATTCAOCA MS TGTCTGTTCAGGCCACrrAACGGGACTAACrrCTGATTCCGATCTGAAATCTATCCAGaATaAAATTCAGCA MT TGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGOATGAAATTCAGCA MO TGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCC(3ATCTC5AAATCTATCCAGGATGAAATTCAGCA OR TGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCA RO XGTCTGTTCAGGCCACTAACGGGACTAACTCTGATTCCGATCTC5AAATCTATCCAGGATGAAATTCAGCA SE TGTCTGTTCAGGCCACTAACGGGACTAACTCTC3ATTCCGATCTGAAATCTATCCAGGATGAAATTCAGCA HI C TGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTCSACCTCGACTCCATCCAGGCTaAAATCACCCA HI D TGGCGGTTCAGTCTGCTAACGGTACTAACTCCCAGTCTGACCTTGACTCTATCCAGGCTGAAATCACCCA HI T TGGCGGGTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCGACTCCATCCAGGCTaAAATCACCCA H i ' i TGGCGGTTCAGTCTGCTAACAGCACCAACTCCCAGTCTGACCTCaACTCCATCCAGGCTGAAATCACCCA E COLI TGACGGTACAGGCCACTACCGGTACTAACTCTGAGTCTGATCTGTCTTCTATCCAGGACGAAATTAAATC
105
3 7 5 4 0 0 + +
HI - A CK:CK:CTGAAC(5AAATC(3ACCGTGTATCCS3GTCAGACTCaVGTTCAACX3GCGTGAAAGTCCrrG<3<:GCAGGAC AD ACGTC7rc;GAAGAAATCGATCCK:GTTTCn'AATCAGACTCy^TTTAAC(3GTGTTAAAGTCX7rGTC7rCAG(jAC BE ACGTCn'CKSAAGAAATCGATCGCGTTTCrTAATCAGACTCAATTTAACGGTGTTAAAGTCCrTGTCTrCAGGAC BU ACGT<7r(K3AAGAAATCC5ATC(3CGTTTC7rAATCAGACTCAATTTAACGGTGTTAAAGTCXrrGTC:TCAG<3AC CA ACGTCTGC3AAGAAATCX3ATCC3CGTTTC7TAATCAC3ACrTCAATTTAAC:GGTGTTAAAGTCCrrGTCrTCAGGAC CH ACGTC?r<5<3AAGAAATC(3ATC(3<:GTTTC^AATCAGACTCAATTTAACGGTGTTAAAGTCC:TGTC7rCAGGAC DA ACGT(7rG<3AAGAAATCX3ATCGCGTTTCrrAATC»GACrrCAATTTAACGGTGTTAAAGTCC7TGTCTCAGGAC DE ACGTCTrCKSAGCaAAATCGATCGCGTTTCTrAATCAGACTCaATTTAACGGTGTTAAAGTCXTrGTCTCaUXaAC DU ACGTCTTCXSAGCSAAATCGATCCSCGTTTCrTAATCAGACrrCAATTTAACMGTGTTAAAGTCXTGTCTrCAGGAC EN ACGTCrr(3C3AW3AAATCGATCXX:GTTTC7TAATCAGACTCAATTTAACX;GTGTTAAAGTCXrrGTC7rCAG<jAC ES ACGTC7TCXSAAGAAATC(3ATCGCGTTTCTAATCAGACTC:AATTTAACGGTGTTAAAGTC:CTGTCrrCAC3<jAC JE AC6TC7rC3<3AA6AAATC(3ATC(3CGTTT(rTAATC:AGACTC:AATTTAAC(3GTGTTAAAGTCC7r(3TCrTCAG<3AC MS ACOTC7r(3<3AAGAAATCGATCGCGTTTCTAATCAGAC7rc:AATTTAACGGTGTTAAAGTCC7TGTCTCAGGAC MT ACGTC?TGGAAGAAATC:(3ATC(3CGTTTC:TAATCAaACTC»ATTTAACC3GTGTTAAAGTCC7rGTC7rCAGC3AC
MO ACGTC7r(3(3AAGAAATCGATC»X:6TTTCrrAATCAGACrrCAATTTAAC(3C3TGTTAAAGTCCrrGTCn?CAGCSAC
OR ACGTC7T6(3AAGAAATCGATCX3C»3TTTCrrAATCAGACTCAATTTAACG<^GTTAAA6TCCTGTCTCAOGAC
RO ACGTCrrCSGAAGAAATCSSATCGCGTTTCrrAATCAGACTCAATTTAACGGTGTTAAAGTCXrrGTCTrCAQGAC
SE ACX3TC7r(3C3AAGAAATCGATCXK:GTTTC7rAATCAGACTCAATTTAACX3<3TGTTAAAGTCCTGTCTCAGGAC HI - C 6CGTC7rGAACGAAATCX3ACC:GTGTATCC(3GTCAGACTCAGTTCAACCX3C:GTGAAAGTCC7rGGC(XJ^GGAC H l - D (XXSTCrrGAACGAAATCGACCXSTGTATCCCSGTCAGACrrCAGTTCAACXXXXSTaAAAGTCCrrC^GCGCAGGAC HI - 1 (3CGTC7TGAACGAAATC(3ACC6TGTAAAT(;C3CCAGAC7rCAGTTCA<3CGG<:GTGAAAGTCXrrGGCGCAG<SAC HI - R GCX3TCrr(3AACGAAATCGACCGTGTATCCGC3CCAGAC7rCA6TTCAACG<3CGTGAAAGTCXrrCXXXX:AG<3AC E <X)LI CCXSTCrTCKSATCSAAATTGACCXXXSTATCTrCKSTCAGACCXaUSTTCAACaSGCGTGAACGTCXrrGGCAAAAAAT
4 2 5 4 5 0 4 7 5 + + +
H I - A AACACCCrrGACCATCX»GGTTGGT(;CCAACC5ACGC3TGAAACCATTGATATCX3ATC7raAAACAGATCAACT AD AACCAaATC3AAAATCCACX5TTC«3TC3CTAACGATGGTGAAACCATTACCATCGATCT(3CAAAAAATTC3ATG BE AACCAGAT(3AAAATCCAGGTTGGT(3<7rAACGAT(3GT(5AAACCATTACCATCMAT<7r(3CAAAAAATTQATG BU AACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCX3ATC7rGCAAAAAATT(3ATG CA AACCAGATGAAAATCCyU3GTTCK5TGCTAACX3AT<3GTGAAACCATTACCATCX3ATCTGCAAAAAATT(3ATG
AACCAGATGAAAATCCAGGTTCK5TGCTAACX3ATGGTGAAA£XATTACCATCXSAT(rrGCAAAAAATTC3ATG AACCAGATGAAAATCCAGGTTCKSTCWITAACGATGGTGAAACXATTACXIATCXSTTCrrGCAAAAAATTGATG AACCAGATGAAAATCCyU3GTTCKSTCXrrAACGAT(3GTGAAACC:ATTACC:ATC»3AT<?rGCAAAAAATTGATG AACX»aATGAAAATCCa^GGTTGGTGCTAACGATGGTGAAACCATTACXATCC5ATC:TCX»AAAAATTC3ATG AACXAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCCSATCrrGCAAAAAATTaATG AACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATG AACXAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGGAAAAAATTGATG AACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACXy^TCaATCTGCZAAAAAATTQATG
CB DA DE DU EN ES JE \£Q
MT ^cCAGATGAAAATCCAGGTTGGTGCrrAACGATGGTGAAACCATTACCATCCSATCTGCaLAAAAATTGATG MO ;^ACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCC5ATCTGCAAAAAATTGATG OR AACCAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATG RO ;^;^cAGATGAAAATCCAGGTTGGTGCTAACGATGGTGAAACCATTACCATCGATCTGCAAAAAATTGATG SE AACCAGATGAAAATTCAGGTTGGTGCTAACGATGGTCSAAACCATTACCATCaATCTGCAAAAAATTGATG HI C AACACTCTGACCATCCAGGTTGGTGCCAACGACGGTC3AAACTATCC3ATATCGATCTaAAO<:AGATCAACT HI n AACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATTGATATTGATTTAAAAGAAATTAGCT HI I T X.CACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATCQATCTQAAGCAGATCAACT HI - i VTcAcCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATCQATCTaAAGCAGATCAACT E TOLI GGCTCCATGAAAATCCAGGTTGGCGCAAATGATAACCAGACTATCACTATCGATCTGAAGCAflATTGATG
106
500 525 + abcdef ghi jlcliiinop+ ab<»l
HI - A CrrCAGACCX:TGCK3TCTC3<3ATACGCrrC3AATGT(3CA (5AAAAAATATC3ATGTGAA AD T(3AAAAGCC7rTCXXX:TTaAT(3GGTTCAATGTTAAT(3C3GCCAAAAGAAGCGACAGTGGGTGATCrrGAA BE T(jAAAAGCCrTT(K3CCrrTGATGGGTTCAATGTTAATG(3GCCAAAAGAAGCaACAGTGGGTaATC7TGAA BU TGAAAAGCC:TTGGCCrrTGAT(K3GTTC:AATGTTAATG(3GCCAAAAGAAGCaACAGTG(K;TaATC7rGAA C:A T(3AAAAGCCTTCKX:CTT(SATG<3GTTCAATGTTAAT(K3<X:CAAAAGAAGCC3ACAGTG(^TaATCT(3AA CH TGAAAAGCC7rTC3C3CCrrTGATGGGTTCAATGTTAAT(3C;GCCAAAAGAAGCGACA6TGGGTaATC7rCSAA DA TGAAAAGCC7rTC3GCCrrTGAT(;(K3TTCAATGTTAAT(3G<K:CAAAAGAAGCaACAGTCX3GTaATC:TaAA DE TGAAAAGCC7rT(3GCC7rTGAT(3C3GTTCAATGTTAATG(3(3CCAAAAGAAGa3ACAGTC;GGTGATC7raAA DU TGAAAAGCCTTG(3CCTTGATGGGTTCAATGTTAATCX3GCCAAAAGAAGCGACAGTGGGTGATCrrGAA EN TC5AAAAGCCrrTGGCCrrTC3ATGGGTTCAATGTTAATGGGCCAAAAGAAGC(3ACAGTGGGTGATC7raAA ES TGAAAAGCCTTCSCSCCrrTGATGGGTTCAATGTTAATGCKXrCAAAAGAAGCGACAGTGGGTaATCTrGAA JE TGAAAAGCCTTGGCCrrTC3AT(KK3TTCaU^TGTTAATGC3<3CXAAAAaAAGCX3AC»OTG<3GTGATC:TGAA MS TGAAAAGCCn'T(;<3CCrrTGATGGGTTCAATGTTAATGG<3<XAAAAGAAGCGACAGTG<3GT(3ATCTaAA MT TGAAAAGCCrrTC;<3CCTTGATGGGTTCAATGTTAAT(3<KX;CAAAAGAAGCX5ACAGTGGGTGATCTC5AA MO TGAAAAGCC7rTC3(X:C7rTGATGGGTTCAATGTTAATCXK3CXAAAAGAAGCGACAGTGGGTaATC7raAA OR TGAAAAG<Xn'T(3<3CC7rTaATCSC3GTTCAATGTTAAT<KKS<XAAAAGAAGCaACAGTGGGTGATC7rGAA RO TGAAAAGCCrrTGGCCTTGATCS<3GTTC»ATGTTAATG<3C3CCAAAAGAAGC»ACAGTGGGTGATC:TGAA SE TGAAAAGCCrrTCK3CC7rTC3AT(3GGTTCAATGTTAAT(3<XX:c:AAAAaA»U3CX3ACAGT(KK3TGATCTaAA H l - C CrrCyU3ACCCrr<3C3<^CrrAGATACGCTGAATGTGCA GAAAAAATATGATGTGAG HI - D CTAAAACACrrGGCSACrrTCJATAAGCrrTAATGTCXA (WATGCCrTACACCCCGAA HI - 1 CTCAGACCCT(3<3GTCrr<X3ATAC(5CTC3AATCrrG<a^ ACAAAAATATAAGGTCA3 H l - R C7rC»GACCCrrGC3GTCrrC5C3ATAC(3CTGAATGTC3CA ACAAAAATATAAGGTCAO E <X>LI C7rAAAACrr<?rTCK3CCTT(3ATGGTTTTAGCGTTAA AAATAACGATACAGTTACCAC
550 575 efg. .abcle + +bc(iefg abccSefgh
Sl-p, GAG CGAAGTCAC(X:CTTCC3GCTACATTAAGCAC TACTTGCACTTGATGGTGC AD 6TC CAGCTTCAAGAATGTTACX3GGGTATGATAC CTATGCAGCGGGTGCCGATAAATAT BE GTC CAGCTTCAAGAATGTTACGGGGTATCJATAC CTATGCTGTTGGTGCCAATAAATAT BU ATC C»GCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT Q^ GTC CAGCTTCAAGAATGTTACG<»^TATGATAC CTATGCTGTTGGTGCCAATAAATAT CH — -ATC—-CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT Uj j TC CAGCrrTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT DE — -GTC—CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGk3TGCCGATAAATAT DU — -ATC— -CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT EN — -ATC-—CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT ES —ATC—CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT JE — -ATC- —CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT
—GTC—CAGCTTCAAGAATGTTACGGGGTATGATAC CTATGCTGTTGGTGCCAATAAATAT ATC- —CAGCTTCAAGAATGTTACGGGTTATGACAC CTATGCAGCGGGTGCCAATAAATAT -ATC—CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT -ATC—-CAGCTTCAAGAATGTTACGGGGTATGATAC CTATGCTGTTGGTGCCAATAAATAT -ATC- - -CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGOTGCCGATAAATAT ATC- - -CAGCTTCAAGAATGTTACGGGTTACGACAC CTATGCAGCGGGTGCCGATAAATAT
—CGA—TACTGCTGTAGCTGCTTCCTATTCCGACTC GAAACAGAATATTGCTGT —AGAAACTGCTGTAACCGTTGATAAAACTACCTATAA AAATGGTACAGATACTAT - -CGA- - -TACGGCTGCAACTGTTACAGGATATGCCGATACTACGATTGCTTTAGACAATAG - -CGA- - -TACGGCTGCAACTGTTACTGGCTATACAGATTCTGCTACTGCTATTGACAAATC
TAGTGC—TCCAGTAACTGCTTTTGGTGCTACCACCAC AAACAATATTAAACTTAC
MS MT MO OR RO SE Hl-C Hl-D Hl-I Hl-R E COLI
107
6 0 0 6 2 5 6 5 0 i i l t l m + + + aj3C
HI - A TCX^CTCAAAACCMGAACCGGTTCrrACAACTGATACTCKSTTCAATTAAGCjATGGTAAGGTTTA— AD CGTGTAGATATTAATTCC(3GTCX7rGTAGTCSACTGATCK:CGCAGCACCGAA TAAAGTATA- -BE C(^GTG<SATGTCAACTCAGGG<3CGGTAGTAACTGACACCACTC»^CCAACTGTTCCTGATAAAGTATA— BU CGTGTAGATATTAATTCC(3GT(3CTGTAGT(3ACTGAT(3CAGCAGGACCGGA TAAAGTATA— CA CGCGTGGATGTCAACTCAGGGGCCK3TAGTAACTGACACCACTGCTCCAACTGTTCCT(3ATAAAGTATA- -CH CGTGTAGATATTAATTCCGGT(3CTGTAGTGACT(3ATGCCGGAGCACCCK5A TAAAGTATA— DA CGTGTAGATATTAATTCCGGTCKrrGTAGTGACTGATGCCGCAGCACCCSCaA TAAAGTATA- -DE C G T G T A G A T A T T A A T T C C ( 3 G T < X : T G T A G T G A C T ( 3 A T ( 3 C A G T A G C A C C G A A TAAAGTATA- -DU CGTGTAGATATTAATTCC(3GTCXrrGTAGT(3ACTGATCX:AGTAGCACCGGA TAAAGTATA— EN CGTGTAGATATTAATTCC(3GTGCTGTAGTC3A(rTCjATGCAGCAGCACCC3GA TAAAGTATA- -ES CGTGTAGATATTAATTCCGGTGCrrGTAGTCjACT(5ATGCCG<:»GCACC(3<3A TAAAGTATA- -JE CGTGTAGATATTAATTCC<3GT(3CTGTAGTGACTGAT(X:CGCAGCACCC3<3A TAAAGTATA— MS C C J C G T ( K 3 A T G T C A A C T C A G G G G C G G T A G T A A C T G A C A C C A C T G C T C C A A C T G T T C C T C 3 A T A A A G T A T A - -
MT CGTGTAGATATTAATTCAGGT(XrrGTAGTAACTGATGAT(3<:»GCACCG<3A TAAAGTATA— MO CX5TGTAGATATTAATTCC(3GT(SCTGTAGTGACTGATCXyU3CAGCACCGaA TAAAGTATA— OR C ( 3 C G T G G A T G T C A A C T C A G C 3 C 3 C 3 C ( K ; T A G T A A C T G A C A C C A C T G C T C X A A C T G T T C C : T C 3 A T A A A G T A T A —
RO CXSTGTAGATATTAATTCCCSGTCKrrGTAGTGACTGATGCAGTAGCACCGGA TAAAGTATA— SE CGTGTAGATATTAATTCAGGTCXrrGTAGTAACTGATC3ATG<»GCACCC;QA TAAAGTATA- -H l - C TCCTGATAAAACAGCTATTACTCKa^AAAATTCKSTGCAGCAACCAGTGGTGGTGCTCXWATAAA- -H l - D TACAGCCCAGAGCAATACTGATATCC»AAC:TCX:AATTGGC(X5TCSGTGCAACCXK3GGTTACTGG- -
H l - I TACTTTTAAAGCCTC(;C3CTACTGGTCTTGGTGGTACTC3ACGAGAAAATTGATGG<MATTTAAA- -H l - R TACGTTT(3CTC3CaVTCAC3CAACTACC:TTAGGTGGTACTCCT<XrrATTAC TGGTGATCTC3AA- -E COLI T(XSAATTACCCTTTC:TAC(MAAGCAGCCACTGATAC:TGC;CG<3AACTAACXXyUXrrTCAATTGAGG
6 7 5 7 0 0 d e f g + . . . . a b c d e f g +
HI - A (^TATAACyUSCACCTrcrrAAAAATTA TTATGTTGAAGTAGAATTTACCC3ATGCX3ACCGATCA AD TGTAAAT(3CAG<y^AAC(X;TCAGTT AACAACT(SACGAT<K:<X3AAAATAACAC:TGCGGTTaA BE CX3TAAACC3CTGCAAACGGTCAGTT AACAACTGACCJATGCGGAAAATAACACTGCGGTTGA
BU TGTAAATGCAGCy^AACCKSTCAGTT G A C A A C T G A C G A T < X : G G A G A A T A A C A C T ( X X ^ T T A A
CA CGTAAAC(Xrr(X»AACGGTCAGTT GACAACTGCCGATCXrGCAAAATAACACOSCGGTTGA CH TGTAAATGCAGCAAACGGTCAGTT AACAACTGACGAT(3CGGAAAATAACACTCXXK5TTGA DA TGTAAATGCAGCAAACGGTCyUSTT AACAACTGACGATGCGC3AAAATAACACT0CGGTTGA DE TGTAAATCXy^GCAAACGGTCy^TT AACAACT(3ACGATGCGGAAAATAACAC:TGCGGTTGA
DU TGTAAATGCAGCAAACGGTCAGTT AACAACTGACGATGC<3GAAAATAACACTGCa<5TTGA EN TGTAAATGCAGCAAACGGTCAGTT AACAACTGACGATGCGGAAAATAACACTGCGGTTGA ES TGTAAATGCAGCAAACGGTCAGTT AACAACTGACC5ATGCGGAAAATAACACTGCGGTTGA JE TGTAAATGCyyGCAAACGGTCACSTT AACAACTGACXSATGCCMAAAATAACACTaCGGTTGA j ^ CGTAAAOXrrGCAAACGGTCAGTT GACAACTGCCGATGCG<:»AAATAACACC»CGGTTaA j ^ TGTAAATGCAGCAAATGGTCAGTT GACAACTGACGATGCGGAAAATAACACTGCGGTTAA MO TGTAAATGCAGCAAACGGTCAGTT AACAACTGACGATGCGGAAAATAACACTGCGGTTGA OR CGTAAACGCTGCAAACGGTCAGTT GACAACTGCCCSATGCGCAAAATAACACCGCGGTTGA RO TGTAAATGCAGCAAACGGTCAGTT AACAACTGACGATGCGGAAAATAACACTGCGGTTGA SE TGTAAATGCAGCAAACGGTCAGTT GACAACTGACGATGCGGAGAACAACACTGCGGTTAA jj l _C AGCAGATATTAGCn-TTAAAGATGGGAAGTATTACGCGACTGTCAGTGGATACGATGATGCCGCAGA
QGCTGATATCAAATTTAAAGATGGTCAATACTATTTAGATGTTAAAGG- - -CGGTOdrTTCTGCTOG ATTTGATGATACGACTGGAAAATA TTACGCCAAAGTTACCGTTACGGGGGGAACTGGTAA GTTTGATGATACTACTGGAAAATA TTACGCTGATGTTTC AOGTAC
H l - D H l - I H l - R E COLI GTGTTTATACTGATAATGGTAATGATTA- -CTATGCGAAAATCAC CCXJTGG
108
725 750 + abcdefghijJtlmnopqrs +. . . . abed
H I - A AACCAACAAAGGCGGATTCTA TAAAGTTAATGTTGCTGA—TGATGGTGCA AD CCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCAAAGCG BE CCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCAAAGCG BU CCTCTTTAAGACCACTAAATC TACTGCTGGTACCGATGA—AGCCAAAGCG CA TCT6TTTAAATCTACTAAATC TACTGCGGGTACTGACGA—GGCCAAGGCT CH TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCT(3A—AGCCAAAGCG DA TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCAAAGCG DE CCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCAAAGCG DU CCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA- - -AGCCAAAGCG EN TCTCTTTAAGACCACTAAATC TACTGCrTGGTACCGCTGA- - -AGCCAAAGCG ES TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA- - -AGCCAAAGCG JE TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCyuWXJG MS TCTGTTTAAATCTACTAAATC TGCTGCGGGTACTGACGA- - -GGCCAAGGCT MT CCTATTCAAGACGACTAAATC TGCTGCTGGTACCGAT(3A-—AGCCAAAGCG MO TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA—AGCCAAAGCG OR TTTGTTTAAATCTACTAAATC TGCTGCAGGTACTGACGA—TGCCAAGGCT RD TCTCTTTAAGACCACTAAATC TACTGCTGGTACCGCTGA AGCCAAAGCG SE CCrrATTCAAGACGACTAAATC TACTCKTrGGTACCGATGA AGCCAAAGCG H l - C T A C A G A T A A A A A T G C J A A C C T A TC3AAGTCACTGTTGCCGC AGATACAGGA HI - D TGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGATACGACTGA TAAAACTCCG H l - I AGATCKSCrrATTA TGAAGTTTCCGTTGATAAGACQAACGGTGAG H l - R TACGCKTTAAAGATGGTGTTTA TCSAAGTAACAGTTGGAGC TGATCSCJAAAA E CLOI T ( 3 A T A A C G A T G ( 3 G A A 6 T A T T A C(XyU3TAACAGTT(X:TAA TCSATGGTACA
775 800 825 + + abcdef g h i j +.
HI - A GTCACAATGACTCSCGCXTTACCACCAAAGAGCSCTACAACTCC TACAGGTATTACTGAAGTTA AD ATAGCTCKST(3CCATTAAG<3GTGGTAAGGAAC3GAGATACCTTTGATTA TAAAGGCGTGACTTTTACTA BE ATAGCrTGGTGCCATTAAGGGTGGTAAGGAAGGAGATACCrrTTGATTA TAAAGGCGTGACTTTTACTA BU ATAGCT(K3T(X:CATTAAG<3GTGGTAAGGAAGGAGATACCTTTGATTA TAAAGGTGTGTCTTTTACTA CA ATCGCAACATCTATCAAAGGCCSGAAAAGTTGGTGATACCn'TTGATTA TAAAGGTGTGTCTTTTACTA CH A T A G C T G G T G C C A T T A A G C 3 G T ( ; G T A A G G A A G < 3 A G A T A C C T T T G A T T A TAAAGGCGTGACTTTTACTA
DA ATAGCT(X3TGCCJVTTAA(»3GTG<3TAAGGAAGGAGATACCTTTGATTA TAAAGCSCGTGACTTTTACTA DE A T A G C T G G T ( X : C A T T A A G < 3 G T ( 3 G T A A G < J A A G G A G A T A C C T T T G A T T A TAAAGGCGTGACTTTTACTA DU ATAGCTCKiTCSCCATTAAGGGTCSGTAAGGAAGCSAGATACCTTTGATTA TAAAGGCrGTGACTTTTACTA EN ATAGCTGGTGCCATTAAAGGTCXSTAAGGAAGGAGATACCTTTGATTA TAAAGC^CGTGACTTTTACTA ES A T A G C T G G T C X ; C A T T A A G G G T G G T A A G < 3 A A G G A G A T A C C T T T C J A T T A TAAAGCXX5TGACTTTTACTA JE ATACXrPC3GT(3CCATTAAG<3GTGGTAAGC3AAG<3AGATACCTTTCSATTA TAAAOC3COTGACTTTTACTA MS ATCCXamCATCTATCAAAGGCCX3AAAAGTT(3GTC3ATAC(rrTTGATTA TAAACK5TGTGTCTTTTACTA MT ATAGCT(X»T(3CCATTAAG<3GTGGTAAG<3AAG<3AGATACCTTTGATTA TAAAGC3CGT(3ACTTTTACTA MO ATAGCTCX3TGCCATTAAAGGT(3GTAAGCjAAGGA(5ATAC<7rTTC3ATTA TAAAGGCGTGACTTTTACTA OR ATCX3<ZAACTTCrrATCAAAGGCG<3AAAAGTTGGTCjATACCTTTC5ATTA TAAAGGTGTGTCTTTTACTA RO A T A G C T G G T ( K : C A T T A A A G G T < S G T A A G < 5 A A ( 3 G A G A T A C C T T T G A T T A TAAACKSCGTGACTTTTACTA S £ ATAGCTAGTCX:CATTAAG<3GTGGTAAGGAAG<3AGATACCrrTTGATTA TAAAGGTGTCSTCTTTTTACTA H l - C GCAGTTACTTTTGCGACTAGACCAACAGTGGTTGACTTACC AACTGATGCAAAAGCAGTTT HI - D TTAGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCAC TATAACCCACAACCAAATTG H l - I GTGACTCTTGCTCXZGGTCACTCXCGCTACAGTGACTACTGC GACAGCAACTGAOGATGTGA HI - R GTCACTTTAACTGCSCACACCAACAGGACCAATTACTGCTCXSCTTCCCTTCAACTCKIAACAAAAGATGTTA E CLOI GTGACAATGGCGACTGGAGCAACGGCAAATGCAACTGTAAC TGATGCAAATACTACTAAAG
109
850 875 + +
HI - A CTCAAGTCCa^AAAACCTGTGCXrrGCTCCAGCTGCTATCCAGGCTCAGTTGACTGCTGCCCATCyrGACCGG AD TTGATACAAAAACTGGCAATGACCSGTAATGGTAAGGTTTCTACTACCATCAATGGTGAAAAAGTTACGTT BE TTGATACAAAAACTCSGCAATGACGGTAATGGTAAGGTTTCTACTACCATCAATGGTGAAAAAGTTACGTT BU TTGATACAAAAGCTGGTAATGAC(3GTAAT(3GTAC<3GTTTCTACTACTATCAATGGTaAAAAAGTTACGTT CA TTGATACAAAAGCT(3GTGATGACGGTAATGGTAC(X»TTTCTACTACCATCAATGGTGAAAAAGTTACATT CH TTGATACAAAAACT<3(3CAATGACGGTAATGGTAAGGTTTCTACTACCATCAATGGTGAAAAAGTTACGTT DA TT(3ATACAAAAACTC3GCAATC3ACCK3TAAT(3GTAAGGTTTCTACTACCATCAATC«3TGAAftAAGTTACGTT DE TTGATACAAAAACTCSGTAATCSACGGTAATGGTAAGGTTTCrrACTACCATCy^TC^GTGAAAAAGTTACXSTT DU TTC3ATACAAAAACTC;GTAAT(3ACGGTAATC;GTAAGGTTTC7rACTACCATCAATC;GTGAAAAAGTTACGTT EN TTGATACAAAAACTTGGTGATGACCSGTAATCXSTAAGGTTTCTACTACCATCAATGGTaAAAAAGTTACGTT ES T T G A T A C A A A A A C T G G C A A T G A C ( 3 G T A A T ( X 3 T A A G G T T T C T A C T A C C : A T C A A T G G T G A A A A A G T T A C G T T
JE T T G A T A C A A A A A C T ( 3 C 3 C A A T G A C G G T A A T G G T A A G G T T T C T A C T A C C A T C A A T G G T G A A A A A G T T A C G T T
MS TTCsATACAAAAGCrrCSGTGATGACGGTAATGGTACGGTTTCTACTACCATCAATGGTGAAAAAGTTACATT MT TTGATACAAAAACT(K3TGATGAC(3GTAATGGTAAGGTTTCTACTACCATCAATGGTGAAAAAGTTACGTT MO TTGATAC:AAAAACT(3GTGAT(3GC(K3TAAT(K3TAAGGTTTCTACTACCATCAATGGTaAAAAAGTTACGTT OR TTGATACAAAAGCTCK3TGATGACGGTAATGGTACGGTTTCTACTACCATCAATCX>TGAAAAAGTTACATT RO TTGATACAAAAACTGGTGATGACGGTAATGGTAAGGTTTCTACTACCATCAATGGTGAAAAAGTTACGTT SE T T G A T A C A A A A G C T G G T A A T G A C G G T A A T C 3 G T A C ( 3 G T T T C T A C T A C T A T C A A T ( ; G T G A A A A A G T T A C G T T
H l - C C A A A A G T T C A A C A G A A T G A T A C T G A A A T A G C A G C A A C A A A T G C G A A A G C
H l - D C T G A A G T A A C A A A A G A G ( 3 G T G T T G A T A C G A C C A C A G T T G C G G C T C A A C T T G C T G C T G C A G G < ; G T T A C T G G
H l - I AAAATGTACAAGTT(X:AAAT(3CTGATTTGACAaAGGCTAAAGCC(3CATTGACAGCAG<:j^GGTGTTAC(:XX> H l - R AACAAACTCAGCAAGAAAACGCTGATTTGACAGA6GCCAAAGCCGCATTGACAGCAGCG(3GTGTTGCAGC E COLI CTACAACTATCACTTCAGGCCXSTACACCTGTTCAGATTGATAA TACTGCAGGTTCCGCAACTGCCAA
9 0 0 9 2 5 9 5 0 . . . + . a b c d e f g + a b e d . . +
HI - A CaCTOK TACT(3<:rr<3AAAT(3GTTAAGATGTCTTATAa3GATAAAAACGGTAA GACTATTGAT AD AACTGT CGCTGATATTACCGCTTCSGTGCGGCGAATGTTGATGCTGCTACCTTACAATCAAGCAAA BE AACTGT CGCrTGATATTACCCSCrrCXSTGCCSGCGAATGTTGATCXrrGCTACCTTACAATCAAGCAAA BU AACTGT CGCTGATATTACCCXrTGGTGCAGCGAATGTTAATGATGCCACCrrTACAATCAAGCAAA CA AACAAT TAGTGATATTGGCCSCGAGTCXaVACAGACGTAAATAGCCSCGAAGATTCAATCAAGTAAA CH AACTGT cGCTGATATT(3CCACTCK3CGCGACG<3ATGTTAAT<3C7rGCTACCTTACAATCAAGCAAA DA AACTGT c(3C7rGATATT(3CCACTC3GCGCGACGC5ATGTTAAT<3CTGCTACCTTACAATCAAGCAAA DE AACTGT cGCTGATATTACC(3GTCK3TGCGGCGAATGTTGATC3CTGCTACCrrTAC:AATCAAGCAAA DU AACTTCST CGCTGATATTACC<3GT<3GT(3CG<3CGAATGTTGATG<n?CX?rACCTTACAATC:AAOCAAA EN AACTGT cGCTGATATTGCCACTGGCGCGACGGATGTTAATGCrTGCTACCTTACAATCAAGCAAA ES AACTTGT CGCTGATATTGCCACTGGCGCGACGGATGTTAATGCTGCTACCTTACAATCAAGCAAA JE AACTGT CGCTGATATTGCCACTGGCGCGACGGATGTTAATGCTGCTACCTTACAATCAAG<:AAA
MS AACAAT TAGTGATATTGGCGCGAGTGCAACAGACGTAAATAGCGCGAAGATTCAATCAAGTAAA MT AACTGT CGCTGATATTGCCACTGGCGCGACGGATGTTAATGCTGCTACCTTACAATCAAGCAAA MO AACTGT CGCTGATATTGCCACTGGCGCGACGAATGTTAATGCTGCTACCTTACAATCAAGCAAA OR AACAAT TAGTGATATTGGCGCQAGTGCAACAGACGTAAATAGCGCGAAGATTCAATCAAGT AAA RD AACTGT CGCTGATATTGGCATTGGCGCGGCGGATGTTAATGCTGCTACCTTACAATCAAGCAAA SE AACTGT CGCTGATATTACCGCTGGTGCAGCGAATGTTAATQATGCCACCTTACAATCAAGCAAA HI c — T O A TACAGCTACTTTAGTGAAAATGTCTTATACAGATAATAATGGCAA—AGTTATTGAT
TGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAA-—GGTTATTGAT - AGCATCTGTTGTTAAGATGTCTTATACTGATAATAACGGTAA AACTATTGAT H l - D
H l - I CAC HI - R c;<3CTcx;
-CCACAGATCTGTTGTTAAGATGTCTTATACTGATAATAACGGTAA AACTATTGAT
E^COLI CCTTGG TGCTGTTAGCTTAGTAAAACTGCAG GATTCCAAGGGTAA—TGATACCGAT
110
975 1000 +. abed +abcd
HI - A C3C3C(K3TTTC(;GTGTTAAAGT T(;C3<3GCTGATATTTATCX:T(3CAAC AAAAAATAAAGATGGATCOT
AD AATCSTTTATACATCriGTAGT (SAACCKSTCAGTTTACTTTTCSATCJA TAAAACCAAAAACGACJAGTG
BE A A T G T T T A T A C A T C T G T A G T CJAACCK3TCy^TTTACTTTTC3ATGA TAAAACCAAAAACC3AGAGTG BU AATGTTTATACATCTGTAGT GAAOSGTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG CA GATGTTTATACTTCCGTTGT AAGC(3GTCAGTTTACTTTTGCTGA TAAAACCAAAAACC5AGAGTG CH AATGTTTATACATCTGTAGT GAACGGTCJ^TTTACTTTTGATGA TAAAACCAAAAACGAGAGTG DA AATGTTTATACATC?TGTAGT GAAC(3GTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG DE AATGTTTATACATCTGTAGT (3AAC<K3TCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG DU AATGTTTATACATCTTGTAGT (3AACGGTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG EN AATGTTTATACATCTGTAGT GAACGGTCy^TTTACTTTTGATGA TAAAACCAAAAACGAGAGTG ES AATGTTTATACATCTGTAGT (3AACCK5TCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG JE AATGTTTATACATCTGTAGT (5AAC(K5TCAGTTTACTTTTGATGA TAAAACCAAAAACC3AGAGTG MS GATGTTTATACTTCCGTTGT AAGCCSGTCAGTTTACTTTTCXrrOA TAAAACCAAAAAOSAGAGTG MT AATGTTTATACATCTGTAGT GAACGGTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG MO AATGTTTATACATCTTGTAGT GAACCSGTCAGTTTACTTTTGATGA TAAAACXAAAAACGAGAGTG OR GATGTTTATACTTCCGTTGT AAGCGGTCAOTTTACTTTTCXTTGA TAAAACCAAAAACGAGAGTG RO AATGTTTATACATCTGTAGT GAACCXSTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG SE AATGTTTATACATCTTGTAGT (SAACCSGTCAGTTTACTTTTGATGA TAAAACCAAAAACGAGAGTG H l - C GGTGGGTTCCSCATTTAAGAC CTCCUBGTCKSTTATTATGCAGCATC TGTTGATAAATCrrCXXXX»C HI - D (X3TC«3CTATCX:AGTGAAAAT (X3C3CGACGATTTCTATGCCX3CTAC ATATGATGAGAAACAGGTAC H l - I CK3T(K3TTTAGCAGTTAAGGT AGGCGATGATTACTATTCTG<»AC TCAAGATAAAGATGGTTCCA HI - R GGTCK5TTTAGCAGTTAAGGT AGGCCSATGATTACTATTCTGCAAC TCAAAATAAAGATGGTTCCA E COLI AC ATATGaSCTTAAAGATACAAATGGCAATCTTTACGCTGCCMATGTGAATGAAACTACTGGTGCTG
1 0 2 5 1 0 5 0 1 0 7 5 _ _ . + + . a b e d . . a b e d + a b e d
HI - A TCAGCATTAACACCaUTTGAATATACCCSATAA AGA CX3GCAACACTAAAACTC3<:ACTAAACCA AD CC3AAACTTTCTC5ATTTGGAAGCAAACAAT(3C TOTTAAGCS<3CC3AAAGTAAAATTAC»GTAAATOO BE CGAAACTTTCTGATTTCMAAGCAAACAATGC TGTTAAG<3GCGAAAGTAAAATTACASTAAATGG BU C G A A A C T T T C T G A T T T G G A A G C A A A C : A A T ( 3 C TGTTAAG<3GCGAAAGTAAAATTACAGTAAAT(X} CA CGAAACTTTCTGATTTCSGAAGCAAACAATGC TGTTAAG<3GC<3AAAGTAAAATTACAGTAAAT(K; CH CGAAACTTTTCTGATTTC^GAAGCAAACAATCK: TGTTAAGGGCGAAAGTAAAATTACAGTAAATGG DA CXSAAACTTTCrrGATTTGGAAGCAAACAATGC TGTTAAGGC3CGAAAGTAAAATTACAGTAAATGG DE CGAAACTTTCTGATTTCMAAGCAAACAATCX: TGTTAAAGGCGAAAGTAAAATTACAGTAAACGO DU CCSAAACTTTTCTGATTTGCAAGCAAACAATCK: TGTTAAGGGCGAAAGTAAAATTACAGTAAAOSO EN CGAAACTTTCTGATTT(K5AAGCAAACyU^TGC TGTTAAGC5C3CGAAAGTAAAATTACAGTAAATGG ES CQAAACTTTCTGATTTGGAAGCAAACAATGC TGTTAAGCX3CGAAAGTAAAATTACAOTAAATGG JE CGAAACTTTCTGATTTCKSAAGCAAACAATGC TGTTAAGC3GCGAAAGTAAAATTACAGTAAATGG MS CGAAACTTTCTGATTTGGAAGCAAACAATGC TGTTAAGC^GCXSAAAGTAAAATTACAGTAAATGG MT CGAAACTTTCTGATTTGGAAGCAAACAATGC TGTTAAGGGCGAAAGTAAAATTACAGTAAATGG MO CGAAACTTTCTGATTTGGAAGCAAACAATGC—TGTTAAGCXXX5AAAGTAAAATTACAGTAAATGG— OR CGAAACTTTCTGATTTGGAAGCAAACAATGC—-TGTTAAGGGCGAAAGTAAAATTACAGTAAATGG- - -RO CGAAACTTTCTGATTTGGAAGCAAACAATGC- - -TGTTAAGGGCGAAAGTAAAATTACAGTAAATGG- - -SE CGAAACTTTCTGATTTGGAAGCAAACAATGC-—TGTTAAGGGCX3AAAGTAAAATTACAGTAAATGG- - -HI - C GTAGCTTGAAAGTTACTAGCTACGTTGACGCTACCAC— -TGGTACCGAAAAAACTGCTGCGAATAA- - -H l - D AATTACTGCTAAACAACCACTATACAGATGG—TGC—TGGCGTGCTCCAAACTGGAGCTGTGAA— H l - I TAAGTATTQATACTACGAAATACACTGCAGA-—TAA- - -CGGTACATCCAAAACTGCACTAAACAA- - -HI - R TAAGTATTAATACTACGAAATACACTGCAGA- —TAA- -CGGTACATCCAAAACTGCACTAAACAA- - -E COLI TTTCTGTTAAAACTATTACCTATACTGACTC—TTC CGGTGCCGCCAGTTCTCCAACCGCGOTCAA
111 1100 1125
abed + +. abed HI - A AC TC GGTCSG C(3CAGACGGTAAAACTGAAGTTGTTTCTATCGACGG TAAAACCTACAATGCCAGC AD G G C T G A A T A T A C T ( 3 C T A A C G C C C X : G G G T G A T A A G G T C A C C T T A G C T ( ; G CAAAACCATGTTTATTGAT
BE GGCTGAATATACTGCTAAC(3CCGCGGGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAT BU GGCTGAATATACTGCrrAACCarCGCCSGGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAT CA GC3CT<3AATATACT<3CTAAC<3CCC3CC;<3GTGATAAGGTCACCTTAGCT(»3 CAAAACCATGTTTATTGAT CH G < 3 C T G A A T A T A C T C 5 C T A A C ( 3 C C A C C K ; G T G A T A A G A T C A C C T T A G C T G G CAAAACCATGTTTATTGAT DA GC3CTGAATATACT(3CTAA DE GGCTGAATATACTC3CTAAC(3CCa^CC3GGTGATAAGGTCACCTrTAGCAGG CAAAACCATGTTTATTGAC DU G<3CTGAATATACT(3CTAACGCCAC(3GGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAC EN G<3CT(3AATATACTCKrrAACC3CCACG<3GTGATAAGATCACCTTAGCTGG CAAAACCATGTTTATTGAT ES GGCTGAATATACTGCTAACGCCZACCSGGTGATAAGATCACCTTAGCTGG CAAAACCATGTTTATTGAT JE GGCTGAATATACTGCTAACGCCACGGGTGATAAGATCACCTTAGCTGG CAAAACCATGTTTATTGAT MS GGCTGAATATACTGCTAACGCCGCGCSGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAT MT GGCTGAATATACTGCTAACGCCGCGGGTGATAAGGTCACCTTAGCTGG CAAAACCJVTGTTTATTGAT MO GGCTQAATATACTGCTAACGCCACGGGTGATAAGATCACCTTAGCTGG CAAAACCATGTTTATTGAT OR GCSCTGAATATACrrGCTAACGCCGCGGGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAT RD G<K:rrGAATATACTC3CTAACC3CCACGC3GTGATAAGATCACC:TTAGCTC3G CAAAACCATGTTTATTGAT SE G<3CTGAATATACTGCTAACGCC(5CGGGTGATAAGGTCACCTTAGCTGG CAAAACCATGTTTATTGAT H l - C ATTAGGT(K3 CGCAGACC3GTAAAACCGAAGTTGTTACTATCGACGG TAAAACCTACAATCSCXAGC H l - D ATTTGGTGG CCSCAAATGGTAAATCTQAAGTTGTTACTGCTACCGTAGGTAAAACTTACTTAGCAAGC H l - I PiCTGGGTGG CGCAGACGCSCAAAACXGAAGTCGTTACTATCGACGG TAAAACCTACAATGCCAGC HI - R ACTGGGT66 C:X3CAGACGGCAAAACCGAAGTTGTTTCTATT(3GTGG TAAAACTTACGCTGCAAGT E COLI ACTGGGCCSG AGATGAT(3<X»AAACAGAAGTGGTCGATATTGATGG TAAAACATACGATTCn-GCC
1 1 5 0 1 1 7 5 1 2 0 0 - . . + a b e d e f g h i j k l m n q p . . + + .
HI - A AAAGCCCKTTCSGTCACAACTTTAAACX: ACAGCCAGAGCTCK3CT(3AAGCGGCTGCTG AD AAAACAGCTTCTCMCGTTAGTACATT AATCAATGAAGACGCTGCCXXTTGCCAAGA BE AAAACAGCTTCTGC3CGTTAGTACATT AATCAATGAAGAC(3CT(XX»CTGCCAAGA BU AAAACAGCTTCTGC3CGTTAGTACATT AATCAATGAAGAC(3CT(X:C(X7rCX:CAAGA CA AAAACAGCTTCTGGCGTAAGTACATT AATCAATGAAGACGCTGCCGCAGCCAAGA CH AAAACAGCrTTCTTGCSCGTAAGTACATT AATCAATGAAGAaXTTGCCCX^GCCAAGA DA DE AAAACAGCrrTCT<3<3CGTTAGTACATT AATCAATGAAGAC(3CTGCCGCTGCTAAGA DU AAAACAGCTTCTGC5CGTTAGTACATT AATCAATGAAGACCKTrGCCGCTGCTAAQA EN AAAACAGCTTCrrGGCGTAAGTACATT AATCAATGAAGACGCTGCCGCAGCCAAGA ES AAAACAGCTTCTGGCGTAAGTACATT AATCAATGAAGACCXTTGCCCXy^OCCAAGA JE AAAACAGCTTCTGGCGTAAGTACATT AATCAATGAAGACGCTGCXX3CAGCCAAGA MS AAAACAGCrrTCT(K3CGTAAGTACATT AATCAATGAAGACCKTrGCOSCAGCCAAGA MT AAAACAGCTTCTGGCGTTAGTACATT AATCAATGAAGACGCTGCCCXauSCCAAGA MO AAAACAG<rrTCTG<K;GTAAGTACATT AATCAATGAAGAC(3<rTCXXX3CAGCCAAGA OR AAAACAGCTTCTGGCGTAAGTACATT AATCAATGAAGACCKTTGCCGCAGCCAAGA RD AAAACAGCTTCT(3GCGTAAGTACATT AATCAATGAAGACCSCTCXXTGCyyGCCa AGA SE AAAACAC3CTTCTC3C5CGTTAGTACATT AATCAATCSAAGACGCTGCCrC^CTCjCCAAGA H l - C AAAGCCC;CT(X3GCACAACTTCAAAGC ACAGCCASA(KrrG<3C<3<3AACGGGCTGCTA HI - D GACCTTGACAAACATAACTTCAGAAC AGCSCGGTGAGCTTAAAGAGGTTAATACAG H l - I AAAGCCCXrTGGTCATGATTTCAAAGC AGAACCAGAGCTGGCGCSAACAAGCCMCTA HI - R AAAGCCGAAGGTCACAACTTTAAAGC ACAGCCTGATCTCKX:CK3AAGCGGCTCXrrA E COLI GATTTAAATG<3C(3GTAATCTG<aUUVCAGGTTT<3ACTGCTC3GT(K5TaA£3<3CTCTGACTGCTGTT(3CAAATG
112
1 2 2 5 1 2 5 0 + +
H I - A CAACCACCGAAAACCCGCT(3GCTAAAATTGAT(3CCGCCXrrGGC(Xa^GGTTGATGCCGTGCGTTCTGACTT AD AAAGTACCCXTTAACCCACTGGCTTCAATTCSATTCTGCATTGTCAAAAGTGGACGCyUSTTCGTTCrrTCTCT BE AAAGTACCGCTAACCCACTCKX:TTCAATTGATTCT(Xa^TTGTCyUJ^AGTGGACGCAGTTCGTTCTTCTCT BU AAAGTACCGCTAACCCa^CT(3GCTTCAATTGATTCTGCATTGTCAAAAGTGGAa3CAGTTCGTTCTTCTCT CA AAAGTACCGCTAACCCACTCKXrTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCT CH AAAGTACCCXrrAACCCACT<3GCTTCAATTGATTCTCXa^TTGTCAAAAGTGGAC(3CAGTTCGTTCTTCTC:T DA CCCACTC5GCTTCAATTGATTCT(3CATTGTCAAAAGTG<3ACGCAGTTCGTTCTTCTCrr DE AAAGTACCCXrrAACCCACT(3GCTTCAATTGATTCT(«:ATTGTGAAAAGTGGACGCAGTTCGTTCTTCTCT DU AAAGTACCCXTTAACCCACTGGCTTCAATTGATTCTCSCATTGTCAAAAGTCMACGCAGTTCGTTCTTCTCT EN AAAGTACCX3CTAACCCACTGGCTTCAATT(5ATTCT(3CATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCT ES AAAGTACCGCTAACCCACTG<3CTTCAATTGATTCTGCATTGTCAAAAGTG<3ACGCAGTTCGTTCTTCTCT JE AAAGTACCGCTAACCCACT(3<3CTTCAATTC3ATTCT(Xa^TTGTCAAAAGTG<3AC<3CAGTTCGTTCTTCTCT MS AAAGTACCGCTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGGACGCAGTTCX5TTCTTCTCT MT AAAGTACCCXrTAACCCACTGGCTTCAATTGATTCTCSCATTGTCAAAAGTGGACGCAGTTCGTTCTTCTCT
MO AAAGTACCCKrTAACCCACTGGCTTCAATTGATTCTGCATTGTCAAAAGTGCSACCKIAGTTCGTTCTTCTCT
OR AAAGTACCMCTAACCCACT(3C3<rrTCAATTGATTCTGCATTGTCAAAAGTGC3ACCKaU3TTCGTTCTTCTCrr
RD AAAGTACCGCTAACCCACTGCSCTTCAATTGATTCTCXa^TTGTCyVAAAGTGGACGCAGTTCGTTCTrTCTCT
SE AAAGTACC(KrrAACCCACTC;<3CTTCAATTC3ATTCT<3<aVTTGTCAAAAGT<3GAC<3<»GTTCGTTCTTCTCn' H l - C CAACCACTGAAAACCCGCTGCAGAAAATTGATGCTGCTTTGGCCSCAGGTC^GATCXXKTrGCGTTCTGACCT H l - D ATAAGACTGAAAACCCACTGCAGAAAATTGATGCn^GCCTTGGCACAGGTTGATACACTTCGTTCTaACCT H l - I AAACCACCGAAAACCCGCTGCAGAAAATTGATGCTGCTTTGGCACAGGTTGACACGTTACGTTCTGACCT HI - R CAACCACCGAAAACCCGCTC»:AGAAAATTGATGCTGC?rTTG<K:ACAGGTTGACACGTTACGTTCTGACCT E COLI G T A A A A C C A C C K S A T C C G C T G A A A G C G C T G C J A C G A T C X T T A T C G C A T C T G T A G A C A A A T T C C G T T C T T C C C T
1 2 7 5 1 3 0 0 1 3 2 5 . . . + + +
HI - A ( M G T ( 3 C ( K 5 T T C A G A A C C G T T T C A A C T ( : X : ( 3 C T A T C A C C A A C C T C X X X » A T A C C G T A A A T A A C C T ( 3 T C T T C T
AD GG<3<3<5CAATTCAAAACCGTTTTGATTCAGCCyVTTACCAACCTT<5CSCAATAC(3GTAACCAATCrrGAACTCC BE GC3G<KX»ATTCAAAACCGTTTTGATTCAG<XATTACCAACCTTGGCAATACGGTAACXAATCrrGAACTCC BU G<3G<3G<»ATTCAAAACCGTTTTGATTCy^GCCATTACC»AC(7rTGGC:AATAC(SGTAACCAATCTGAACTCC CA GGGGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGC3CAATACCX5TAACCAATCTGAACTCX: CH GG<3G<3CAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGC3CAATAC<SGTAACCAATCrrGAACTCC DA GC^GGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTC^GCAATACGGTAACCAATCrrGAACTCC DE G(3GGG<ZAATT(»AAACCGTTTTGATTCAGCCATTACCAACCTT(XK»ATACGGTAACCAATCTaAACTCC
GG<3<3<3C;ATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTG<3CAATACGGTAACCAATCrrGAACTCC GC3GG<3CAATTCyj^AACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACT(X GGGCK3CAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCC QQQQQCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTCSAACTCX: GGGGGCAATTCAAAACCGTTTTGATTCAGCCATTAa»ACCTTCKK»ATAC<3GTAACC:AATCTGAACTCC GGGCSGCAATTCAAAACCGTTTTGATTCAGCCyVTTACCAACCTTCSCSCAATACGGTAACCAATCTGAACrrCC GGGG<3CAATTCAAAACCGTTTTGATTCAGCCATTACC»ACCTTG<XyU^TACGGTAACCAATCTGAACTCX: QQQQQCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCC GGGC3GCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGGAATACGGTAACCAATCTGAACTCC GC3GGGCAATTCAAAACCGTTTTGATTCAGCCATTACCAACCTTGGCAATACGGTAACCAATCTGAACTCC GGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCT GGGTGCGGTACAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCT
HI T QQQTGCGGTACAGAACCGTTTCAACTCCGCTATTACCAACCTGGGCAACACCGTAAACAACCTGTCTTCT HI i GQGTGCGGTACAGAACCGTTTCAACTCCGCTATTACCAACCTGGGCAACACCGTAAACAACCTGACTTCT E COLI CGGTGCGGTGCAAAACCGTCTGGATTCCGCGGTTACCAACCTGAACAACACCACTACCAACCTGTCTGAA
DU EN ES JE MS MT MO OR RO SE H l - C H l - D
113
1 3 5 0 1 3 7 5 1 4 0 0 + + +
HI - A GCCCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTTTCCAACATGTCTCGCGCCXIAGATCCTGC AD CX:(3CGTAGCCGTATCGAAGAT(5CTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC BE GCC5CGTAGCCGTATCC3AAGATC»CTGACTAT(5CAACG<3AAGTTTCTAATATGTCTAAAGCGCAQATTCTGC BU GCGCGTAGCCGTATTGAAGATC3CTGACTAT(3CAACC;<3AAGTTTCTAATATGTCTAAAGCGCAGATTCTGC CA GC(3CGTAGCCGTATCGAAGAT(3CrrGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC CH GCCSCGTAGCCGTATCGAAGATGCTTGACTATGCAACGCSAAGTTTCTAATATGTCTAAAGCGCAGATTCTCX: DA GC(3CGTAGCCGTATCGAAGATGCTGACTATCXa^ACCMAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC DE C3CC3CGTAGCCGTATCGAAGATGCTGACTATC3CAACCK3AAGTTTCTAATATGTCTAAAGCGCAGATTCrrGC DU GCGCGTAGCCGTATCGAAGATGCTGACTATG<::AACGGAAGTTTCTAATATGTCTAAAGCG<:y^GATTCTGC EN GCCKJGTAGCCGTATCGAAGATGCTGACn'ATGCAACGGAAGTTTCrrAATATGTCTAAAGCGCAGATTCTGC ES (3CCX:GTAGCCGTATCGAAGAT<3CTGACTATG<:aWVC(MAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC JE CSCGCGTAGCCGTATCGAAGATCSCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC MS (3C(K:GTAGCCGTATCGAAGATGCTGACTATGCAAC(KSAAGTTTCTAATATGTCTAAAaCGCAGATTCTGC MT CSCCX:GTAGCCGTATCGAAGATGCTGACTATG<a^AC<K3AAGTTTC?rAATATGTCTAAAGC<3CAGATTCTGC
MO (;CGCGTAGCCGTATCGAAGAT(X7rGACrrATG<:AACGGAAGTTTCTAATATGTC:TAAAGCGCAGATTCT(X:
OR GCGCGTAGCCGTATCaAAaATGCTGACTAT(X:AAC(;GAAGTTTCrrAATATGTCCAAAG<::GCAGATCCTCX:
RO GCGCGTAGCCGTATCGAAGATGCrrGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCaCAGATTCTGC
SE GCGCGTAGCCGTATCGAAGATGCTGACTATGCAACGGAAGTTTCTAATATGTCTAAAGCGCAGATTCTGC HI - C GCCCGTAGCCGTATCGAAaATTCCGACTACGCGACCGAAGTTTCCAACATGTC:TCGC<3CC3CAGATTCTC3C HI -D C5CCCGTAGCCGTATCGAAGATTCCGACTACGCGACCGAAGTCTCCAACATGTCTCCX;G<:GCyU3ATTCTGC H l - I C X : C C G T A G C C G T A T C ( 3 A A G A T T C C G A C T A C G C G A C C < 3 A A G T C T C C A A C A T G T C T C < X : G C ( 3 C A G A T T C T C X :
HI - R (X:CCGTAGCCGTATC(3AAGATTCCGACrTACGCGACC(3AGGTTTCCAACATGTC7rCCX:GCGCAGATTCTGC E COLI GCC3CAGTCCCGTATTCAGC3ACGCCGACrrATCX:C3ACCGAAGTGTCC»ATATGTCGAAAGCG<a^GATCATCC
1 4 2 5 1 4 5 0 1 4 7 5 + + +
HI -A AGCAGC3<:S GGTACCrrC(33TTCT(3GCCK:AG<K:GAACCAGGTTCXX3CAAAACC5TCCTC:TCTTTACTC3CGT AD AGCAG<SCTCK5TACTTCCGTTCrr(X3CCKy GGCGAACCAGGTTCCGCAAAACGTC(rrCTCn'TTACT(X:GT BE AGCAGGCTCKSTACTTCXrGTTCTCaCXJCSCAGGCGAACCZAGGTTCCGCAAAACGTCCTCTCrrTTACTGCGT BU AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCrrCTCTTTACTGCGT CA AGCAG<3CTCSGTACTTCCGTTCTGGCGCAGG<rrAACCAGGTTCCGCAAAACGTCCTCTCTTTACrrGCGT CH AGCAGGCTGGTACTTCCXSTTCTCXSCGCyUXKrrAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT DA AGCAGGCTGGTACTTCCGTTCTGC3CGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT DE AGCAGGCTGGTACTTCCGTTCTGGCC5CAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT DU AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCG<::AAAACGTCCTCrPCTTTACTGCGT EN AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT ES AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCrrAACCAGGTTCCGCAAAACGTCCTCTCrrTTACTGCGT JE AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCXST MS AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCCXaiAAACGTCCTCrrCTTTACTGCGT MT AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT MO AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCrrCTCTTTACTGCGT OR AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT RD AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT SE AGCAGGCTGGTACTTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT HI C AGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT HI -n AGCAGGCCGGTACCTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT HI - T AGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT HI i AGCAGGCCGGTACCTCCGTTCTGGCGCAGGCTAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGT E COLI AGCAGGCCGGTAACTCCGTGTTGGCAAAAGCTAACCAGGTACCGCAGCAGGTTCTGTCTCTOCTGCAG
APPENDIX B
AMINO ACID ALIGNMENT OF 17 MEMBERS OF THE SALMONELLA
FliC FLAGELLIN g... SERIES AND c, d, i, AND rAND THE
E. COLI K-12 FliC FLAGELLIN WITH FliC FLAGELLIN a
Abbreviations: H1-A, S. paratyphi (^e\ and Joys. 1985); AD, S. adelaide; BE, S.
berta; BU, S. budapest, CA, S. califomia; CH, S. chaco; DA, S.danysz; DE, S. derby;
DU, S. dublin; EN, S. enteritidis; ES, S. essen; MS, S. monschaui; MT, S. montevideo;
MO, S. moscow; OR, S. oranienberg; RO, S. mstock, SE, S. senftenberg; Hl-C, S.
cholerae-suis (Wei and Joys. 1985); Hl-D, S. muenchen (Wei and Joys. 1985); Hl-I, S.
typhimurium (Joys, 1985); Hl-R, S. mbislaw (Wei and Joys, 1986); E. coli, E. coli K-12
(Kuwajima et al., 1986).
0 10 20 30 40 50 60 70 + + + + + + + +
HI -A AQVINTNSLSLLTQNNUQCSQSALGTAIERLSSGLRINSAKDDAAOQAIANRFTANIKGLTQASRNANDO AD AQVINTNSLSLLTC2NNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLT0ASRNANDG BE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKOLTOASRNANDG BU AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG CZA AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKOLTQASRNAHDG CH AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG DA AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTOASRNANDG DE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG DU AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG EN AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGIiRINSAKDDAAGQAIANRFTSNIKGLTOASBNANDG ES AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAG<2AIANRFTSNIKGLTQASRNANDO JE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MS AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MT AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MO AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG OR AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG RO AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAaQAIANRFTSNIKGLTQASRNANDG SE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKOLTQASRNANDG HI -C AQVINTNSLSLLTQNNLNKSQSALGTAIEKLSSGLRINSAKDDAAC30AIANRFTANIKGLT0ASRNANDG HI -D AQVINTNSLSLLTQNNI2nCSQSALGTAIERLSSGLRINSAKDDAA£30AIANRFTANIKGLTOASRNANDG Hl-I AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDG HI -R AQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDG E COLI AQVINTNSLSLITQNNINKNQSALSSSIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQAARNANDG
114
115
80 90 100 110 120 130 140 + + + + + + +
HI-A ISIAQTTEGAUTEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQD AD ISIAQTTEGALNEINNNLQKVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDKVSNQTQFNGVKVLSQD BE ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD BU ISIAQTTEC3ALNEINNNLQRVRELSV0ATNGTNSDSDLKSIQDEIQQRLEE1DRVSNQTQFNGVKVLSQD CA ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNOVKVLSQD CH ISIAQTTEGAUJEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD DA ISIAQTTE(SALNEINNNLQRyRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNC3VKVLSQD DE ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDK7SNQTQFNGVKVLSQD DU ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD EN IS IAQTTEGALNEINNNLORTRELSVQATNGTNSD SDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD ES ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD JE ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNC^VKVLSQD MS IS I AQTTEGAUn INNNLQRVRELSVQATNGTNSD SDLKS I QDE I QQRLEE IDRVSNQTQFNCTVKVLSQD MT ISIAQTTE(SALNEINNNLQRVRELSVOATNGTNSDSDLKSIQDEIQQRLEEIDRySNQTQFN(;VKVLSQD MO ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD OR IS IAQTTEGAU^ INNNLQRVRELSVQATN6TNSDSDLKSI QDE I QQRLEE IDRVSNQTQFNGVKVLSQD RD ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD SE ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD Hl-C ISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQD Hl-D ISIAQTTEGALNEINNNLQR7RELAVQSANGTNSQSDLDSIQAEITQRLNEIDR7SGQTQFNGVKVLAQD Hl-I ISIAQTTEGALNEINNNLQRVRELAGQSANSTNSQSDLDSIQAEITQRLNEIDRVNGQTQFS(;VKVLAQD Hl-R ISIAQTTEGAU7EINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQD E COLI ISVAQTTEGALSEINNNLQRVRELTVQATTGTNSESDLSSIQDEIKSRLDEIDRVSCSQTQFNGVNVLAKN
150 160 170 180 190 + + + ... abedef +beab +abe abed
HI-A NTLTIQVGANDGETIDIDLKQINSQTLGLDTLNV QKKYDV—K-SEVTPSATLS—TTALDG AD NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATV<H)L—K-SSFKNVTGYD—TYAAGADKY BE NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCa)L—K-SSFKNVTGYD—TYAVGANKY BU NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVOJL—K-SSFKNVTC^YD—TYAAGADKY CA NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATV(H)L—K-SSFKNVTGYD—TYAVGANKY CH NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaJL—K-SSFKNVTGYD—TYAAGADKY DA NQMKIQVGANDGETITIVLQKIDVKSLGLDGFNVNGPKEATVCajL—K-SSFKNVTGYD—TYAAGADKY DE NQMKIQVGANDGETITIDLQKIDVKSLGLD6FNVNGPKEATV(a)L—K-SSFKNVTGYD—TYAAGADKY DU NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVOJL—K-SSFKNVTGYD—TYAAGADKY EN NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVODL—K-SSFKNVTGYD--TYAAGADKY ES NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaJL—K-SSFKNVTGYD--TYAAGADKY JE NQMKIQVGANDGETITIDLQKIDVKSLGII>GFNVNGPKEATV(roL--K-SSFKNVTGYD—TYAAGADKY MS NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCT)L--K-SSFKNVTGYD--TYAVaANKY MT NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDL--K-SSFKNVTGYD--TYAAGANKY MO NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDL- -K-SSFKNVTGYD - -TYAAGADKY OR NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaJL- -K- SSFKNVTGYD—TYAVGANKY RD NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVC3)L—K-SSFKNVTGYD—TYAAGADKY SE NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaJL—K-SSFKNVTGYD—TYAAGADKY Hl-C NTLTIQVGANDGETIDIDLKQINSQTLGLDTLNV QKKYDV--S-DTAVAASYSD--SKQNI A—-Hl-D NTLTIQVGANDGETIDIDLKEISSKTLGLDKLNV QDAYTP—KETAVTVDKTTY—KNGTDT— Hl-I NTLTIQVGANDGETIDIDLKQINSQTLGLDTLNL QQKYKV—S-DTAATVTGYADTTIALDN— Hl-R NTLTIQVGANDGETIDIDLKQINSQTLGLDTLNV QQKYKV—S-DTAATVTGYTDSATAIDK— E COLI GSMKIQVGANDNQTITIDLKQIDAKTLGLDGFSV KNNDTVTTS-APVTAFGATT—TNNIKL—
116
200 210 220 230 240 250 e.. + + abe. + abe. . . + +. . . .abedefg.... +ab....
HI -A -AGLKTGTGSTTDTGSIKDGKV—YYNSTSKN—YYVEVEFTDATDQTNKGGF YKVNVA-DDGA AD KVDINSGAWTDAAAP NKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA BE RVDVNSGAWTDTTAPTVPDKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA BU RVDINSGAWTDAAAP DKV—YVNAANGQ—LTTDDAENNTAVNLFKTTK STAGTD-EAKA CA RVDVNSGAWTDTTAPTVPDKV—YVNAAN(3Q—LTTADAQNNTAVDLFKSTK STAGTD-EAKA CH RVDINSGAWTDAAAP DKV—YVNAANCSQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA DA RVDINSGAWTDAAAP DKV—YVNAANC^Q—LTTDDAENNTAVDLFKTTK STAGTA-EAKA DE RVDINSGAWTDAVAP NKV—YVNAANC^—LTTDDAENNTAVDLFKTTK STAGTA-EAKA DU RVDINSGAWTDAVAP DKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA- EAKA EN RVDINSGAWTDAAAP DKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA ES RVDINSGAWTDAAAP DKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA JE RVDINSGAWTDAAAP DKV—YVNAAN<3Q—LTTDDAENNTAVDLFKTTK STAGTA-EAKA MS RVDVNSGAWTDTTAPTVPDKV—YVNAANC3Q—LTTADAQNNTAVDLFKSTK SAAGTD -EAKA MT RVDINSGAWTDDAAP DKV—YVNAANC^l—LTTDDAENNTAVNLFKTTK SAAGTD-EAKA MO RVDINSGAWTDAAAP DKV—YVNAANGQ—LTTDDAENNTAVDLFKTTK STAGTA-EAKA OR RVDVNSGAWTDTTAPTVPDKV—YVNAANCSQ—LTTADAQNNTAVDLFKSTK SAAGTD-DAKA RD RVDINSGAWTDAVAP DKV—YVNAAHCK2—LTTDDAENNTAVDLFKTTK STAGTA-EAKA SE RVDINSGAWTDDAAP DKV—YVNAANC^Q—LTTDDAENNTAVNLFKTTK STAGTD-EAKA Hl-C -VPDKTAITAKIGAATSGCSAGI—KADISFKDGKYYATVSGYDDAADTDKNGT YEVTVA-ADTG Hl-D -ITAQSNTDIQTAIGGGATGVT—GADIKFKDC3QYYLDVK-GGASAGVYKATYDETTKKVNIDTT-DKTP Hl-I -STFKASATGL<3GTDEKID(3DL—KFDDTTGK—YYAKVTVTGGTG KDGY YEVSVDKTNGE Hl-R -STFAASATTLGGTPAI-T(H)L—KFDDTTGK—YYADV SGTTAKDGV YEVTVA-ADGK E COLI -TGITLSTEAATDTGGTNPASIEC^VYTDNGND—YYAKI T<K3DND(3KY YAVTVA-NDGT
260 270 280 290 300 310 . . . . + abcd. + + + +bc + . . . .ab. . . .
HI - A VTMTAATTKEATT PTGITEVTQVQKPVAAPAAIQAQLTAAHVTGA—DTAEMVKMSYTDKNG-KTID AD IAGAIK(3GKECa}TFD-YK(;VTFTIDTKTGNDGNCaCVSTTINGEKVTLT—VADITAGAANVDAATLQSSK BE IAGAIKG<aCE<aDTFD-YKC5VTFTIDTKTGNDQ«GKVSTTINGEKVTLT--VADITAGAANVDAATLQSSK BU IAGAIK(K5KEGOTFD-YK<3VSFTIDTKACaroGNGTVSTTINGEKVTLT—VADITAGAANVNDATLQSSK CA lATSIKGCSKVGDTFD-YKGVSFTIDTKAGDDCa^GTVSTTINGEKVTLT—ISDIGASATDVNSAKIQSSK CH lAGAIKGCSKECTOTFD-YKCSVTFTIDTKTGNDOJCSKVSTTINGEKVTLT—VADIATGATDVNAATLQSSK DA IAGAIKG<3KECT)TFD-YK(3VTFTIDTKTCaro<aJC3KVSTTINGEKVTLT—VADIATGATDVNAATLQSSK DE IAGAIKG<»KECa)TFD-YK(5VTFTIDTKTCaro(aJGKVSTTINGEKVTLT—VADITGGAANVDAATLQSSK DU lAGAIKGGKECaJTFD-YKGVTFTIDTKTOTOGNCaCVSTTINGEKVTLT—VADITGC3AANVDAATLQSSK EN IAGAIKG<aCEGDTFD-YK(?VTFTIDTKTCaDDGaiGKVSTTINGEKVTLT—VADIATGATDVNAATLQSSK ES IAGAIKG<2«EGDTFD-YKGVTFTIDTKTGNDGNGKVSTTINGEKVTLT—VADIATGATDVNAATLQSSK JE lACSAIKGCaOSCSDTFD-YKGVTFTIDTKTCaroOICaCVSTTINGEKVTLT—VADIATGATDVNAATLQSSK MS iATSIKG<2CVCHJTFD-YK(3VSFTIDTKACH)D(aJGTVSTTINGEKVTLT—ISDIGASATDVNSAKIQSSK MT iAGAIK<3C*KE(»DTFD-YKC5VTFTIDTKTCa)DGNCaCVSTTINGEKVTLT—VADIATGATDVNAATLQSSK MO iAGAIK<K5KECa>TFD-YKGVTFTIDTKTCT)G<aJGKVSTTINGEKVTLT—VADIATGATNVNAATLQSSK OR iATSIK(3GKVCaDTFD-YK6VSFTIDTKACa)D(aJGTVSTTINGEKVTLT—ISDIGASATDVNSAKIQSSK RD lAGAIKGCSKEGDTFD-YKGVTFTIDTKTCTODGNGKVSTTINGEKVTLT—VADIGIGAADVNAATLQSSK SE iASAIK(3GKECaDTFD-YKGVSFTIDTKA£aJD(aJGTVSTTINGEKVTLT--VADITAGAANVNDATLQSSK HI - c AVTFATRPTWDL PTDAKAVSKVQQ NDTEIAATNAK-A- -DTATLVKMSYTDNNG-KVID HI - D LATAEATAIRGTA T I T H N Q I A E V T K E G V D T T T V A A Q L A A A G V T ( J A D K D N T S L V K L S F E D K N G - K V I D
H l - I V T L A A V T P A T V T T A T A T E D V K N V Q V A N A D L T E A K A A L T A A G V T G TASWKMSYTDNNG-KTID
HI - R VTLTGTPTGPITAGFPSTATKDVKQTQQENADLTEAKAALTAAGVAAA—GHRSWKMSYTDNNG-KTID E COLI VTMATGATANATV T D A N T T K A T T I T S < ; G T P V Q I D N T A G S A T A N L - - G A V S L V K L - - Q D S K G - N D T D -
117
320 330 340 350 360 370 380 + ....ab.... + ..ab + +bab -t-b. . ab +.... ab.... + . .
H I - A CK3FC»VK-VGADIYAA-TKNKDGSFSINTTEYTD-K-D(arrKTAUI-QLG-GADGKTEWSID-GKTYNAS AD NVYTSV-VNCSQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAAaJKVTLA-GKTMFID BE NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAAtaDKVTLA-GKTMFID BU NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAACTIKVTLA-GKTMFID CA DVYTSV-VSGQFTFA-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAAODKVTLA-GKTMFID CH NVYTSV-VNC3QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANATCT)KITLA-GKTMFID DA NVYTSV-VNC3QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTAN DE NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-C3AEYTANATCH)KVTLA-CSKTMFID DU NVYTSV-VN(KlFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAT(a)KVTLA-(5KTMFID EN NVYTSV-VNC^QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANATGDKITLA-GKTMFID ES NVYTSV-VNC;QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANATCa)KITLA-GKTMFID JE NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANATCaSKITLA-GKTMFID MS DVYTSV-VSGQFTFA-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAACTOKVTLA-CaCTMFID MT NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAACaJKVTLA-GKTMFID MO NVYTSV-VN(3QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANATGDKITLA-(5KTMFID OR DVYTSV-VSGQFTFA-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAAtS)KVTLA-(5KTMFID RD NVYTSV-VNC3QFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-6AEYTANAT(aDKITLA-(5KTMFID SE NVYTSV-VNGQFTFD-DKTKNESAKLSDLEANN-AVKGESKITVN-GAEYTANAACTOKVTLA-GKTMFID H l - C GGFAFK-TSGGYYAA-SVDKSGARSLKVTSYVDAT-TGTEKTAAN-KLG-GADGKTEWTID-GKTYNAS H l - D GGYAVK-MODDFYAA-TYDEKQVQLLUWHYTD -G-AGVLQTGAV-KFG-GAN<3KSEVVTATV(3KTYLAS H l - I GGLAVK-VCH)DYYSA-TQDKDGSISIDTTKYTA-D-NGTSKTALN-KLG-aAD(5KTEVVTID-CnCTYNAS HI - R GGLAVK-V<a)DYYSA-TQNKDGSISINTTKYTA-D-NGTSKTAIiJ-KLG-aAD(SKTBWSIG-SKTYAAS E COLI TYALKDTNCan,YAADVNETTGAVSVKTITYTD-S-S<3AASSPTAVKLG-<a)DCSKTEVVDID-(SKTYDSAD
3 9 0 4 0 0 4 1 0 4 2 0 4 3 0 4 4 0 + b e d e f + + + + +
HI -A KAAGHNFK AQPELAEAAAATTENPLAKIDAALAQVDAVRSDLGAVQNRFNSAITNLCaTTVNNLSS AD KTASCSVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGftlTVTNLNS BE KTAS(3VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLOrrVTNI^S BU KTAS6VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNS CA KTASCSVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSL(3AIQNRFDSAITNL(»nVTNLNS CH K T A S C T V S T LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNS DA PLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCanVTNLNS DE KIASGVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCaTTVTNLNS DU KTASC^VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCaJTVTNLNS EN KIASaVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNS ES KTASC;VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCaJTVTNLNS JE KTAS<3VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLOITVTNLNS MS KTASGVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNS MT KTAS(3VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLGNTVTNLNS MO KTAS(3VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCanvrNLNS OR KTASGVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLfflTrVTNLNS RD KTASGVST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLGAIC^TOFDSAITNLCanvrNLNS SE KTAS(5VST LINEDAAAAKKSTANPLASIDSALSKVDAVRSSLCSAIQNRFDSAlTNLCaiTVTNLNS HI -C KAAGHNFK AQPELAERAATTTENPLQKIDAALAQVDALRSDLCSAVQNRFNSAITNLCan'VNNLSS HI -D DLDKHNFR TGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLQJTVNNLSS Hl-I KAAGHDFK AEPELAEQAAKTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAI TNLCanVNNLSS HI -R KAEGHNFK A Q P D L A E A A A T T T E N P L Q K I D A A L A Q V D T L R S D L G A V Q N R F N S A I T N L G N T V N N L T S E COLI LNGCaiLQTGLTAGGEALTAVANGKTTDPLKALDDAIASVDKFRSSLGAVQNRLDSAVTNLNNTTTNLSEA
118
450 460 470 480 490 .. + + + + + ...
HI -A ARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR AD ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR BE ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR BU ARSRI EDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR CA ARSRI EDAD YATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR CH ARSRI EDAD YATEVSNMSKAQI LQQAGTSVLAQANQVPQNVLSLLR DA ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR DE ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR DU ARSRI EDAD YATEVSNMSKAQI LQQAGTSVLAQANQVPQNVLSLLR EN ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR ES ARSRI EDADYATEVSNMSKAQI LQQAGTSVLAQANQVPQNVLSLLR JE ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPfflJVLSLLR MS ARSRI EDAD YATEVSNMSKAQI LQQAGTSVLAQANQVPQNVLSLLR MT ARSRI EDAD YATEVSNMSKAQI LQQAGTSVLAQANQVPQNVLSLLR MO ARSRI EDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR OR ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQ^TVLSLLR RO ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR SE ARSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLR Hl-C ARSRI EDSDYATEVSNMSRAQI LQQAGTSVLAQANQVPQNVLSLLR Hl-D ARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR Hl-I ARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR Hl-R ARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLR E COLI QSRIQDADYATEVSNMSKAQI IQQAGNSVLAKANQVPQQVLSLLQG
APPENDIX C
AMINO ACID SEQUENCE OF 17 MEMBERS OF THE
SALMONELLA FliC FLAGELLIN g... SERIES
Abbreviations: AD, S. adelaide; BE, S. berta; BU, S. budapest, CA, S.
califomia; CH, S. chaco; DA, S.danysz; DE, S. derby; DU, S. dublin; EN, S. enteritidis;
ES, S. essen; MS, S. monschaui; MT, S. montevideo; MO, S. moscow; OR, S.
oranienberg; RO, S. mstock, SE, S. senftenberg.
0 25 50 + + +
AD AQVINTNSLSLLTQNNUQCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG BE AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG BU AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG CA AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG CH AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG DA AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG DE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG DU AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG EN AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG ES AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG JE AQVINTNSLSLLTQNNUIKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MS AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MT AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG MO AQVINTNSLSLLTQNNUnCSQSSLSSAIERLSSGLRINSAKDDAAOQAIANRFTSNIKGLTQASRNANDG OR AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRKANDG RD AQVINTNSLSLLTQNNUJKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG SE AQVINTNSLSLLTQNNLNKSQSSLSSAIERLSSGLRINSAKDDAAGQAIANRFTSNIKGLTQASRNANDG
75 100 125 + + +
AD ISIAQTTEGALNEINNNLQRVRELSVQATNOTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD BE ISIAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNCJVKVLSQD BU ISIAQTTEC3ALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNQFVKVLSQD CA isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNCJVKVLSQD CH isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD DA isiAQTTEGAI2IEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFN(3VKVLSQD DE I SI AQTTEGALNE INNNLQRVRELSVQATNOTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNC3VKVLSQD DU isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD EN ISI AQTTEGALNE INNNLQRVRELSVQATNGTNSDSDLKSI QDE I QQRLEE IDRVSNQTQFNGVKVLSQD ES isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD JE IS I AQTTEGALNE INNNLQRVRELSVQATNGTNSDSDLKS I QDE I QQRLEE IDRVSNQTQFNGVKVLSQD MS isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD MT isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD MO ISI AQTTEGALNE INNNLQRVRELSVQATNGTNSDSDLKS I QDE I QQRLEE IDRVSNQTQFNGVKVLSQD OR isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD RD isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNOVKVLSQD SE isiAQTTEGALNEINNNLQRVRELSVQATNGTNSDSDLKSIQDEIQQRLEEIDRVSNQTQFNGVKVLSQD
119
120
150 175 200 + + +
AD NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDIN BE NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAVGANKYRVDVN BU NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGELKSSFKNVTGYDTYAAGADKYRVDIN CA NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaJLKSSFKNVTGYDTYAVGANKYRVDVN CH NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVOJLKSSFKNVTGYDTYAAGADKYRVDIN DA NQMKIQVGANDGETITIVLQKIDVKSLGLDGFNVNGPKEATVC3DLKSSFKNVTGYDTYAAGADKYKVDIN DE NQMKIQVGANDGET IT IDLQKIDVKSLGXJ5GFNVNGPKEATV(H5LKSSFKNVTGYDTYAAGADKYRVD IN DU NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDIN EN NQMKI QVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCTOLKS SFKNVTGYDTYAAGADKYKVD IN ES NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDIN JE NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVGDLKSSFKNVTGYDTYAAGADKYRVDIN MS NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCTOLKSSFKNVTGYDTYAVGANKYRVDVN MT NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATV(35LKSSFKNVTGYDTYAAGANKYRVDIN MO NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATV(3)LKSSFKNVTGYDTYAAGADKYRVDIN OR NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVajLKSSFKNVTGYDTYAVGANKYRVDVN RD NQMKIQVGANDGETlTIDLQKIDVKSLGLDGFNVNGPKEATVCaJLKSSFKNVTGYDTYAAGADKYRVDIN SE NQMKIQVGANDGETITIDLQKIDVKSLGLDGFNVNGPKEATVCaDLKSSFKNVTGYDTYAAGADKYKVDIN
2 2 5 2 5 0 2 7 5 + + +
AD SGAWTDAAAPNKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGOCEGDTFDYKGV BE SGAWTDTTAPTVPDKVYVNAANCSQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKEGDTFDY BU SGAWTDAAAPDKVYVNAANGQLTTDDAENNTAVNLFKTTKSTAGTDEAKAIAGAIK(KxKE(roTFDYK(>V CA SGAWTDTTAPTVPDKVYVNAANGQLTTADAQNNTAVDLFKSTKSTAGTDEAKAIATSIKGCSKVaDTFDY CH SGAWTDAAAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKEOSTFDYKC^V DA SGAVVTDAAAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKG<3KECT>TFDYKC;V DE SGAWTDAVAPNKVYVNAANCKJLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGCSKECHJTFDYKC^ DU SGAWTDAVAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKECZiTFDYKCJV EN SGAVVTDAAAPDKVYVNAAN(3QLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIK(K3KE(a}TFDYKGV ES SGAWTDAAAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKEGDTFDYKCSV JE SGAWTDAAAPDKVYVNAANGQLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGCSKECSTFDYKGV MS SGAWTDTTAPTVPDKVYVNAANGQLTTADAQNNTAVDLFKSTKSAAGTDEAKAIATSIKGGKVGDTFDY MT S G A V V T D D A A P D K V Y V N A A N < 3 Q L T T D D A E N N T A V N L F K T T K S A A G T D E A K A I A G A I K C J G K E ( 2 ) T F D Y K G V
MO SC3AVVTDAAAPDKVYVNAAN(3QLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKGGKE(a3TFDYKGV
OR SGAWTDTTAPTVPDKVYVNAANCSQLTTADAQNNTAVDLFKSTKSAAGTDDAKAI A T S IKG<2CVCa)TFD Y RD SGAVVTDAVAPDKVYVNAAN(3QLTTDDAENNTAVDLFKTTKSTAGTAEAKAIAGAIKCK3KECa3TFDYKGV SE sGAWTDDAAPDKVYVNAAN(3QLTTDDAENNTAVNLFKTTKSTAGTDEAKAIASAIKO<3KE(2)TFDYKGrV
3 0 0 3 2 5 3 5 0 + + +
AD TFT IDTKTCarocaJCaCVSTT INGEKVTLTVADITAGAANVDAATLQSSKNVYTSWNCKJFTFDDKTKNESA BE KGVTFTIDTKTGNDGNGKVSTT INGEKVTLTVAD ITAGAANVDAATLQSSKNVYTSWNGQFTFDDKTKN BU sFTIDTKAGNDCaJGTVSTTINGEKVTLTVADITAGAANVNDATLQSSKNVYTSWNCSQFTFDDKTKNESA CA KGVSFTIDTKACaDDaJGTVSTTINGEKVTLTISDIGASATDVNSAKIQSSKDVYTSWSGQFTFADKTKN CH TFTIDTKTCarocaJCSKVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSWNGQFTFDDKTKNESA DA TFTIDTKTCarocaJCSKVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSWNC^QFTFDDKTKNESA DE TFTIDTKTCaroC2«3KVSTTINGEKVTLTVADITCK3AANVDAATLQSSKNVYTSVVNGQFTFDDKTKNESA DU TPTIDTKTCarocaJGKVSTTINGEKVTLTVADITGGAANVDAATLQSSKNVYTSWNGQFTFDDKTKNESA EN TFTIDTKTCT)DGN(3KVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSVVNGQFTFDDKTKNESA ES TFTIDTKTCarocaiCaCVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSWNGQFTFDDKTKNESA JE TFTIDTKTCaroOIGKVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSWNGQFTFDDKTKNESA MS KGVSFTIDTKATODCaiGTVSTTINGEKVTLTISDIGASATDVNSAKIQSSKDVYTSWSGQFTFADKTKN MT TFTIDTKTCTODGNGKVSTTINGEKVTLTVADIATGATDVNAATLQSSKNVYTSWNGQFTFDDKTKNESA MO TFTIDTKTfflJGGNGKVSTTINGEKVTLTVADIATGATNVNAATLQSSKNVYTSWNGQFTFDDKTKNESA OR KGVSFTIDTKAOJDCaiGTVSTTINGEKVTLTI SDIGASATDVNSAKIQSSKDVYTSWSGQFTFADKTKN
RD TFTIDTKTGDDGNGKVSTTINGEKVTLTVADIGIGAADVNAATLQSSKNVYTSWNGQFTFDDKTKNESA
SE sFTIDTKAGNDGNGTVSTTINGEKVTLTVADITAGAANVNDATLQSSKNVYTSWNGQFTFDDKTKNESA
121
BE BU CA
375 400 + +
AD KLSDLEANNAVKGESKITVNGAEYTANAAODKVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLAS ESAKLSDLEANNAVKGESKITVNGAEYTANAACH)KVTLAGKTMFIDKTAS6VSTLINEDAAAAKKSTANP KLSDLEANNAVKGESKITVNGAEYTANAACHJKVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLAS ESAKLSDLEANNAVKGESKITVNGAEYTANAACTOKVTLAGKTMFIDKTASCSVSTLINEDAAAAKKSTANP
CH KLSDLEANNAVKGESKITVNGAEYTANATCaJKITLAGKTMFIDKTASC^VSTLINEDAAAAKKSTANPLAS DA KLSDLEANNAVKGESKITVNCSAEYTANPLASIDSALSKVDAVRSSLGAIQNRFDSAITNLCan'VTNLNSA DE KLSDLEANNAVKGESKITVNCJAEYTANATODKVTLAGKTMFIDKTASCSVSTLINEDAAAAKKSTANPLAS DU KLSDLEANNAVKGESKITVNGAE YTANAT(H5KVTLA£2CTMF IDKT ASCTVSTLINEDAAAAKKSTANPLAS EN KLSDLEANNAVKGESKITVNGAEYTANATCTOKITLAGKTMFIDKTASC^STLINEDAAAAKKSTANPLAS ES KLSDLEANNAVKGESKITVNGAEYTANATCaDKITLACTCTMFIDKTASGVSTLINEDAAAAKKSTANPLAS JE KLSDLEANNAVKGESKITVNGAEYTANATCTOKITLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLAS MS ESAKLSDLEANNAVKGESKITVNGAEYTANAACTJKVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANP MT KLSDLEANNAVKGESKITVNGAEYTANAACT>KVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLAS MO KLSDLEANNAVKGESKITVNGAEYTANATCH)KITLAGKTMFIDKTAS(3VSTLINEDAAAAKKSTANPLAS OR ESAKLSDLEANNAVKGESKITVNGAEYTANAACSKVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANP RD KLSDLEANNAVKGESKITVNGAEYTANATCHJKITLAGKTMFIDKTASC^STLINEDAAAAKKSTANPLAS SE KLSDLEANNAVKGESKITVNGAEYTANAACTOKVTLAGKTMFIDKTASGVSTLINEDAAAAKKSTANPLAS
425 450 475 .... + + +
AD IDSALSKVDAVRSSLGAIQNRFDSAITNLCaiTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA BE LASIDSALSKVDAVRSSLGAIQNRFDSAITNLG»nVTNIllSARSRIEDADYATEVSNMSKAQILQOAGTS BU IDSALSKVDAVRSSLGAIQNRFDSAITNL(»TTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA CA LASIDSALSKVDAVRSSLGAIQNRFDSAITNLOrTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTS CH IDSALSKVDAVRSSLCSAIQNRFDSAITNLCarrVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA DA RSRIEDADYATEVSNMSKAQILQQAGTSVLAQANQVPQNVLSLLRZ DE IDSALSKVDAVRSSLGAI{9niFDSAITNL(»TTVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA DU IDSALSKVDAVRSSLGAIQNRFDSAITNLOITVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA EN IDSALSKVDAVRSSLGAIQNRFDSAITNLCarrVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA ES IDSALSKVDAVRSSLGAIQNRFDSAITNL(an'VTNU«SARSRIEDADYATEVSNMSKAQILQQAGTSVLA JE IDSALSKVDAVRSSLGAIQNRFDSAITNLCarrVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA MS LASIDSALSKVDAVRSSLGAIQNRFDSAITNLOrrVTNI^SARSRIEDADYATEVSNMSKAQILQQAGTS MT IDSALSKVDAVRSSLGAIQNRFDSAITNLCarrVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA MO IDSALSKVDAVRSSLGAIQNRFDSAITNLOrrVTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA OR LASIDSALSKVDAVRSSLGAIQNRFDSAITNLCan^rrNLNSARSRIEDADYATEVSNMSKAQILQQAGTS RO IDSALSKVDAVRSSLCSAIQNRFDSAITNLCan'VTNI^SARSRIEDADYATEVSNMSKAQILQQAGTSVLA SE IDSALSKVDAVRSSLGAIQNRFDSAITNLOn'VTNLNSARSRIEDADYATEVSNMSKAQILQQAGTSVLA
AD BE BU CA CH DA DE DU EN ES JE MS MT MO OR RD SE
500 +
QANQVPQNVLSLLR VLAQANQVPQNVLSLLR QANQVPQNVLSLLR VLAQANQVPQNVLSLLR QANQVPQNVLSLLR
QANQVPQNVLSLLR QANQVPQNVLSLLR QANQVPQNVLSLLR QANQVPQNVLSLLR QANQVPQNVLSLLR VLAQANQVPQNVLSLLR QANQVPQNVLSLLR QANQVPQNVLSLLR VLAQANQVPQNVLSLLR QANQVPQNVLSLLR QANQVPQNVLSLLR