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A Molecular Basis for Erythromycin Sensitivity andResistance in Escherichia ColiHarold S. ChittumEast Tennessee State University

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A molecular basis for erythromycin sensitivity and resistance in Escherichia coli

Chittum, Harold S., Ph.D.

East Tennessee State University, 1093

UMI300 N, Zecb Rd,Ann Arbor, MI 48106

A MOLECULAR BASIS FOR ERYTHROMYCIN SENSITIVITY AND RESISTANCE IN ESCHERICHIA COLI

A Dissertation Presented to

the Faculty of the Department of Biochemistry James H. Quillen College of Medicine

East Tennessee State University

In Partial Fulfillment of the Reguirments for the Degree

Doctor of Philosophy in Biomedical Sciences

byHarold S. Chittum December 1993

APPROVAL

This is to certify that the Graduate Committee of

Harold S. Chittum

met on the

Thirtieth day of September. 1923.

The committee read and examined his dissertation, supervised his defense of it in an oral examination, and decided to recommend that his study be submitted to the Graduate Council and the Associate Vice-President for Research and Dean of the Graduate School, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Bicmedical Sciences.

C 7

Signed on behalf of the Graduate Council Associate Vice-President for

Research and Dean, School of Graduate Studies

ii

ABSTRACTA MOLECULAR BASIS FOR ERYTHROMYCIN

SENSITIVITY AND RESISTANCE IN ESCHERICHIA £QLX

byHarold Chittum

The effect of erythromycin on the 50S riboscanal subunit during cell growth has been extensively investigated.Sucrose density gradient analysis of ribosomes formed in the presence and absence of the drug revealed a 50S specific assembly defect is partially responsible for erythromycin's inhibitory effects on wild type cells. Examination of two erythromycin-resistant mutants of E. coli {N281 and N282) revealed that mutant N281 (L22 mutant) but not N282 {L4 mutant) was assembly defective in the presence of the drug, although only at much higher drug concentrations (300 ug/ml vs. 75 ug/ml for wild type cells).

The altered genes from each mutant have been isolated and sequenced. The L22 mutant was found to contain a 9 bp deletion which eliminated codons 82-84, and the sequence Met- Lys-Arg from the protein. The L4 mutant had an A to G transition mutation in codon 63 resulting in a Lys to Glu change in the protein. Complementation of each mutant by their respective wild type genes resulted in an increased sensitivity to the drug in the partial diploid strains.

Two other macrolide antibiotics (oleandomycin and spiranycin) were also examined but revealed no apparent assembly effect on wild type cells. The MLS antibiotics also appeared to be unable to effect assembly. However, the erythromycin derivative azithronycin showed a similar effect on assembly to that of the parent compound although clarithromycin (another erythromycin derivative) did not. These resultB suggest erythromycin and azithronycin effect assembly through rlbosomal protein L4 and to a lesser extent through protein L22.

iii

ACKNOWLEDGMENTS

I would like to express ny sincere appreciation to the many people who have made it possible for me to complete this degree. First, X would like to thank the members of my committee, Drs. Phil Musich, Lee Pike, Jill Suttles and Paul Monaco. Their advice and encouragement was essential to the successful completion of this degree.

X would also like to express my sincere appreciation to my advisor Dr. Scott Champney. His patience and good nature made working in his lab a pleasure as well as a learning experience. Without his encouragement, advice, and friendship I moBt likely would never have pursued such lofty goals.

My heartfelt thanks to Karen and Ray for the many things they do for biochemistry graduate students, which sometimes go unappreciated. My thanks to all the former graduate students (especially Bonnie) who provided the much needed advice and encouragement we all need occasionally in graduate school.

X would also like to take the time to thank Dumi for being a great friend and often times advisor. You're wise beyond your years; may all the best be yours.

Finally, I would like to thank my parents and grandparents for the encouragement and support they gave, even though they weren't really sure what a ribosome was.

iv

CONTENTSPage

APPROVAL .................................... iiABSTRACT .................................... iiiACKNOWLEDGMENTS ............................... ivCONTENTS .................................... VLIST OF TABLES ............................... viiLIST OF FIGURES ............................. viiiABBREVIATIONS ............................... XCHAPTER

1. INTRODUCTION .......................... 12. MATERIALS AND METHODS ................. 17

Materials ............................ 17Bacterial Strains ..................... 18Media ............................... 18Methods ............................. 18

Growth Experiments ................. 18Preparation of Competent Cells ....... 20Bacterial Transformations .......... 20Screening Transformants ............ 21Isolation of Plasmid DNA ............ 21Isolation of Genomic DNA ............ 24Polymerase Chain Reaction .......... 25Electroelution of PCR Products from

Agarose Gels ................... 27Agarose Gel Electrophoresis ......... 27Restriction Enzyme Digests .......... 28DNA Sequencing by Primer Extension ... 28

v

CHAPTER - Page7 0S Ribosome Isolation 30Rlbosomal Protein Isolation ......... 31Two-Dimensional Polyacrylamide Gels . . 32Rlbosomal Subunit Assembly Analysis . . 34Ribosomal Subunit Degradation Analysis . 35Erythromycin Binding Reaction ....... 36

3........... RESULTS ..................... 37Experimental Results ....................... 37

Two-Dimensional Electrophoresis . . . . 37sequencing the rpiv and rplD Genes . . 38complementation Experiments 44Ribosomal Degradation Analysis . . . . 55Rlbosomal Assembly Analysis 61Assembly Analysis of Mutant Cells ... 65Assembly Effects of Related Drugs . . . 65

4. DISCUSSION ..................... 76BIBLIOGRAPHY ........................... 89VITA .......... 100

vi

LIST OF TABLESTable Page1. Bacterial Strains and Recombinant Plasmids. . . 192. Growth Rates of Mutant and Plasmid-Complemented

Cells in the Presence and Absence of Erythromycin............................ 48

3. Quantitation of Ribosomal Proteins fromComplemented Cells ................... 52

4. Quantitation of Plasmid DNA from ComplementedCells ............................. 54

5. [i4C]-Erythromycin Binding to 70S Ribosomes . . 566. Characteristics of Ribosomal Subunits from

Cells Labeled in the Absence ofErythromycin ..................... 60

7. Characteristics of Ribosomal Subunit Assemblyin the Presence of Erythromycin ........ 64

8. Characteristics of Ribosomal Subunit Assemblyin the Presence of Other Drugs ....... 74

9. Ribosomal Protein L4 Homologies ....... 8010. Ribosomal Protein L22 Homologies ....... 81

vii

LIST OF FIGURESFigure Page1. Chemical Structure of Erythromycin ......... 42. Nucleotide Alterations in 23S rRNA Leading to

Erythromycin Resistance in Escherichia coliand Other Organisms ................... 11

3. Assembly Map of EL. coll SOS Ribosomal Subunit . 144. Two-Dimensional Electrophoresis of Mutant

Ribosomal Proteins ..................... 395. Two-Dimensional Electrophoresis of Wild Type

Ribosomal Proteins ..................... 406. Sequence of rplV PCR Products .............. 417. Sequence of rplD PCR Products................ 428. Purification of PCR Products .............. 439. DNA and Ribosomal Protein Sequence Changes in

Mutants N282 and N281 4510. Origin of pLF Plasmid Inserts ............. 4611. Two-Dimensional Electrophoresis of Rlbosomal

Proteins from Conqplemented Cells ....... 5012. Plasmid Isolation from Complemented Cells . . . 5313. Ribosomal Subunit Degradation Analysis . . . . 5814. Ribosomal Subunit Assembly Analysis ....... 6215. Sucrose Gradient Analysis of Strain N281

Ribosomes . . . . . . . . . . . . . . . . . . 6616. Sucrose Gradient Analysis of Strain N282

Ribosomes . . . . . . . . . . . . . . . . . . 6817. Sucrose Gradient Analysis of Ribosomes Formed

During Growth in Oleandomycin ............ 7118. Sucrose Gradient Analysis of Ribosomes Formed

During Growth in Spiramycin .............. 7219. Sucrose Gradient Analysis of Ribosomes Formed

During Growth in Lincomycin andVirginiairycin Mi ......................... 73

viii

Figure Page20. Sucrose Gradient Analysis of Ribosomes Formed

During Growth in Azithromycin andClarithromycin ........................ 75

21. Proposed Secondary Structure for the rplv GeneDeletion Region ........................ 78

22. Macrolide Drug Structures ................ 87

ix

ABBREVIATIONS A site aminoacyl sitearo E gene coding for dehydroshikimate reductasebp base pairBSA bovine serum albuminCD circular dichroismdH20 deionized waterDNA deoxyribonucleic acidDNAse deoxyribonucleaseDTT dithiothreitol£. £Qli Escherichia collEDTA ethylenediaminetetraacetic acidETTSH 2 -mercaptoethanolg gravitygm gramsHPLC high performance liquid chromatographykb kilobaBe pairMLS Macrolide Lincosamide StreptograminmRNA messenger ribonucleic acidNMR nuclear magnetic resonanceP site peptidyl sitePAGE polyacrylamide gel electrophoresisPCR polymerase chain reactionPEG polyethylene glycolpmol picamolePPO 2,5-diphenyloxaz oleR buffer 10 nw Tris-HCl pH 7.6, 10 rrM magnesium

acetate, 50 mM NH.Cl, 0.2 mM ETSH

x

RNA ribonucleic acidRNAse ribonucleaserplD L4 ribosomal protein generplV L22 ribosomal protein generRNA ribosomal ribonucleic acidS Svedberg unitSDS sodium dodecyl sulfateSET 20% sucrose, 50 mM Tris-HCl pH 8.0,

50 mM EDTA spp. speciesSTET 0.1 M NaCl, 10 TOM Tris-HCl pH 8.0,

1 mM EDTA, 5% Triton X-100 TjEq.! 1 mM Tris-HCl pH 8.4, 0.1 mM EDTAT10EX 10 nlM Tris-HCl pH 8.4, 1 mM EDTATAE 40 nM Trie acetate pH 8.5, 2 mM EDTATB tryptone brothTBE 89 mM Tris base, 89 mM boric acid,

2 mM EDTA, pH 8.3 TCA trichloroacetic acidTEMED N,N,N' ,N' - tetramethylethylenediaminetRHA transfer ribonucleic acidTris tris (hydroxymethyl) aminomethaneuCi microcurieug microgramul microliterUV ultraviolet

xi

Chapter 1 Introduction

The study of antimicrobial agents has long been of interest to researchers due to the medically relevant knowledge which can result from their examination. Equally as important is the role these drugs play as molecular tools for probing the structural and functional domains of their cellular targets. Examples include the use of rifaitpicin in the study of RNA polymerase {Junjin et al., 1988), the isoflavones, flavones and coumarins in the examination of topoisomerase II (DNA gyrase) (Robinson et al., 1993) and vancomycin as a means of studying the cell wall synthesis machinery (Nagarajan, 1991). It seems that virtually every major conponent of the bacterial cell is a target for one antibiotic or another, making the use of antibiotics a powerful means of probing many cellular processes. The ribosome 1b no exception as it is the target of many antibiotics: the macrolides, lincosamides, streptogrammins, puromycin and chloramphenicol to name a few (Vasguez, 1979). For this reason antibiotics which target the ribosome can be extremely useful in unraveling the details of ribosome structure and function relationships. A case in point is the construction of a model for the binding sites of puronycin and chloramphenicol which helped to define the location of the peptidyltransferase center of the large subunit (Moazed and Noller, 1987). The macrolides may be equally useful in this regard pending a more through understanding of their

1

2mode(s) of action.

Recently, the macrolide antibiotics have gained new attention due to their therapeutic importance in combating increasingly prevalent pathogens such as Legionella spp., Campylobacter leiuni. and Chlamydia trachomatis (Kirst and sides, 1989}. The macrolides are an old and well established group of drugs which still play a critical role in fighting infectious diseases. The fact that they account for 10-15% of the worldwide oral antibiotic market makes this point clear (Kirst, 1991). In 1950 Brockmann and Henkel isolated the first macrolide, pikromycin. The name was chosen due to the bitter taste associated with the drug. The chemical structures of pikromycin and other related antibiotics were later shown to possess a common feature, a macrolactone ring. Hence, the term "macrolide" was coined to describe this new class of antibiotics. The macrolides are usually grouped according to the size of the aglycone compound as 12, 14, or 16-membered ring macrolides. Most macrolides also contain amino or neutral sugar moieties. So far, five amino sugars and 11 neutral sugars have been associated with the the structures of these drugs. To date only three drugs have been found which belong to the 12-menibered ring group of macrolides. However, 23 compounds have been isolated which belong to the 14-membered ring group and as of 1982, 71 compounds had been identified in the 16-membered ring macrolide group. Erythromycin shows the greatest antibacterial activity on cultured bacteria of the macrolide antibiotics which are utilized clinically (Gmnra, 1984}. in

3fact, the erythromycins are probably the most well investigated members of the 14-membered ring group (Hardy et al., 1968).

As it occurs naturally, erythronycin is produced as a mixture of three isomers: erythromycins A, B, and C (Omura, 1984) (Figure 1). The process employs a series of fatty acid synthase-like enzyme modules to synthesize the antibiotic (Donadio et al., 1991). The predominant component of the resulting mixture, erythromycin A (Omura, 1984), has been used clinically and possesses broad spectrum activity against both Gram positive and some Gram negative bacterial cells (Gale et al., 1981; Nakayama, 1984). However, one disadvantage of the use of erythromycin is it's low stability in an acid environment such as that of the stomach. Under conditions of low pH the drug degrades to an inactive hemiketal derivative. Therefore, chemical modification of erythromycin A began with the goal of resolving this problem as well as Improving the water solubility and taste of the drug (Omura, 1984). The result was a considerable wealth of information concerning the structure-activity relationships associated with the drug.

Conformational studies using NMR, CD, and X-ray crystallography data, demonstrated that the orientation of the hydrophilic groups (the ketone carbonyl, lactone carbonyl, and hydroxyl groups at positions C-6 and C-ll) and the hydrophobic groups (the methyl groups at the C-4, c-8, and C-12 positions and the ethyl group at the c-13 position) on the macrolactone ring and the desosamine sugar moiety play

Figure 1. Chemical Structure of Erythromycin. The three molecular forms of erythromycin which occur naturally are indicated below.

AGLTCOHZ

HEU1SAL SUGAB

Erythromycin A OH CH3Erythromycin B H CH3Erythromycin C OH H

5a key role in the biological activity of the drug (Omura,1984). Mao and Putterman have proposed that the 2' -hydroxyl and 3' -dimethylamino groups (on desosamine) form hydrogen bonds with the 23S rRNA in the ribosome. The 11,12-hydroxyl,9-carbonyl, and 3"-methoxyl, and most likely the 6-hydroxyl groups of erythromycin A also form additional hydrogen bonds (Mao and Putterman, 1969).

Although there have been extensive investigations of the mechanism by which erythromycin and other macrolides inhibit protein synthesis over the last quarter of a century, progress in elucidating the exact process has been slow and indecisive. The inhibitory effects of erythromycin and other related compounds have probably been most thoroughly investigated in Escherichia coli (Corcoran, 1984; Vazquez, 1979). In this organism, erythromycin interacts with components of the 50S subunit associated with the peptidyltransferase activity (Vogel et al., 1971).Association of subunits in the presence of the antibiotic appears to proceed normally, producing ribosomes which are capable of peptide bond formation (catalyzed by peptidyltransferase). Polyribosomes and elongating ribosomes also seem to function normally in the presence of the drug (Andersson and Kurland, 1987; Tai and Davis, 1979), although their distribution on mRNA is apparently influenced by the drug as observed by their altered mobilities in agarose- polyacrylamide conposite gels (Dahlberg et al., 1973). These observations have led to the suggestion that erythromycin inhibits a step in translation between initiation and

elongation (Andersson and Kurland, 1987; Tai and Davis, 1979; Vester and Garrett, 1987), or effects the P site*dependent binding of peptidyl tRNA (Chinali et al., 1988). In cell* free translation systems where polypeptides are synthesized in response to synthetic polynucleotide mRNAs, there have been many reports of differential actions, depending on the polynucleotide errployed in the assay. For example, in the translation of polyadenylic acid (coding for polylysine) erythromycin stimulated the production of di, tri, and tetrapeptides, but inhibited formation of oligo or polypeptides (Omura, 1984; Tanaka and Teroka, 1966). in a polyuridylic acid directed system (coding for polyphenylalanine), erythromycin failed to inhibit polyphenylalanine formation unless the free 50S subunits were first preincubated with the drug (Oleinick and Corcoran,1969). One hypothesis proposes that erythromycin may bind in the ribosomal "tunnel" (located between the two subunits) consequently irrtpeding the passage of nascent peptides as translation proceeds (Contreras and Vazquez, 1977). Unfortunately, a consensus as to the exact mechanism by which macrolides inhibit protein synthesis has not been reached (Cundliffe, 1980, 1990).

It has been noted that certain differences exist between the inhibitory effects produced by various groups of macrolides. Therefore, some researchers have suggested that the macrolides should be grouped into two categories according to their effects on protein synthesis (Arevalo et al., 1988; Seigrist et al., 1985; Tejedor and Ballesta,

1985a, 1985b, 1986). The erythromycin group is characterized by the presence of monosaccharides on the aglycone ring, while the spiramycins are characterized by the presence of disaccharides on the aglycone ring, as mentioned previously, the erythromycins inhibit chain elongation, but only after the formation of several peptide bondB has been completed.The spiramycins, on the other hand, are thought to be more typical peptidyltransferase inhibitors. For example, spiramycin is an excellent inhibitor of both the puroniycin reaction and the so called * fragment reaction" while erythromycin is a poor inhibitor of either unless specific aminoacyl-tRNAs are used as a substrate (Arevalo et al.,1989). The identification of erythromycin-resistant mutants which are sensitive to spiraitrycin provides sane evidence that the binding sites of the two macrolides may not be identical either. Further evidence for this difference lies in the fact that the binding site for erythromycin on the 5OS subunit is thought to be close to, but not at, the peptidyltransferase center (Cooperman et al., 1990). Fluorescence energy transfer experiments have suggested a distance of 2.3 nm (Wells and Cantor, 1980).

Studies involving ribosomes from Staphylococcus aureuB (Mao and Putterman, 1969), Bacillus subtilis (Oleinick and Corcoran, 1969) and E. coli (PeBtka, 1974; Teraoka, 1970) have allowed the binding parameters of erythromycin to be defined and have suggested features of the compound important for binding interactions (Mao and Putterman, 1969; PeBtka, 1974). Erythromycin was shown to bind tightly and

stoichiometrically in a X:l ratio to the 50S ribosomal subunit from all three organisms (Oleinick and Corcoran,1969; Mao and Putterman, 1969; Pestka, 1974). These studies also revealed that erythromycin is a competitive inhibitor for the binding of virginiamycin S (deflethune and Nierhaus, 1978) and prevents the binding of chloramphenicol, lincomycin and Bpiramycin to the 50S ribosomal subunit (Vazquez, 1979). These compounds all inhibit peptide bond formation reinforcing the idea that erythromycin does interact with ribosomal components at or near the peptidyltransferase center. Protein L22 of the large subunit has been shown to interact directly with virginiamycin S (DiGiambattista et al., 1990). Other work has shown that two different photoreactive derivatives of erythromycin specifically bound to protein L22 and more weakly to proteins L2, L4 and L15 (Arevalo et al., 1988). A combination of polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography (HPLC) approaches were used to identify the labeled proteins. In both cases hZZ labeling was inhibited by the presence of the unlabeled drug indicating that the labeled drugs bound the same region of the ribosome.

Other studleB have focused on the spiramycin-type macrolides (Tejedor and Ballesta, 1985a, 1985b, 1986). Reduction of the aldehyde group present on each drug allowed the production of 3H-labeled dihydro derivatives of carbomycin A, niddamycin, and tylosin. All three of these derivatives are capable of light-independent incorporation into ribosomal proteins, with the first two showing photo-induced

incorporation as well. All five labeling reactions consistently showed protein L27 as the major labeled protein. Site specificity was demonstrated by the ability of the unlabeled macrolides to inhibit covalent binding. Some have proposed the idea that these differences can be attributed to the different glycosidic substituents present on each drug group (Arevalo et al., 1969).

Reconstitution experiments have been useful in identifying key components of the erythromycin binding site. One study identified proteins L15 and L16 as critical for erythromycin binding to the 50S subunit and demonstrated weak binding of the antibiotic to isolated protein L15 (Teraoka and Nierhaus, 1976}. Each of these proteins has been shown to be essential for reconstitution of the peptidyltransferase activity of the SOS subunit (Hampl et al., 1961}. in fact, recent studies have identified the location of these proteins at a common site on the 50S subunit structure (Cooperman et al., 1990; Nag et al., 1991).

Genetic studies are another important way in which interactions between erythromycin and the ribosome have been examined. Resistance to erythromycin can be achieved in several wayB. One of the moBt extensively examined has been resistance produced by sequence changes in the 23S ribosomal RNA. Although resistance to macrolides can occur as a result of mutational changes in ribosomal components, the most common type of resistance is defined as MLS (Macrolide, Lincosamide and streptogramin) resistance (Goldman and Kadam, 1989). The resistance mechanism involves the dimethylation

io

of a single adenine residue at position 2058 of the 23S rRNA by a plasmid-encoded methyItransferase. This mechanism has been shown to confer drug resistance in several different Gram positive organisms (Skinner et al., 1983). Hie methylation results in the production of 50S ribosomal subunits which are unable to bind the MLS antibiotics (Goldman and Kadam, 1989}. Nucleotide alterations at this same site (2058) and in adjacent residues have been shown to lead to drug resistance as well (Douthwaite, 1992; Ettayebi et al., 1985; Sigmund and Morgan, 1982; Vannuffel et al.,1992; Vester and Garrett, 1987) or hypersensitivity (Douthwaite, 1992} (Figure 2). Sequence analysis and protection studies have indicated that changes in either domain II or V of the secondary structure of 23S rRNA can give rise to erythromycin resistance (Douthwaite et al.,1985; Moazed and Noller, 1987). In the case of domain V, the changes come in the form of point mutations as Figure 2 indicates. The only domain II change found to produce resistance to erythromycin was the deletion of nucleotides 1219 to 1230. This has been interpreted as evidence of a functional tertiary interaction between these two domains of the 23S rRNA (Douthwaite et al., 1985). The GTPase- associated center of the 23S rRNA which is also located in domain II has previously been implicated in antibiotic binding. The two drugs involved, thiostrepton and micrococcin, both block processes associated with the ribosomal A-site (Egebjerg et al., 1989). This would seem to lend further credence to the idea of domain II's

Figure 2. Nucleotide Alterations in 23S rRNA Leading to Erythromycin Resistance in Escherichia coli and Other Organisms. Abbreviations: Ans, anisomycin; Cam, chloramphenicol; Cld, clindamycin; Ery, erythromycin; Lnc, lincarnycin; Spr, spiramycin; r, resistant; hs, hypersensitive; Cc, Chlamydomonas chloroplasts; E.c, £. coli; H, Halohacterla: Hu, human mitochondria; M, mouse mitochondria; T, Tetrahvmena cytoplasmic ribosomes; tc, tobacco chloroplasts; Y, yeast mitochondria.

an{*1 lncr | t c ) | C u r • C id ' lE .c l

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A G AC U

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| | \ I.UI Cldr » Eryr(E.c|

ki C*«r * Eryr|E.c)

C \ f v i Spt'rtt\0i Ery‘ * tprr|Cc,r|

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n-rki tw G C UC G G

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12interaction with the peptidyltransferase loop of domain V (Douthwaite 1992}.

Another way in which resistance to erythromycin can he produced is through alterations in a number o£ ribosomal proteins. Several different ribosomal protein mutants of Escherichia ££2li resistant to high levels of erythromycin have been isolated and characterized (Apirion, 1967; Otaka et al., 1970; Pardo and Rosset, 1974; Pardo and Rosset, 1977; Takata et al., 1970; Wittmann et al., 1973). Mutants altered in proteins L4 and L22 have been the most extensively investigated (Pardo and Rosset, 1977a; Pardo and Rosset,1977b; Wittmann et al., 1973). Ribosomes from one mutant containing an altered L4 protein were shown to bind erythromycin poorly and had a reduced peptidyltranBferase activity, interestingly, the distribution of polysomes in these cells waB unaltered by growth in the presence of the drug. Curiously, none of these features was found for ribosomes from another mutant which contained an altered L22 (Wittmann et al., 1973). Mutants dependent on erythromycin for growth have also been isolated and studied (Dabbs, 1979; Sparling and Blackman, 1973; Wild, 1988). Protein L25 was found to be responsible for the dependence in one of these strains (Wild, 1988). it has also been shown that some of the revertants from this dependence mutation in Escherichia coli were altered in ribosomal subunit assembly and were missing protein L19 (Wild, 1988). In fact, several erythromycin dependent mutants have been described with either protein S20, Lll, L15, L28, L29, or L30 missing (Dabbs, 1979) .

13Intersubunit interactions in erythromycin binding have

been implied by the observation that 1*4 erythromycin- resistant mutants become sensitive to the drug if the genes for streptomycin or spectinamycin-resistance (affecting proteins S12 and S5) are present in the same cell as well (Apirion and Saltzman, 1974; Saltzman and Apirion, 1976).Drug binding to the 50S subunit has also been shown to be dependent on the types of 3OS subunit proteins present. This suppression of drug-resistance suggests an interaction between the subunits in the action of the antibiotic. Altogether this information suggests that erythromycin binding and inhibition involves a collection of ribosomal components including 50S proteins L4, L15, L16, L22, specific 23S ribosomal RNA sequences and certain macromolecules of the 30S subunit.

Virtually all of the extensive work on erythromycin and related 50S inhibitors has focused on their mechanism of translational inhibition. With the exception of the suggestion by Vester and Garrett (1987) that erythromycin may stimulate 50S subunit degradation, effects on the formation or breakdown of the large subunit have not been investigated. This point seems worth considering since the 23S rRNA, and ribosomal proteins L4 and L22 are all involved in the earliest parts of SOS subunit assembly, interacting at the 5' end of the 23S rRNA (Herold and NierhauB, 1987) (Figure 3). Two observations suggest that erythromycin may have an additional effect on growing bacterial cells. It has been pointed out that the molar concentration of erythromycin

14Figure 3. Assembly Map of £. coll SOS Ribosomal Subunit.The upper bar represents the 23S rRNA while numbered circles designate large ribosomal subunit proteins. Thiele arrows indicate strong binding and thinner arrows indicate weaker binding.

10 i1000 2000 i200423 S 3'

pQlOTi tlMflUltorAf}alO formation

p r t t tn a o tiam U l tor 6SrRNA ta a g ru o n

(Herold and Nierhaus, 1987)

required to affect translation in vitro is significantly greater than the measured inhibitory concentrations in vivo (Corcoran, 1964), suggesting a second effect of the compound in cells. In addition, ribosomes from an erythrontycin- resistant mutant strain of Escherichia coli with an alteration in ribosomal protein L22 could still bind the antibiotic and showed normal peptidyltransferase activity. Both of these activities were altered in another resistant mutant with an L4 protein change (wittmann et al., 1973). These data Buggest that a different target for erythromycin has been effected in the L22 mutant strain compared with that in the L4 mutant strain.

When one considers the conplexity of the mRNA translation process, it seems apparent that molecular tools like erythromycin can be important in unraveling the process. The Escherichia coli ribosome with its 53 proteins and 3 ribosomal RNAs no doubt has many nuances yet to be discovered. The main focus of previous genetic research into the mechanism(s) of erythromycin resistance has focused on 23S rRNA alterations. Although certain ribosomal protein mutants (L4 (rplD) and L22 (rpiv)) have been shown to exhibit this phenotype, their examination has not been as extensive.A complete understanding of the mechanism(s) is not likely to be forth coming without consideration of these types of mutants. For that reason, the erythromycin resistant mutants N281 and N282 were obtained from Dr. David Apirion (Apirion, 1967). Hopefully this work will allow a better understanding of a small part of this process as well as to aide in the

16design of better and more effective antibiotics for the future. Key to these objectives is the development of a model for the mechanism(s) by which erythromycin produces its inhibitory effects on bacterial growth. This work describes the development of Buch a model.

Chapter 2 Materials and Methods

MaterialsAmpicillin, pancreatic deoxyribonuclease I (DNAse I),

erythromycin, ethidium bromide, lincomycin, lysozyme, oleandomycin, 2,5-diphenyloxazole (PPO), ribonucleaBe (RNAse), spiramycin, and virginiamycin Ml were purchased from Sigma Chemical Corporation. Acrylamide, agarose, methylene bis-acrylamide, Triton x-100, and N,N,N’ ,N’ - tetramethylethylenediamine (TEMED) were purchased from BioRad. Acetic acid, mercaptoethanol, chloroform, ethylenediaminetetracetic acid (EDTA), isoamyl alcohol, isopropanol, trichloroacetic acid, and toluene were purchased from Fisher Scientific. Restriction enzymes and buffers, phenol, sucrose, and urea were purchased from International Biotechnologies, inc. Hydrolink Long Ranger gel was purchased from AT Biochemical, Inc. Coomassie Brilliant Blue R-250 stain was purchased from Kodak. All culture media used in this study were purchased from Difco Laboratories. Radioisotopes ([5,6-3H]-uridine (40.7 Ci/mmol), [N-methyl- 14C]-erythronycin (54 mCi/mmol), and 35S-dATP (1355 Ci/mmol) were purchased from New England Nuclear Products. The Taq polymerase kit was purchased from Promega Corporation. Proteinase K was purchased from E.M. Biochemicals. Pyronine Y was purchased from Allied Chemical. The Sequenase 2.0 kit was purchased from United States Biochemical Corporation. Azithrorrrycin and clarithromycin were gifts from Pfizer and

17

Abbott Laboratories respectively.18

Bacterial Strains and Plasmids All bacterial strains and recombinant plasmids used are

described in Table 1.

MediaBacterial strains were stored at -80°C in 1 ml aliquots

of L broth containing 20% glycerol. Cells were grown in L broth or tryptone broth (TB). L broth consists of 10 g bacto-tryptone, 10 g Had, 5 g yeast extract, and 1.5 ml 1 N NaOH dissolved in l liter of deionized water (dHao). Tryptone broth contains 13 g tryptone and 7 g NaCl dissolved in 1 liter of dH20 {Miller, 1972}. Plates were made with the same media containing 1.6% agar.

MethodsGrowth Experiments

Small-scale growth experiments were done in a shaking water bath. Ten milliliters of L broth were inoculated with 0.2 ml of an overnight culture in a 125 ml side-arm flask.The cell density was determined using a Klett-Summerson colorimeter with a red {Number 66) filter. The appropriate drug was added at a cell density of 2 x I0a cells/ml. Large- scale growth experiments were done on a shaking platform in a Bally environmental room at 32°C. cell densities for these cultures were determined by reading the absorbance at 600 nm

19Table 1. Bacterial Strains and Recombinant Plasmids.

Strain Mo. Genotype Source

SK901 F", aroE+, malA", thi", ery6

Kushner et. al, 1977

N281 Hfr, mal+, thi+, eryR

Apirion, 1967

M282 Hfr, mal+, thi+, eryR

Apirion, 1967

Plasmid Genotype Source

pLF 1.0 l% Ecori fragment of Xfus2 subcloned into pBR325

Watson and Surzycki, 1983

pLF 4.6 4.6% EcoRl fragment of Xfus2 subcloned into pBR325

Watson and Surzycki, 1983

20

in a Beckman Model 25 spectrophotometer.

Preparation of Competent CellsThis procedure was done according to the method of

Silhavy et al. (1984). Cells were grown in L broth to an absorbance of 0.2 at 600 nm. The cells were then chilled on ice for 20 minutes followed by centrifugation at 6400 x g, for 20 minutes at 4°C in a Beckman JA-10 centrifuge. The cellpellet was resuspended in 200 ml of cold 100 mM CaCl2. Next! the suspended cells were incubated on ice for 20 minutes and repelleted as before. Finally, the cells were resuspended in 5 ml of cold 100 mM CaCl2, made 10% in glycerol and stored at 4°C overnight. Competent cells were used immediately for transformation or frozen at -80°C for use later.

Bacterial TransformationsTransformation of competent cells followed the method of

Silhavy et al. (1984). First, 0.5 ug of plasmid DMA (in a volume of 10 ul or less) was added to a 0.2 ml aliquot of competent cells in a 1.5 ml microfuge tube. The suspension was allowed to stand on ice for 1 hour, followed by a two- minute heat shock at 37°C. Next, the mixture was diluted with1 ml of L broth and incubated for 1 hour at 37°C for phenotypic expression. The cells were pelleted in a microfuge and resuspended in o.l ml of L broth. Fifty microliters of the transformation mix was plated on an L broth plate containing 50 ug/ml of ampicillin and placed at

2137°C overnight to select for transformed cells.

Screening TransformantsAmpicillin-resistant colonies were picked from the

overnight transformation plate and placed onto a second ampicillin plate. This plate was allowed to grow at 37°C overnight and then replica plated onto a third ampicillin plate containing 500 ug/ml erythromycin. Complemented mutant cells are unable to grow at this concentration of erythromycin, while noncomplemented mutants grow slowly. The complemented colonies were selected and then used to make freezer stockB.

Isolation of Plasmid DNAPlasmid DNA. was isolated by one of two methods. The

first method employed alkaline lysis and PEG precipitation steps. The second method involved a boiling miniprep procedure followed by further purification using a commercially available "glass milk" type DNA purification kit.

Method One. This miniprep procedure was provided by Dr. Phillip Musich (Musich and Chu, 1993). Cells containing the desired plasmid were used to inoculate 20 ml of L broth containing 50 ug/ml of ampicillin. The cultures were then incubated on a platform shaker at 32°C overnight. Cells were collected by centrifugation at 1,000 x g for 10 minutes. The

cell pellet was resuspended in 0.6 ml of SET buffer (20% sucrose, 50 mM Tris-HCl pH 8) by vortexing. The suspension was transferred to a 1.5 ml microfuge tube. The culture tube was then rinsed with an additional 0.7 ml of SET buffer and transferred to the microfuge tube. The cells were pelleted by centrifugation for 20 seconds in a microfuge. The supernatant was poured off and discarded. The remaining pellet was resuspended in the residual liquid by vortexing until creamy. The suspension was mixed with 250 ul of SET buffer by vortexing. Cell lysis was initiated with the addition of 0.5 ml of alkaline lysis solution (0.2 N NaOH and 1% SDS) and gentle mixing by tube inversions. Lysis was canqpleted by incubation at 65°C for 30 minutes with one or two tube inversions during the incubation. Following incubation, 375 ul of 5M KCl, pH 5.2, was added with gentle mixing.Next, the tubes were placed on ice for at least 20 minutes, insoluble material was pelleted by spinning the tubes in a microfuge for 15 minutes. The supernatant was transferred to a clean tube and spun for an additional 10 minutes to assure that all debris was removed. Then the supernatant was transferred to a clean microfuge tube and 300 ul of 27% polyethylene glycol (PEG) in 3.3 M NaCl was added and mixed by inversions. The tubes were placed on ice for 2 hours and the DNA precipitate was pelleted in a microfuge by spinning 15 minutes. The pellet was washed with 1 ml 70% ethanol and repelleted as before. The pellet was then dried in a speed vac and redissolved in 200 ul of T^E* (10 rrtM Tris-HCl pH 8.4,1 mM EDTA). A 5 ul aliqout of DNA was removed and examined

23for purity on an agarose gel of the appropriate percentage.To further purify the DNA., 200 ul of 5 M anmonium acetate and 400 ul of isopropanol were added with mixing by inversion.The samples were chilled on ice for 20 minutes followed by a 15 minute spin in a microfuge. The DNA pellet was washed twice with 1 ml aliquots of 70% ethanol and repelleted as before. The pellet was vacuum dried and resuspended in the appropriate volume of TjEq (l mM Tris-HCl pH 8.4, 0.1 mM EDTA) .

Method Two. The boiling miniprep and "glass milk* portions of this method were preformed according to the procedures provided in an Elu-Quik DNA purification kit purchased from Schleicher and Schuell. CellB containing the desired plasmid were used to inoculate 5 ml of L broth containing 50 ug/ml of an$>icillin. A 1.5 ml portion of the culture was transferred to a 1.5 ml microfuge tube and pelleted in a microfuge for 1 minute. The supernatant was removed and the pellet was resuspended in 350 ul of STET buffer (0.1 M NaCl, 10 irM Tris-HCl pH 8.0, 1 m EDTA pH 8.0 and 5% Triton X-100). To this, 25 ul of freshly prepared lysozyme (10 mg/ml in 10 mM Tris-HCl pH 8.0) was added and mixed by vortexing. The cells were lysed by placing the tube in a boiling water bath for 40 seconds. Cell debris was pelleted by centrifugation in a microfuge for 10 minutes.The supernatant was placed in a clean 1.5 ml microfuge tube and the plasmid purification process was continued using the Elu-Quik DNA purification kit and the procedure provided by

24the manufacturer. Purified DNA was placed in an appropriate volume of T1E0ii and examined for purity as described in the previous section.

Isolation of Genomic DMAGenomic DNA. was prepared according to the method of

Ausubel et al. (1969). First, a 5 ml overnight culture grown in L broth at 37°C was divided into three 1.5 ml microfugetubes. Next, the cells were pelleted by centrifugation for 2 minutes in a microfuge and resuspended in 567 ul of T^E^ To this, a volume of 30 ul of 10% SDS was added, followed by 3 ul of 20 mg/ml proteinase K. The sample was then mixed by inversion and incubated at 37°C for 1 hour. Following incubation, 100 ul of 5 M NaCl and 80 ul of CTAB (0.7 M NaCl, 10% hexadecyltrimethyl ammonium bromide) were added, and the tubes were placed in a 65°C water bath for 10 minutes. Next, the sample was extracted with an equal volume of chloroform:isoamyl alcohol (24:1) by inverting the tube repeatedly. The sample was separated into two phases by centrifugation for 4 minutes in a microfuge. The aqueous layer waB transferred to a fresh microfuge tube and extracted again with an equal volume of phenol: chloroform: isoairyl alcohol (25:24:1). The DNA-containing aqueous phase was removed and precipitated by the addition of 0.6 volumes of isopropanol. Next, the DNA was spooled from the precipitation interface onto a sealed Pasteur pipet and transferred into 1 ml of 70% ethanol for washing by

25inversion. The DNA was pelleted by centrifugation in a microfuge for 10 minutes and rewashed a second time aB before. Finally, the DNA pellet was dried in a speed vac and resuspended in 100 ul of Concentration and puritywere determined by measuring the absorbance at 260 and 280 ran.

Polymerase Chain ReactiontPCRlThe nucleotide sequence of the S10 operon was retrieved

from Genebank (accession number X02613) (Zurawski and zurawski, 1985). The sequence was examined for suitable oligonucleotide primers using the oligonucleotide analysis program "Oligo* (National Biosciences). The program identified two sets of forward and reverse primers appropriate for the amplification of the rpiv and rplD genes. The sequences of the primers were sent to Dr. Kenton L.Lohman (Southwest Foundation for Biomedical Research; San Antonio, Texas) for synthesis. The sequences of the rpiv forward and reverse primers were 5'-CGG TGG AAA GCG GAG ACAAGA AGC C-3' and 5'-ACC AGT TTT GCG TCC AGT TCA GGC T-3',respectively. The sequences of the rplD forward and reverse primers were 5'-GGC AAG AAA ATG GCA GGT CAG ATG G- 3' and 5'-TTC CAT CGC AGT AGA CGC TTT TTC A- 3', respectively. Uponarrival the primers were cleaned by n-butanol extraction.The concentrations of the rpiv primers were determined by Dr. Phillip Musich (ETSU) using agarose gel analysis with known DNA standards. The concentrations of the rplD primers were determined by measuring their absorbance at 260 ran. PCR

26reactions were carried out £or the rpiv gene with the wild type control strain and the rpiv mutant strain (N281), using the appropriate primers. Reactions for the rplD gene were performed using the same control strain and the rplD mutant strain (N282) in conjunction with their designated primers. EnzymeBi nucleotides, and buffers were purchased as a kit from Promega. The following ingredients were placed into a 0,5 ml microfuge tube : 1.0 ug genomic DNA, 20 ul of 10X buffer (0.5 M KC1, 0.1 M Tris-HCl pH 8.4, 25 nM MgCl2, 1 mg/ml gelatin, 0.1% Tween 20, and 0.1% NP-40), 4 ul of dNTP mix (from 10 mM solutions), 40 pmol each of the forward and reverse primers, 4 ul of sequencing grade Taq polymerase (10 Units) and dH20 to a final volume of 200 ul. Twenty-five microliters of paraffin oil was then layered on top of the sample to prevent evaporation of sample during the reaction process. The Bamples were placed in a Precision Scientific GTC-l thermal cycler for thirty cycles under the conditions of a predetermined PCR program scheme (Ehrlich et al., 1988). The program consisted of the following steps in order of occurrence: denaturation at 94°C for 2 minutes, annealing of primers to target DNA at 55°C for 2 minutes, and polymerization at 72°C for 5 minutes. Three microliters of the PCR reaction product from each set of reactions was examined on a 1.14% agarose gel for spurious bands. Finally, the remaining portion of each reaction was run on another 1.14% agarose gel and the desired fragment electroeluted from the gel in to purify the PCR products from contaminating

fragments, nucleotides and primers.27

Electroelution of PCR Products from Agarose GelsPCR products were electrophoresed in a 1.14% agarose gel

containing 0.5 ug/ml ethidium bromide. The DNA bands of interest were located by fluorescence using a UV transilluminator. The bands were then excised with a razor blade and placed into the elution wells of an electroelutionapparatus (International Biotechnologies, Inc.) Then, 150 illof 7.5 M ammonium acetate/0.01% xylene cyanol solution was placed into the V-shaped collection channels of the apparatus and the DNA was eluted from the gel slice at 50 volts for 1 hour in tae buffer (40 nM TriB acetate, 2 nM EDTA}. The eluted DNA in the ammonium acetate/xylene cyanol solution was recovered by alcohol precipitation.

Agarose Gel ElectrophoresisElectrophoresis was carried out in a Hoefer submarine

minigel apparatus according to the procedure adapted by ManiatiB et al. (1982). The gel solution consisted of an agarose concentration appropriate to produce fragment resolution and 30 ml of running buffer. The running buffer was comprised of 89 nM Tris-borate and 2 nM EDTA, pH 8.3 (TBE). The agarose solution was heated in a boiling water bath until melted, cooled to 55°C, and poured into a gel mold containing an 8 well comb (well dimensions = 1 x 6 x 8 mm). After the gel had solidified, the comb and gel tray were

28removed from the molded gel and samples were loaded. The gel was then electrophoresed at 50 volts until the Orange 6 tracking dye reached the desired point, in some instances ethidium bromide was added to the gel and running buffer solution at a concentration of 0.5 ug/ml. Otherwise, the gels were stained in a solution of 0.5 ug/ml ethidium bromide following the electrophoresis. DNA bands were visualized by placing the gel on a UV transilluminator. Photographs were taken using a Polaroid MP-4 Land camera, a red #23A UV filter and Polaroid type 55 film.

Restrictton Enzyme DigestsRestriction digests of plasmid DNA were performed

according to the method of Maniatis et al. (1982).Restriction enzyme buffer waB added to the DNA and diluted to a final concentration of IX with water. The DNA was digested using 10 units of the appropriate restriction enzyme per microgram of DNA. The digests were incubated at 37°C overnight. Samples were then heated at 70°C for 5 minutes to inactivate the enzyme. The digests were then examined on an agarose gel of the appropriate percentage.

DNA Sequencing bv Primer ExtensionPCR products from the rpiv and rplD genes were sequenced

using a dideoxynucleotide chain-termination method (Sanger et al., 1977). This procedure was performed using materials and instructions provided in a Sequenase 2.0 DNA sequencing kit purchased from united states Biochemical corporation. The

29

PCR products and primers were generated as described above. Primers for sequencing the rplD gene were the same as those used to generate the PCR product, but were used individually. However, a second set of primers (15-mers) were generated by Dr. Kent Lohman for sequencing the rpiv gene. The sequence of this forward primer was 5' -GCG ACG CTG CTG ATA- 3'. The sequence of the reverse primer was 5f-CCC AGG CGA ATA CCA- 3'.

Templates and primers were annealed according to the method of Ausubel et al. (1989). The annealing reactions consisted of 1 ug of PCR product, 1 pmol of primer (either forward or reverse), and 2 ul of Sequenase reaction buffer brought to a final volume of 10 ul with water. The labeling mix was diluted 5-fold with water and the enzyme was diluted 1:8 with the provided dilution buffer. One microliter of 35S- dATP was used per reaction set. The reactions were conducted according to the Sequenase protocol provided by the manufacturer. Sanples were preheated to 75°C for two minutes and one-half of the reaction volume was loaded onto either a 6% or 8% polyacrylamide gel containing 42% urea or a 6% Hydrolink Long Ranger gel in 89 iflM TBE (lx) poured using 0.4- 0.8 mm wedge spacers. The sequencing buffer was IX TBE. The sequencing gel was pre-run at, constant power (45 watts) for 30 minutes. The saiqples were loaded and electrophoresed for an appropriate time, then the second half of the reaction volume was loaded and electrophoresed to the desired end point. Running and loading timeB were dependent on the sequence position which was to be read. Position was

30determined by following the migration of the xylene cyanol and bromophenol blue marker dyes. After electrophoresis was completed, the gel was removed and placed on Whatman 3 mm filter paper, covered with plastic wrap, and dried using a gel drier apparatus. Autoradiograms were prepared from the dried gel by exposure to Kodak diagnostic X-GMAT AR film for periods of 3 days or more, developed and read manually.

70S Ribosome IsolationRibosomes were isolated according to Traub et al.

(1971). A milliliter of overnight culture was used to inoculate 1 liter of L broth. Cultures were grown at 32°C until the absorbance at €00 run reached l.o. The cells were pelleted at 8700 x g at a temperature of 4°C for 20 minutes. The cell pellets were washed with 10 ml R-buffer (10 nM Tris- HCl pH 7.6, 10 mM magnesium acetate, 50 mM NH4C1, 0.2 mM mercaptoethanol (ETSH)), repelleted and stored frozen at -80°C until used. Upon thawing, the cells were resuspended in 5 ml of R-buffer. Freshly prepared lysozyme was added to a concentration of 30 ug/ml, followed by a 15 minute incubation at room terrperature. The suspension was then frozen in an ethanol/dry ice bath and thawed in warm water 3-4 times until cell lysis was canplete. Next, pancreatic DNAse I (l mg/ml in 10 mM Tris-HCl pH 7.9, 10 mM MgS04) was added to a concentration of 10 ug/ml. Lysates were then allowed to stand for 10 minutes until digestion of DNA was complete.Cell debris was pelleted at 12,000 x g for 20 minutes at 4°C.

The supernatant was layered onto 5 ml of ribosome wash I (10% sucrose, 2 M NH4Cl, 10 nM MgClz, 10 nM Tris-HCl pH 7.9) and spun at 134,800 x g, for 4 hours, at 4°c in a Beckman model L5-65 ultracentrifuge. The ribosomal pellets were resuspended in 1 ml R-buffer overnight at 10°C. Any insoluble contaminants were removed by a 2 minute centrifugation in a microfuge. Ribosomal purity and concentration were determined by measuring the absorbance ratio at 260 and 280 nm. The ribosomal pellets were stored frozen at -80°C.

Ribosomal Protein isolationRibosomal proteins were isolated using an acetic acid

extraction method (Hardy et al., 1969). An aliquot of ribosomes equal to 20 a260 units was removed from the -80°C freezer and placed in a 1.5 ml microfuge tube for ribosomal protein extraction. To this, a one tenth volume of 1 M magnesium acetate and 2 volumes of acetic acid were added to extract the proteins and precipitate the rRNA. This solution was placed at 4°C and precipitation allowed to proceed for 2 hours. Next, the rRNA was pelleted by centrifugation in a microfuge for 2 minutes. The supernatant was placed in a fresh microfuge tube and ribosomal proteins were precipitated with 5 volumes of cold acetone at -20°C for 4 hours (Barritault et al., 1976). Ribosomal protein was pelleted by spinning the saiqples for 5 minutes in a microfuge. The supernatant was removed and the protein pellet was allowed to dry inverted overnight. Finally, proteins were resuspended

32in the appropriate buffer for electrophoresis.

Two-Dimensional Polyacrylamide GelsRibosomal proteins were separated according to the

method of Kenny et al. (1979). This method employs an acidic first-dimensional separation technique.

First -Dimension. Gel tubes measuring 12 cm in height and 2 mm in diameter were used for the first-dimension tube gel electrophoresis. The gel tubes were filled to a 10 cm mark with first-dimension separating gel containing 4% (w/v) acrylamide, 0.066% (w/v) methylene bis-acrylamide, 6 M urea; and 38 mM bis-Tris adjusted to a pH of 5.5 with acetic acid.Gel polymerization was initiated by the addition of 1 |il of

N,N,N' ,N'-TEMED and 5 ul of 10% (w/v) amonium persulfate per milliter of gel solution. A flat interface was achieved by the addition of a 100 ul of water. After polymerization (approximately 20 minutes) the water was removed. Ribosomal proteins were then dissolved in 50 ul of first-dimension sample buffer containing 8 M urea, 40 mM mercaptoathanol, and 0.1% (w/v) pyronine Y. The samples were loaded on top of the polymerized gels and the gels were placed in an electrophoresis apparatus containing 250 ml bis-Tris acetate at pH 3.7 (upper reservoir) and pH 7.0 (lower reservoir).The proteins were then stacked at the gel-sample interface by electrophoresis at 0.5 milliamps per gel (constant current) for 30 minutes. Finally, the current was increased to 1 milliamp per gel until 45 minutes after the tracking dtye ran

I

33off the gel {3.5-4 hours).

Second-Dimension. The second dimension electrophoresis was performed according to the method of Kaltschmidt and Wittmann (1970). The first-dimension tube gels were removed using a water filled syringe and positioned on top of freshly poured 18% slab gels. Second-dimension gel solution (pH 4.5) consisted of: 18% (w/v) acrylamide? 0.25% (w/v) methylene bis-acrylamide; 48 mM KOH; 6 M urea? 0.92 M acetic acid; and 0.58% (v/v) TEMED. Slab gels were polymerized by the addition of 20 pi of 10% (w/v) amonium persulfate per

milliter of second-dimension gel solution. The gel slab was14 cmx 12 cm x 2,5 m and held approximately 30 ml of gel solution. The IX running buffer (pH 4.0) contained 140 g of glycine and 15 ml of acetic acid dissolved in 1 liter of water. The upper (cathode) and lower (anode) reservoirs contained IX (400 ml) and 0.33X (1200 ml) running bufferrespectively. Pyronine Y was used as the tracking dye. Theelectrophoresis consisted of a 30 minute stacking at 40 volts followed by an increase to 100 volts (constant voltage) for15 hours or until the tracking dye was approximately 2 cm from the bottom of the gel. Proteins were visualized by staining for l hour with 0.1% (w/v) Cocxnassie Brilliant Blue R-250, 50% (v/v) methanol, and 7% (v/v) acetic acid. Gels were destained in a solution of 10% (v/v) ethanol and 5%(v/v) acetic acid until protein spots were visible against aclear background. Photographs of stained gels were taken UBing a Polaroid mp-4 Land camera, Polaroid type 55 film, and

34a #8 yellow filter.

Ribosomal Subunit Assembly AnalysisAssembly experiments were done according to the method

of Champney (1979). Cells were grown in 10 ml of tryptone broth at 37°C on a shaker water bath to a Klett density of 20 followed by addition of the desired antibiotic to test cultures. Next, the cells were labeled with 1 uCi 3H-uridine and 2 ug uridine per milliliter of culture 1 hour after the addition of the drug. Incubation continued to a Klett density of 100 for control cellB or until growth stopped in cultures containing antibiotics (8-10 hours). After growth was complete, the cells were collected by centrifugation at 2000 x g for 20 minutes. The cell pellet was resuspended in 0.2 ml S buffer and 30 ug lysozyme was added. S buffer consists of 10 ium Tris-HCl, pH 7.6, 0.5 irM magnesium acetate, 50 inM NH4C1, and 6 mM mercaptoethanol. The suspension was then frozen in an ethanol/dry ice bath and thawed in warm water 2-3 times until cell lysis was complete. After lysis was complete, 10 ug of pancreatic DNAse I was added and the cells were placed on ice for 5 minutes. Cell debris was removed by centrifugation at 12,000 x g for 15 minutes at 4°C. The supematantB were then layered onto 5-20% sucrose gradients in S buffer (no mercaptoethanol) and centrifuged at 189,000 x g for 2 hours at 4°C in a swinging bucket rotor (SW50.1). Typically, 30-35 fractions of three drops each were collected and diluted with 1 ml of water. The absorbance at

260 nm was measured for each fraction. An equal volume of 10% TCA and 100 ug of BSA were added and the precipitated materials were collected on Whatman type GF/A glass microfibre filter by vacuum filtration. Finally, the filters were dried and counted in in a Beckman LS 3801 scintillation counter. The 3H counting efficiency was 82.0% in a toluene0.25% (v/v) PPO solution. The optical density and radioactivity in counts per minute tcpm) from an equal number of fractions surrounding both the 50S and 30S peaks were used to calculate the specific activity ratios for each gradient.

Ribosomal Subunit Degradation AnalysisCells were grown in 8 ml of tryptone broth at 37°C on a

shaker water bath to a Klett density of 60 in the presence of 0.4 uCi 3H-uridine per milliliter of culture. The cultures were subsequently chased with 10 ug uridine per milliter of culture and split into two 4 ml cultures. An additional 6 ml of tryptone broth and erythromycin at 75 ug per milliliter of sanple were added. Incubation continued to a Klett density of 100 for control cells or until growth stopped in the erythromycin culture (8-10 hours). After growth was canplete, the cells were collected by centrifugation at 2000 x g for 20 minutes. Subunits were collected and counted as previously described for ribosomal subunit asseirbly analysis. The optical density and radioactivity in counts per minute (cpm) from an equal number of fractions surrounding both the 50S and 30S peaks were used to calculate the specific

activity ratios for each gradient.36

Ervthromvcin Binding ReactionErythromycin binding reactions were performed

essentially according to the method of Teraoka (1970).Binding reactions took place in 125 pi of reaction mixture

containing 3-4 A260 units of 70S ribosomeB, 40,000 cpm [14C] - erythromycin, 50 mM Tris-HCl pH 7.8, 16 mM magnesium acetate, and 80 nW KCl. The samples were incubated at 37°c for 15 minutes, immediately immersed in an ice bath and diluted with 3 ml of cold wash buffer. WaBh buffer contained 25 mM Tris- HCl pH 7.8, 60 mM KCl, and 20 mM magnesium acetate. The diluted mixture was then vacuum filtered onto a 0.45 um Millipore type HAWP filter prewetted with wash buffer. The filters were then washed five times with wash buffer, dried, and counted as previously described for ribosomal subunit assembly analysis. The [MC] counting efficiency was 91.6%.

Chapter 3 Results

Experimental Results

Two-dimensional ElectrophoresisPreviously, Wittmann et al. (1973) found that mutants

N281 and N282 contained alterations in ribosomal proteins L22 and L4 respectively. These findings were initially based on genetic mapping studies. Later, two-dimensional electrophoretic analysis of the ribosomal proteins of the mutants revealed that strain N281 had an altered electrophoretic mobility for protein L22. The other mutant did not appear to exhibit any clear mobility changes in the gel system used. However, the researchers were able to confirm the previous mapping results (which implicated protein L4) using a chromatographic separation technique.That technique revealed that protein L4 of mutant N282 had an altered elution profile from a carboxymethyl cellulose column at pH 5.6 (Osawa et al., 1969). When conpared to the pH 8.6 first-dimension separation used in the previous polyacrylamide gel electrophoresis, it seemed likely that the L4 protein from this mutant might have a detectably altered mobility if the electrophoresis was at or near pH 5.6. The two-dimensional electrophoresis procedure of Kenny et al. (1979) employs a first-dimension separation at pH 5.5. Therefore, it seemed likely that the L4 protein from this mutant (as well as the L22 mutant) might be better resolved

37

38in that gel system.

To investigate this possibility, ribosamal proteins from both mutants were electrophoresed using the separation method of Kenny et al. (1979). As Figures 4A and B indicate, the mutant proteins from each strain showed a significantly decreased electrophoretic mobility in the first-dimension gel at pH S.5. This indicates that the altered protein from each mutant has a more negative overall charge at pH 5.5 than do the wild type proteins from strain SK901 (Figure 5). In addition, the mobility of L4 was normal in the L22 mutant strain as was the L22 protein in the L4 mutant strain (Figure 4).

Sequencing the rolv and rnlD genesIn order to deteimine the exact nature of the mutation

responsible for erythrontycin resistance in these mutants it was necessary to sequence the altered genes. Therefore, the rpiv (L22) and xplD (L4) genes were anplified from the genomic DNA isolated from the mutant and wild type strains using the (PCR) and a series of oligonucleotide primers 25 nucleotides in length. The L22 and L4 genes were contained in 926 bp and 846 bp PCR products, respectively (Figures 6 and 7). Purification of products away from spurious bands, primers and nucleotides was accomplished by electrophoresis on a 1.0% agarose gel (Figure 8). The desired bands were then sliced from the gel and the DNA electroeluted.

The purified PCR products were then sequenced to determine the mutational changes in the genes responsible for

Figure 4. two-dimensional electrophoresis of mutant RIBOSOMAL PROTEINS. Ribosomal proteinB from strains N282 (A) and N281 (B) separated by the electrophoretic method of Kenny et al. {1979). Arrows indicate the positions of both wild type (N) and mutant (M) ribosomal proteins.

40Figure 5. TWO-DIMENSIONAL ELECTROPHORESIS OF WILD TYPE RIBOSOMAL PROTEINS. ElectrophoreBis of ribosomal proteins from £. coli strain SK901 (Control) by the method of Kenny et al. (1979). Arrows indicate the position of wildtype L4 and L22 proteins.

41Figure 6. SEQUENCE OF rplV PCR PRODUCTS. The positions of primers used for PCR amplification (l»22 F-l and R-l) and sequencing of the rplV gene (D22 F-2 and R-2) are indentified by parallel lines. The wild type sequence indicated below is in accordance with that reported by Zuraweki and Zurawski (1985).

L22_ F-l1 CGGTGGAAAG CGGAGACAAG AAGCCCCTGC GCACFK3GTC CCGTCGTTCA51 ACGATCTTTC CTAACATGAT CGGTITGACC ATOGCTGTCC ATAATGGTCG101 TCAGCACGTT CCGGTATTIG TAACCGACGA AATCGTTGGT CACAAACTGG151 GTQAATTCGC ACCGACTCGT ACTTATCGCG GCCACGCTGC TGATAAAAAA

1*22 F-2201 GCGAAGAAGA AATAAGGTAG GAGGAAGAGA TGGAAACTAT CGCTAAACAT251 CGCCATBCTC GTTCTTCTGC TCAGAAGGTT CGCCITGTTG CTGACCTGAT301 TCGCGGTAAG AAAGTGTCGC AGGCTCTGGA TATTTTGACC TACACCAACA351 AGAAAGCGGC TGTACTGGTC AAGAAAGTTC TGGAATCTGC CATTGCTAAC401 GCTGAACACA ACGATGGCGC TGACATTGAC GATCTGAAAG TTACGAAAAT451 TTTCGTAGAC GAAGGCCCGA GCATGAAGOG CATTATGCCG CGTGCAAAAG501 GT0GTGCAGA TCGCATCCTG AAGCGCACCA GCCACATCAC TGTGGTTGTG551 TCCGATCGCT GAGACTCTGG AGACTAGCAA TGGGTCAGAA AGTACATCCT

L22.:R z2601 AATGGTATTC GCCTGGGTAT TGTAAAACCA TGGAACTCTA CCTGG7TTOC651 GAACACCAAA GAATTCGCTG ACAACCTGGA CAGCGATTFP AAAGTACGTC701 AGTACCTOAC TAAGGAACTG GCTAAAGCGT CCGTATCTCG TATCGTTATC751 GAGCGTCCGG CTAAGAGCAT CCGTGTAACC ATTCACACTG CTCGCCCGGG801 TATCGTTATC GGTAAAAAAG GTGAAGACGT AGAAAAACTG CGTAAGGTCG851 TAGOGGACAT CGCTGGOGTT CCTGCACAGA TCAACATCGC CGAAGTTCGT

L22 R-l901 AAGCCTGAAC TGGACGCAAA ACTGGT

42Figure 7. SEQUENCE OF rplD PCR PRODUCTS. The positions of primers used for PCR amplification and sequencing of the rplD gene {L4 F and R) are indicated by parallel lines. The wild type sequence indicated below is in accordance with that reported by Zurawski and Zurawski (1985).

L4 F1 GGCAAGAAAA TGGCAGGTCA GATGGGTAAC GAACGTGTAA COGTTCAGAG51 CCTTGACGTA GTACGCGTTG ACGCTGAGCG CAACCTGCTG CTGGTTAAAG101 GTGCTGTCCC GGGTGCAACC GGTAGCGACC TGATCGTTAA ACCAGCTGTG151 AAGGCGTAAG GAGATAGCAA TGGAATTAGT ATTGAAAGAC GCGCAGAGCG201 CGCTGACTGT TTCOGAAACT ACCTPCGGTC GTGATTTCAA CGAAGCGCTG251 GTTCACCAAG TTGTTGTrGC TTATGCAGCT GGTGCTCGTC AGGGTACTCG301 TGCTCAGAAG ACTCGTGCTG AAGTAACTGG ttcoggtaaa AAACCGTGGC351 GCCAGAAAGG CACOGGCCGT GCGCGTTCTG GTTCTATCAA GAGCCCGATC401 TGGCGTTCTG GTGGCGTGAC CTTTGCTGCT CGTCCGCAGG ACCACAGTCA451 AAAAGTTAAC AAGAAAATGT ACCGCGGCGC GCTGAAAAGC ATCCTGTCCG501 AACTGGTACG TCAGGATCGT CTGATCGTTG TCGAGAAGTT CTCTGTAGAA551 GCGCCGAAAA CTAAGCTGCT GGCACAGAAA CTGAAAGACA TGGCTCTGGA601 AGATGTGCTG ATCATCACCG GTGAGCTGGA CGAAAACCTG TTCCTGGCTG651 CGCGCAACCT GCACAAGGTT GACGTACGCG ATGCAACTGG TATCGACCCG701 GTTAGCCTGA TOGCCTTOGA CAAAGTCGTA ATGACTGCTG ATGCTGTTAA751 GCAAGTTGAG GAGATGCTGG CATGATTCGT GAAGAACGTC

i,4 nTGCTGAAGGT

801 GCTGCGTGCA CCGCACGTPT CTGAAAAAGC GTCTACTGCG ATGGAA

43Figure 8. PURIFICATION OF PCR PRODUCTS. A three microliter aliquot oC each PCR reaction was examined on a 1% agarose gel. Lanes 1-2: PCR products obtained from theamplification of the rplV (L22) geneB of strains N281 and SK901 respectively. Lanes 4-5: PCR products amplified fromthe rplD (L4) genes of strains N282 and SK901 respectively. Lane 3: Commercially available DNA size standards obtainedfrom Bio Ventures, Inc.

1 2 3 4 5

h « i o o o-«700**500

44

conferring erythromycin resistance. The rplD sequence of strain N282 revealed an A->G transition mutation in the first position of codon 63 (Figure 9A). This change would result in a Lys -> Glu change in the L4 protein and would account for the reduced gel mobility of the mutant protein at pH 5.5. Surprisingly, the rplV gene of strain N281 revealed a 9 bp deletion eliminating codons 82-84 in the gene and resulting in the loss of the tripeptide sequence Met-Lys-Arg from the L22 protein (Figure 9B). This loss would reduce the mobility of the protein at pH 5.5, similar to that of the L4 alteration. Notably, a 4 bp direct repeat (GCAT) was found in the wild type sequence flanking the L22 deletion site.This may be indicative of some type of secondary structure (i.e. stem-loop) participating in the deletion of the effected sequence.

Complementation ExperimentsAntibiotic sensitivity is well known to be genetically

dominant to resistance in bacterial cells which are merodiploid for the resistance gene (Nomura and Engbaek1972). In order to examine this phenomenon as it relates to these mutants, strains N281 and N282 were transformed with plasmids pLF 1.0 and pLF 4.6, respectively. Each of these plasmid constructs are derived from pBR325. Plasmid pLF 1.0 contains the L22 gene on a 0.5 kb EcoRX restriction fragment of bacteriophage Afus2 (which contains the E. coll S10

operon). Therefore, pLF 1.0 is capable of complementing mutations in the gene for protein 122 (Figure 10). In

45Figure 9. dna AND RIBOSOMAL PROTEIN SEQUENCE changes in MUTANTS N282 and N281. (A) The Lys to Glu alteration, foundin the mutant L4 (N282) protein is highlighted in bold. (B) The Met-Lys-Arg sequence deletion in the L22 mutant, N281 is indicated in bold and the two 4-bp repeats (GCAT) are double underlined.

A60 65

SK901 L4 -Trp-Arg-Gln-Lya-Gly-Thr-Gly-SK901 rplD -TGG CGC CAG AAA GGC ACC GGC-

N282 L4 -Trp-Arg-Gln-Glu-Gly-Thr-Gly-N282 rplD -TGG CGC CAG GAA GGC ACC GGC-

B80 85

SK901 1*22 -Pro-Ser-Met-Lys-Arg-Ile-Met-SK901 rplV -COG AGO ATG AAG CGC ATT ATG-

N281 1.22 -Pro-Ser-ile-Met-N281 rplV -CCG AGC ATT ATG-

46Figure 10. ORIGIN OF pLF PLASMID INSERTS. Map of the E- £Oli S10 ribosomal operon contained in the Xfus 2 genome.Arrows indicate the EcoRl sites used to generate the pLF 1.0 and pLF 4.6 inserts used for complementation of the rplD and rplV genes.

1S10 Operon

tfAViVP S10 S10 L3 U: L23 L2 SI9 122 S3 LI6 L29 SI 7VAv.v/j

T T T2.3Kb 0.5KbpLF 4.6 pLF 1.0

47addition, mutations which occur in the gene for protein L4 can be complemented using plasmid pLF 4.6, which contains the L4 gene on a 2.3 kb EcoRI fragment of the Sio operon (Figure 10).

The growth rates of the mutant cells with and without coirplementing plasmids were measured in the presence and absence of erythromycin. As Table 2 indicates, both mutant strains showed a reduced rate of growth with increasing concentrations of erythromycin. The mutants grew about 2.5 times more slowly in the presence of 600 ug/ml of erythronycin. Each complemented strain became more sensitive to erythromycin in the presence of the wild type gene. N281 (pLF 1.0) grew 7.5 times more slowly at this same final concentration, while N2B2 (pLF 4.6) grew 3.7 times more slowly. These results are consistent the known dominance of erythromycin sensitivity to resistance in merodiploid cells (Nomura and Engbaek, 1972). However, N281 (pLF 1.0) was more affected by higher concentrations of the drug compared with N281 than was N282 (pLF 4.6) compared with its plasmid-free parent N282.

Since wild type cells will not grow at an erythromycin concentration of 300 ug/ml or greater (Apirion, 1967) these results indicate both mutant and wild type ribosomes are being assembled in complemented mutants. It seemed likely therefore, that the differences in the degree of growth inhibition between the two complemented mutants might be reflected in differing amounts of mutant protein found under each condition for each complemented mutant. To test this

48Table 2. Growth Rates o£ Mutant and Plasmid-Complemented Cells in the Presence and Absence of Erythromycin.

Strain

Erythromycin (ug/ml)

_Q_ iQjQ m .

M2 81 45 60 90 105N281 (pLFl.O) 40 85 160 300N282 45 55 75 115N282 (pLF4.6) 35 65 70 130

Data points in the table above indicate doubling times in minutes.

hypothesis each complemented mutant was grown in either the presence or absence of 300 ug/ml of erythromycin. Ribosomes were collected from the cells and their proteins were examined on two-dimensional polyacrylamide gels (Figures U A ­DI. in each instance mutant protein predominated in the presence of the antibiotic, while wild type protein was most abundant in the absence of the drug. However, as Table 3 indicates, the ratios between mutant and wild type protein were not the same for each of the merodiploids. These findings are quite consistent with the differences in growth rate found between the two complemented mutants obtained in the previous experiment (Table 2},

One possible explanation for these differences is that plasmid copy number might differ depending on the type of growth conditions used (i.e. erythromycin present or absent). In order to examine this possibility, plasmidB were recovered from equal numbers of each of the complemented cells and cut with EcoRl to release the complementing inserts (Figure 12). Image analysis of the gel negative was then used to compare the amount of plasmid present under each growth condition (Table 4). These analyses revealed essentially no differences in plasmid copy number between a given merodiploid grown tinder either condition. This indicates that differences in incorporation of mutant versus wildtype protein are real and not a phenomenon resulting from plaBmid copy number changes found under the two conditions.

The percent of [14C] - erythromycin binding to 70S ribosomes was used to obtain another estimate of the ratio of

Figure 11. TWO-DIMENSIONAL ELECTROPHORESIS OF RIBOSOMAL PROTEINS FROM COMPLEMENTED CELLS. (A-B) Enlargement of the L22 region of a gel for ribosomal proteins isolated from N281 (pLF 1.0) cellB grown in the presence (A) and absence (B) of erythromycin (300 ug/ml). (C-D) Enlargement of the Li regionof a gel for ribosomal proteins isolated from N282 (pLF 4.6) cells grown in the presence (C) and absence (D) of erythronycln (300 ug/ml). Relative positions of the L4 and L22 proteins are indicated.

52Table 3. Quantitation o£ RiboBomal Proteins from Complemented Cells.

Strain + Plasmid WT L4 M L4 WT L22 M L22

N281 {pLF 1.0) (E) 37.7 62.3N281 (pLF 1.0) (E) 36.1 63.9N281 (pLF 1.0) (NE) 100 *N281 (pLF 1.0) (NE) 100 *

Average (E) 36.9 ± x.13 63.1± 1.13Average (NE) 100 ± o.oo *

N282 (pLF 4.6) (E) 12.7 87.3N282 (pLF 4.6) (E) 19.6 80.4N282 (pLF 4.6) (NE) 73.3 26.7N282 (pLF 4.6} (NE) 73.0 27.0N282 (pl«F 4.6) (NE) 69.0 31.0

Average (E) 16.2 ± 4.B8 83.8 i 4.8BAverage (NE) 71.8 1 2.40 28.2 1 2.40

(E) 300 ug/ml Erythromycin; (NE) No Erythromycin; * = Not Detected; Data for each complementented mutant is summarized from each section of the table aB an average value i one standard deviation.

Figure 12. PLASMID ISOLATION FROM COMPLEMENTED CELLS. All plasmidB used for quantification were digested with EcoRl. Lanes 1*2: Plasmids pLF 1.0 (lane 1) and 4.6 (lane 2)isolated from strain SK901. Lanes 3-4: Plasmid pLF 1.0isolated from strain N281 grown in the absence (lane 3) and presence (lane 4) of erythromycin. Lanes 5-6: Isolation ofplasmid pLF 4.6 from strain N282 grown in the absence (lane 5) and presence (lane 6) of erythromycin. The positions of the vector and insert bands are indicated.

1 2 3 4 5 6

-^Vector

4.6

- * 1.0

Table 4. Quantitation of Plasmid DNA from Complemented Cells.

54

integrated IntensityStrain + Plasmid V/i ratio

pBR325 Insert

SK901 (pLP 1.0) (NE) 0.257 0.092 2.793SK901 (pLF 4.6) (NE) 0.277 0.119 2.328N281 (pLF 1.0) (NE) 0.391 0.113 3.460N281 (pLF 1.0) (E) 0.415 0.140 2.964N282 (pLF 4.6) (NE) 0.317 0.138 2.297N282 (pLF 4.6) (E) 0.358 0.186 1.925

NE = No Erythromycin,* E - 300 ug/ml Erythromycin; V/I Ratio ■ pBR325 intensity/insert intensity

55wild type versus mutant protein in the ribosomes from strain N282 (pLF 4.6) assembled under each condition. This was possible since N282 ribosomes have been reported to have no binding of the drug, while wild type and N281 ribosomes have stoichiometric binding of erythromycin {Wittmann et al.,1973). As Table 5 demonstrates ribosomes from strain N282 (pLF 4.6) grown under each condition bound the expected amounts of radiolabeled drug. This prediction was based on two-dimensional electrophoresis results obtained for the Bame ribosomes and binding data collected for ribosomes from strains SK901 and N282 alone. Since ribosomes from strain N282 showed no binding of the drug, the percentage of wildtype protein from cells grown under each condition (obtained from the electrophoresis results) was multiplied by the percentage of erythromycin binding for wildtype ribosomes. This allowed a drug binding prediction to be made for ribosomes isolated from complemented cells grown under both conditions.

Ribosomal Degradation AnalysisVirtually all of the extensive work on erythromycin has

centered on its mechanism of translational inhibition. The observation that the molar concentration of erythromycin required to affect translation in vitro is significantly greater than that in vivo (Corcoran, 1984) adds support to the concept of a second inhibitory mechanism. The complementation work done in this project supports such a hypothesis as well. Recently, Vester and Garrett (1987) have

56Table 5. t14C]-Erythromycin Binding to 70S Ribosomes.

Strain 2D Gels WT / M

% Erythromycin Bound

PredictedBinding

SK901 100 / 0.0 77.0 + 1.08 NAN281 0.0 / 100 64.5 ± 3.22 1 NAN282 0.0 / 100 0.0 ± 0.00 NAN282 (4.6) (NE) 71.8/28.2 49.7 ± 2.33 55.3N282 (4.6) (E) 16.2/83.8 0.0 + 0.00 12.5

WT = percent wild type protein; M *= percent mutant protein;NA - not applicable; % Erythromycin Bound = pmol [14C] - erythromycin bound/ pmol 70S ribosome x 100; A prediction of the binding percentage for complemented strains was calculated by multipling the percent wildtype L4 found in 2D gels by the percent erythromycin binding for SK901 ribosomes. All binding percentages represent an average of three points t

one standard deviation.

57proposed that erythromycin may stimulate 50S subunit degradation. If true, this would represent a secondary mechanism capable of explaining these observations.

In order to examine this possibility, ribosomes in wild type cells were prelabeled with [3H] uridine and then exposedto an inhibitory concentration of erythromycin. To assure adequate incorporation of label, the cells were grown in tryptone broth, which is a poorer medium than L broth but advantageously contains no uridine. As a result, the cells grow more slowly and a lower concentration of the drug is required for inhibition. Under these conditions 75 ug/ml of erythromycin was sufficient to limit cell growth by about 80%. After cells reached their final densities they were collected and lysed. Ribosomal subunits were then separated on sucrose gradients and sedimentation profiles were constructed using the counts per minute of pH]-uridine and the absorbance at 260 nm per fraction (Figures 13A and B).If degradation of ribosomes occured in the presence of the drug, the 50S subunit radioactivity peak would be expected to disappear or at least decline significantly. However, this did not occur. As Figure 13B shows, ribosomes prelabeled with pH]-uridine in the absence of the antibiotic were fairly stable during growth in the presence of the drug, showing a subunit radioactivity ratio reasonably close to that normally ejected (Table 6). However, the effect of erythromycin on assembly can be seen in the reduced amount of 50S subunits, reflected in the altered A ,, ratio of the subunits. This

58Figure 13. RIBOSOMAL SUBUNIT DEGRADATION ANALYSIS.Ribosomal subunits fran strain SK901 were prelabeled by growth in I3H]-uridine. After dilution and uridine addition, the cells were grown in the presence or absence of erythromycin {75ug/ml). Subunits were then separated on 5-20% sucrose gradients. The A260 (□) and cpm (•) profiles for subunits formed in the absence (A) and presence (B) of erythromycin were plotted.

2500

-2000

-1500

-1000

aQ.o

-500

0.00 00 10 20 30

Fraction Number

A 2

60

59

Fraction Number

60Table 6. Characteristics of Ribosomal Subunits from Cells Labeled in the Absence of Erythromycin.

5OS cum SOS A2£0 50S 3 OS S.A.Sanqple 30S cpm 3OS Aaso cpm/Aaeo cpm/Aaso Ratio

SK901 (NE) 1.70 1.78 4,451 4,683 0.95SK901 (E) 1.45 1.15 5,630 4,468 1.26

cpm/Aaeo = specific activity (S.A.); NE - no erythromycin; E = 75 ug/ml erythrooiycin; S,A Ratio = SOS S.A./30S S.A.; All controls were repeatable within one standard deviation.

61result indicates that the antibiotic has a specific effect on the biosynthesis (and not the degradation) of the large ribosomal subunit in £. coli cells.

Ribosomal Assembly AnalysisThis work was initiated to examine the hypothesis that

macrolides might affect the formation of ribosomes in addition to their well established effects on translation. Ribosomal subunit assembly was monitored using a method previously described to study the assembly of ribosomes in several temperature-sensitive protein synthesis mutants (Champney, 1979).

The analysis was begun by labeling cells with [3HJ - uridine for several generations in the presence or absence of erythromycin (75 ug/ml). The radiolabeled subunits were separated from cell lysates by sucrose gradient centrifugation. Figure 14A shows a control gradient of ribosomal subunits from cells grown without erythromycin. As expected, a constant specific activity (cpm/A3G0) was found for each subunit and the 50S/30S specific activity ratio was 1.0 (Table 7). in contrast, ribosomes formed during growth in the presence of erythromycin revealed a 50S subunit deficit (Figure 14B). The 50S/30S specific activity ratio for those ribosomes fell to 0.73. The fact that the specific activity ratio was less than 1 indicates that the effect was specific to the large subunit. Prolonged growth in the presence of erythromycin prior to labeling made this point even clearer. Gradients from cells labeled in this fashion

62Figure 14. RIBOSOMAL SUBUNIT ASSEMBLY ANALYSIS. Ribosomal subunits from strain SK901 were labeled with [3H] -uridine inthe absence (A) or presence of erythromycin (75 ug/ml) beginning one (B) or five hours (C) after the addition of drug. The A^q (P) and cpm (•) of each fraction was then plotted.

toCM

0,45

0.30-

0.15-

0.00

40000

30000

-20000 J

- 1 0 0 0 0

Fraction Number

atoCM4

30000

20000

-10000

a.u

Fraction Number

63

400006

-30000

4-20000

2 -10000

00 10 3020

Fraction Number

64Table 7. Characteristics of Riboscmal Subunit Assembly in the Presence of Erythromycin.

SampleSOS cum 3OS cpm

50S A26D 30S Aaeo

50Scpm/AsGo

30SCpm/Aaeo

S.A. Ratio

SK901 (NE) 1.84 1.81 43,362 42,706 1.02SK901 (El) 0.98 1.35 27,307 37,475 0.73SK901 (E2) 0.68 1.31 20,291 39,099 0.5215201 (NE) 1.58 1.B2 452,759 523,126 0.87N281 (75) 1.39 1.61 496,768 576,509 0.86N281 (300) 0.79 1.13 199,377 284,505 0.70N281 (450) 0.81 1.25 134,372 207,572 0.65N282 (NE) 1.74 1.84 426,771 450,182 0.95N282 (75) 1.86 1.94 486,404 508,504 0.96N282 (300) 1.72 1.88 242,074 264,341 0.92N282 (450) 1.29 1.47 205,022 233,468 0.88

cpm/Aaeo - specific activity (S.A.); NE = no erythromycin; El = 75 ug/ml erythromycin, 10 hr. label; E2 = 75 ug/ml erythronycin, 6 hr. label; (Number) = ug/ml erythromycin; All controls were repeatable within one standard deviation.

65showed that 50S biosynthesis was depressed even further over time (Figure 14C), showing a specific activity ratio of 0.52 (Table 7).

Assembly Analysis of Mutant RibosomesHaving established that erythromycin specifically

inhibits 50S subunit assembly, ribosomes from the erythromycin resistant mutants N281 and N282 were examined for assembly defects in the same manner as those from the wild type strain SK901. Several different concentrations of erythromycin were used as it was expected that a higher concentration of erythromycin would be required to see an assembly effect, if one occurred.

Unlike wild type cells, neither mutant showed an assembly defect at a concentration of 75 ug/ml erythromycin (Figures 15B and 16B}. At a concentration of 300 ug/ml, a limited effect on assembly was observed in strain N281 (Figure 15C), and at 450 ug/ml a reduction in 50S assembly was found similar to that observed in wild type cells at 75 ug/ml (Figure 15D and Table 7). In contrast, no assembly defect was observed in strain N282 at 300 ug/ml erythromycin (Figure 16C). At the highest erythromycin concentration tested (450 ug/ml), only a limited effect on assembly was observed (Figure 16D and Table 7).

Assembly Effects of Related DrugsIt is well known that certain alterations in the central

loop of domain V of the 23S rRNA secondary structure can

Figure 15. SUCROSE GRADIENT ANALYSIS OF STRAIN N281 RIBOSOMES. Ribosomal subunits from strain N281 were prepared as before to examine assembly. The subunits were labeled in the absence (A) or presence of 75 (B); 300 (C); or 450 ug/ml erythromycin (D). The A ^ (□) and cpm (•) of each fraction was then plotted.

8000001.00

-600000

5 °-5°- <-400000

-200000

10 200 30Fraction Number

6000001.00

0.75- -600000

-400000

-2000000.25-

0.000 10 20 30

Fraction Number

A26

0 A

26

0

67

300000

200000

100000

Fraction Number

0.75 300000

0.50-

0.25- -1 0 0 0 0 0

0.000 10

ao

Fraction Number

Figure 16. SUCROSE GRADIENT ANALYSIS OF STRAIN N282 RIBOSOMES. Ribosomal subunits from strain N282 were prepared as before for assembly analysis. The subunits were labeled in the absence (A) or presence of 75 (B); 300 (C); or 450 ug/ml erythromycin (D). The A260 (□) and cpm (•) of each fraction was then plotted.

0.45 400000

•3000000.30-

e<sCl<

•200000

0.15--100000

0.00100 20 30Fraction Number

0.45 400000

•3000000.30-

©ISCl<

•200000 a

0.15-•100000

0.000 10 3020

Fraction Number

A26

0 A

26

0

69

0.6 300000

0.4- -200000

-100000

0 10 20 30Fraction Number

0.45 200000

1500000.30-

•100000 q.

-50000

0.000 10 20 30

Fraction Number

produce so called MLS antibiotic resistance. To investigate the generality of the assembly effect of erythromycin, two other macrolides, a lincosamide, and a streptogramin were examined for their abilities to inhibit 50S assembly. Oleandomycin and spiramycin were chosen to represent the 14- and 16-membered macrolides, while lincamycin and virginianycin MA were selected to represent the remaining drug groups. Neither of the macrolides was found to effect 50S subunit assembly when tested at concentrations which reduced the rate of growth by 75% or more {Figures 17, 18 and Table 8), Lincamycin (a lincosamide) appeared to have some effect on assembly although it did not seem to be identical to that of erythromycin (Figure 19A and Table 8). Virginiamycin HL (a streptogramin) on the other hand, did not have any noticeable effect on assembly (Figure 19B). These results seemed to indicate that erythromycin's effect on assembly was not generally applicable to all members of the MLS resistance group or, for that matter, to all macrolides (Table 8). However, to test this idea further, two recently developed derivatives of erythromycin A, azithromycin (9-deoxo-9a- methyl-9a-aza-9a-homo-erythranycin A) and clarithromycin (6- O-methylerythromycin) were examined as well. These two drugs have been modified to prevent the breakdown which occurs to erythromycin at low pH. Interestingly, azithromycin was found to be as effective as erythrcnycin in inhibiting assembly of the large subunit (Figure 20A). However, clarithronycin did not inhibit assembly (Figure 20B and Table

Figure 17. SUCROSE GRADIENT ANALYSIS OF RIBOSOMES FORMED DURING GROWTH IN OLEANDOMYCIN. RibOBOmal subunits from strain SK901 were prepared as before for erythromycin assembly experiments. The subunits were labeled with [3HJ- uridine in the presence of 80 (A) or 150 ug/ml (B) oleandomycin. The A260 {□} and cpm {•) of each fraction was then plotted.

0.45 300000

0.30- -200000oIBCM<

0.15- - 1 0 0 0 0 0

0.000 10 20 30

Fraction Number

0,20-

0.15-

0.05-

150000

•100000

50000

Fraction Number

72Figure 18. SUCROSE GRADIENT ANALYSIS OF RIBOSOMES FORMED DURING GROWTH IN SPIRAMYCIN. Ribosomal subunits from strain SK901 were prepared as before for erythromycin assembly experiments. The subunits were labeled with [3H]-uridine in the presence of 80 (A) or 150 ug/ml (B) spiramycin. The A260 (□} and cpm (•) of each fraction was then plotted.

1000000.20

-75000

o« 0.1 0- <

-50000

0.05- -25000

0.0020 300 10

Fraction Number

0.20 100000

*750000.15“

o-50000

0.05- -25000

0.00200 3010

Fraction Number

73Figure 19. SUCROSE GRADIENT ANALYSIS OF RIBOSOMES FORMED DURING GROWTH IN LINCOMYCIN AND VIRGINIAMYCIN M^ Ribosomal subunits from strain SK901 were prepared as before for erythromycin assembly experiments. The subunits were labeled with pH] -uridine in the presence of 75 ug/ml lincamycin (A)or 20 ug/ml Virginiamycin Mi {B). The A260 (0) CP™ (•) of each fraction was then plotted.

0.20 75000

0.15--50000

o« 0.1 0- <

-250000.05-

0.000 10 20 30

Fraction Number

0.45 100000

-750000.30-

-500000.15-

-25000

0.000 10 20 30Fraction Number

74Table 8. Characteristics of Ribosomal Subunit Assembly in the Presence of Other Drugs.

Sample5JQS_CBH 3os cpm

50S Aaco 30S Aaeo

50Scpm/Aaco

3 OS cpm/Aaeo

S.A. Ratio

SK901(080) 1.86 1.91 318,603 327,467 0.97SK901(0150) 1.78 1.84 328,811 340,717 0.97SK901(S80) 1.74 1.85 274,197 291,035 0.94SK901 (S150) 1.89 1.74 295,503 272,382 1.08SK901(L75) 1.30 1.30 205,353 205,111 1.00SK90KV20) 1.77 1.89 82,435 88,433 0.93SK901(A2.5) 0.94 1.36 113,796 163,734 0.70SK901(C20) 1.68 1.70 103,389 103,906 1.00

cpm/Aaco « specific activity; (Letter Number) *= Drug, pg/mlDrug; o = oleandomycin; S = spiramycin; V = virginiamycin Mlf- L » lincamycin; A = azithromycin; C = clarithromycin; All controls were repeatable within one standard deviation.

Figure 20. SUCROSE GRADIENT ANALYSIS OF RIBOSOMES FORMED DURING GROWTH IN AZITHROMYCIN AND CLARITHROMYCIN. Ribosomal subunits from strain SK901 were prepared as before for assembly experiments. The subunits were labeled with [3H] - uridine in the presence of 2.5 ug/ml azithromycin (A) or 20 ug/ml clarithromycin (B). The A2S0 {□} and cprn (•} of each fraction was then plotted.

0.6 150000

-100000otOCM<

0.2 - -50000

0.00 10 20 30

Fraction Number

0.4 8DOOD

0.3- -60000

o* 0,2- CM<

-40000

-20000

0.0300 10 20

Fraction Number

Chapter 4 Discussion

This work began as an attempt to better define the interaction(s) between erythromycin and the £. coll ribosome by identifying ribosomal protein sequences important for resistance. As is often the case in studies of this nature, mutants exhibiting an altered phenotype for the

icharacteristic under examination can frequently be useful in probing any interactions which may exist. The strains (N281 and N2B2) used in these experiments were originally isolated as spontaneous mutants resistant to high concentrations of erythromycin (Apirion, 1967) and, therefore provided a useful tool for the examination of this ribosome-drug interaction.

Previous analysis of ribosomal proteins from mutant N281 revealed that protein L22 possessed an altered electrophoretic mobility at pH 8.6 (Wittmann et al., 1973). However, the protein responsible for resistance in mutant N282 could not be detected using this method. Analysis of the L4 protein from this mutant on a carboxymethyl cellulose column indicated that the protein had an altered elution profile at pH 5.6. Based on the Buccess of this low pH separation, an alternate two-dimensional electrophoresis technique (at similar pH) was employed in this work. As expected, both mutant proteins showed an altered first- dimension mobility in the second gel system.

In order to more precisely define the nature of the alterations in these mutants, the appropriate ribosomal

76

protein genes were sequenced from each resistant mutant. The wild type sequences found for the rplV and rplD genes agreed exactly with those reported by ZurawBki and Zurawski (1985) with one exception. The rplD sequence contained a CAG codon for Gin at position 30 in place of the reported CAA codon which also codes for that amino acid (Zurawski and Zurawski 1965). unlike the the point mutation found in the rplD gene of the L4 mutant, the deletion of nine nucleotides from the rplV gene of the L22 mutant was somewhat unexpected. Close examinination of the deleted region for possible secondary structures using the DMA analysis program *DNASISW revealed that a reasonably stable stem-loop structure (-1.2 kcal/mole) was possible (Figure 21). significantly, the deleted region of the rplV gene was entirely contained in the loop. It seems likely that this area might be susceptible to deoxyribonuclease activity and therefore to deletion.

The altered electrophoretic mobilities detected for these proteins would predict a more negative form of each protein. Both the lysine to glutamate alteration in 1A

(N282) and the Met-Lys-Arg deletion found in L22 (N281) are entirely consistent with this prediction. One apparent inconsistency is the fact that wittmann et al. (1973) were unable to detect an elctrophoretic mobility change for the L4 protein of mutant N282 at pH 8.6. This is most likely explained by the lack of reference points near that protein in the pH 6.6 electrophoretic system. A small change such as the one likely to occur would be difficult to detect. The identification of these sequence alterations is quite

Figure 21. PROPOSED SECONDARY STRUCTURE FOR THE r p l V GENE DELETION REGION. Possible stem-loop structure for the deleted region of the L22 gene. Arrows indicate the boundaries of the deletion. The four base repeats flanking the deletion are enclosed in boxes.

A = T

79significant. Their identification represents the first sequence changes to he reported for ribosomal proteins from an erythromycin-resistant mutant. These studies also identify critical components of the sequences of two different ribosomal proteins necessary for the interaction of erythromycin with the bacterial ribosome. These observations should help in identifying the important amino acid and nucleotide residues involved in the binding and inhibitory effects of the compound.

A comparison of the altered protein sequences from the mutants used in these experiments with the homologous ribosomal proteins of several other organisms revealed that both regions are highly conserved (Tables 9 and 10). The sequence surrounding the L4 mutation site shows canplete identity in 2£. nseudotuberculosis and strong homology with the Mycoplasma homolog (Table 9). The altered region of protein h22 also demonstrated strong homology with sequences from two other prokaryotic L22 hamologs (Bacillus and Mycoplasmal (Table 10). In the four chloroplast genes examined additional significant homology was observed with the region surrounding the L22 deletion site. The fact that ribosomes from these cells and plastids are sensitive to erythromycin (Mets and Bogorad, 1971; Qcmira, 1984) is compelling evidence for the importance of these regions in the erythromycin-ribosome interaction. This point is strengthened by the fact that L22-like sequences from archaebacteria and rat (Arndt et al., 1990; Laine et al.,1991) which show little sensitivity to these drugs (Amils et

Table 9. Ribosomal Protein L4 Homologies80

Organism Sequence ft Homology ReferenceEscherichiacollYersiniapsuedotuber.Mycoplasmacapricolum

PWRQgGTGR 100 (201) (Zurawski and Zurawski 1985)

FWRQXGTGR 94.5 (210) (Gross et al., 1989)

PWKQKGTGL 35.8 (193) (Ohkubo et al, 1987}

Residues altered in erythromycin-resistant ribosomal proteins are double underlined. Identical amino acids from L4 homologs in other organisms are in bold. The percent of overall sequence homology is given along with the total number of amino acids in each homolog which is indicated in parentheses.

81Table 10. Ribosomal Protein L22 Homologies.

Organism

Escherichia£QliMycoplasmacaprlcolumBacillusstearotherm.Liverwort(chloroplast)Red algae (chloroplast)Spinach(chloroplast)Tobacco(chloroplast)Rat liverHalobacteriuromarismortui

Sequence ft.-HOmology Reference

QPSMKRIMP

GPTLKRFRP

GPTLKRFRP

gtffkrfqp

GPKLKRFQP

OITLKKVKP

GTTVKKLKP

APKMRRRSGVGESQGRKP

100 (110) (Zurawski andZurawski, 1985)

54.1 (109) (Ohkubo et al., 1987)

52.8 (108) (Kroner et al., 1990)

41.6 (101) (Fukuzawa et al.,1988)

40.6 (101) (Kao and Hu, 1990)

38.1 (97) (Zhou et al., 1989)

37.6 (101) (Tanaka et al., 1986)

32 (109) (Laine et al., 1991)17.8 (90) (Arndt et al., 1990)

Residues altered in erythromycin-resistant ribosomal proteins are double underlined. Identical amino acids from L22 homologs in other organisms are in bold. The percent of overall sequence homology is given along with the total number of amino acids in each homolog which is indicated in parentheses.

82al., 1990) also have poor homology. These results suggest that ribosomes from other prokaryotes, or from mitochondria that have significant sequence homology to £. coli ribosomal proteins L4 and L22 should be sensitive to inhibition by erythromycin, as was demonstrated previously (Lamb et al., 1968).

A number of recent studies have identified several 23S ribosomal RNA mutations in £. cflli (Douthwaite, 1992;Ettayebi et al., 1985; Sigmund and Morgan, 1982) and other organisms { Vester and Garrett, 1987 and references therein) which also lead to the erythromycin resistance phenotype. These sequence changes are almost exclusively located in domain V of the RNA secondary structure with the exception of one deletion affecting domain II described b y Douthwaite et al. (1985). This is a significant finding when one considers that proteins 14 and L22 interact with each other in binding to 23S rRNA at sequences associated with domain II (Herold and Nierhaus, 1987).

These experiments have also provided evidence that erythromycin has a second mechanism of action. Perhaps the first clue to this was the observation that the expression of wild type and mutant alleles in merodiploid cells was different in the presence and absence of the drug. Two- dimensional electrophoresis of ribosomal proteinB from particles formed under each condition demonstrated this clearly. Those experiments showed that a considerable excesB of wild type protein was present in ribosomal particles from cells grown under permissive conditions (no erythromycin

83present) while mutant protein predominated in the presence of the drug. This difference in protein content waB alBO reflected in the relative sensitivity of the two complemented mutants to increasing concentrations of erythromycin. The L22 diploid was far more sensitive to higher concentrations of the macrolide than it's L4 counterpart was under the same conditions. A quantitative comparison of the relative expression of these genes revealed that plasmid copy number differences were not a factor in the distribution of protein forms.

However, it is conceivable that transcription might be effected in the presence of the L4 plasmid (pLF 4.6) since expression of the S10 operon is under autogenous control of protein L4. Termination of trancription for the operon is specically elevated with increasing concentrations of free L4 (Zengel and Lindahl, 1990). under those conditions one would expect to see a decrease in the ratio of mutant versus wild type protein conpared to that of the L22 proteins, since pLF4.6 transcription would be independent of that regulation. However, this was not the case, possibly this is due to the alteration present in the mutant form of L4. It is possible that the mutant protein is not capable of the same regulatory function or at least is not as efficient. Such questions can only be answered by quantifing mRNA levels from both the plasmid and the chromosomal genes, since the two are under the control of different promoters.

Further evidence for a second mechanism was provided by the fact that differences in binding affinities for the drug

have been observed for each of these mutants. Previous examination of ribosomes from the 1A mutant showed that they bound erythromycin poorly and had a reduced peptidyltransferase activity. Curiously, neither of these features were found for ribosomes isolated from the L22 mutant {wittmann et al., 1973). These results agree with the binding data reported here, although somewhat less binding was found in these experiments compared with that reported by Wittmann et al. (1973). This is most likely a result of the inactivation of a greater percentage of ribosomes during the purification process. Given these results, the basis for resistance in the L22 strain seemed less clear. However, the possibility of a second mode of action for the drug seemed more likely.

The assembly experiments presented here provide strong evidence that erythromycin has two independent but related effects on protein biosynthesis in Escherichia coli cells.The functional effect of this antibiotic is to prevent the elongation of short polypeptide chains during translation, interfering with a step after initiation (Andersson and Kurland, 1987; Vester and Garrett, 1987). This work also shows that an additional structural effect of the compound is to block ribosomal subunit formation, probably early in the assembly process. SOS subunit formation is specifically affected, since breakdown is not stimulated and the 30S subunit is unaffected. These results differ from the conclusion of Vester and Garrett (1987) that erythromycin apparently stimulated the degradation of 5OS subunits in a

85merodiploid strain containing 23S rRNA alleles for sensitivity and resistance to erythromycin. However, differences in strains and experimental conditions makes a direct comparison with these results difficult. It is important to note the the efficacy of erythromycin and azithromycin in vivo is much greater than that of oleandomycin, clarithromycin or spiramycin, consistent with the two site effect of the former compounds (Nakayama, 1984; Whitman and Tunkel, 1992) .

Although the MLS resistance drug group does not appear to effect assembly as a whole, it is still not clear from this work whether other members of this drug group might have an effect, it is clear however, that the macrolide effect on assembly is restricted to erythromycin and azithromycin among the antibiotics examined here. A number of studies have indicated that erythronycin interacts with both sequences in the 23S ribosomal RNA and with ribosomal proteins L4 and L22 (Arevalo et al., 1988; Coopeman et al., 1990; Douthwaite et al., 1985; Ettayebi et al., 1985; Moazed and Noller, 1987; Sigmund and Morgan, 1982; Teraoka and Nierhaus, 1978). Since the assembly defect is significantly reduced in resistant mutantB with an altered L22 or L4 protein, it seems clear that these proteins are part of the binding site for the drug on the 50S subunit.

These results suggest a model for the effect of erythronycin on assenibly, based on two facts. A comparison of the structures of erythromycin and azithrontycin (which prevent assembly) and of oleandomycin and clarithromycin

(which do not), reveals that the former compounds contain a pair of cis hydroxyl groups at positions 6 and 12 of the macrolactone ring (Figure 22). Oleandomycin lacks both of these hydroxyl groups and clarithromycin is a 6-0- methylerythranycin (Nakagawa and Ontura, 1984; Whitman and Tunkel, 1992). This inplies that the 6-OH group is important in promoting the assembly deficiency. In addition, the mutational change in protein L4 conferring assembly resistance involves the loss of a baBic amino acid residue.I propose that erythromycin (and azithromycin) interferes with the binding of L4 to 23S rRNA early in the assembly process and that an electrostatic interaction between the 6- OH group of the drug and Lys 63 of L4 is critical for this effect. Loss of the positive charge (as in the mutant strain) or the absence of the 6-hydroxyl group (as in oleandonycin and clarithromycin) would allow assembly to proceed. Protein L4 is a strong 23S rRNA binding protein associating with the 5f end of the RNA (Nierhaus, 1982). It is also noteworthy that the erythronycin-resistant mutant N282 is cold-sensitive for growth at 20°C, and is 50S subunit assenbly defective under those conditions (Champney, unpublished observations). This subunit assembly defect has been observed for several other cold-sensitive mutants (Bryant and Sypherd, 1974; Guthrie et al., 1969) including a strain with an L22 protein mutation (Pardo et al.r 1979). Apparently the price for resistance to erythronycin in the L4 mutant is ribosomal instability at low tenperature.

These findings suggest two important directions to be

87Figure 22. MACROLIDE drug structures. Molecular structures of erythromycin a , oleandomycin, azithromycin, and clarithromycin. Hydroxyl groups at positions 6 and 12 (as they relate to erythromycin A's structure) are indicated in bold.

HO -------

ERYTHROMYCIN A OLEANDOMYCIN

AZITHROMYCIN CLARITHROMYCIN

pursued. The in vitro reconstitution of the large subunit should be performed in the presence of erythromycin to help define the site and stage in assetribly at which the inhibition occurs. It will also be important to test other microorganisms (especially Gram positive cells) to explore the generality of this novel effect of the macrolide antibiotic drug group on ribosome formation.

Finally, these results indicate a second parameter to be evaluated when new macrolide antibiotics are under review for effectiveness. This pronounced effect on riboscmal assembly indicates that this may be an equally important target for drug activity as is an inhibition of translation. It is also important to be reminded that the idea of "one drug one action" is not necessarily true.

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99Zengel, J.M., L. Lindahl. 1990. Ribosomal protein L4 stimulates in v i t r o termination of transcription at a NusA-dependent terminator in the S10 operon leader.Proc. Natl. Acad. Sci. USA. 87: 2675-2679.Zhou, D. X., P. M. Quigley, 0. Massenet and R. Mache.1989. Cotranscription of the S10- and spc-like operons in spinach chlorpplasts and identification of three of their gene products. Mol. Gen. Genet. 216: 439-445.Zurawski, G. and S.M. Zurawski. 1985. Structure of the Escherichia coll S10 ribosomal protein operon. Nucleic Acids Res. 13: 4521-4526.

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Harold S. ChittumDepartment of Biochemistry, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614 Business Phone: (615) 929-6211Birth: August 4, 1962Place of Birth: Knoxville, TennesseeMarital Status: SingleEast Tennessee State University, James H. Quillen College of Medicine, Johnson City,TN; Ph.D., Biochemistry, 1993East Tennessee State University, College of Arts and Sciences, Johnson City, TN;M.S., Biology, 1988East Tennessee State University, College of Arts and Sciences, Johnson City, TN;B.S., Biology, 1984Experience in protein purification, chromatographic techniques, HPLC, and a variety of protein gel electrophoresis. Extensive training in the chemistry and biochemistry of nucleic acids, and protein- nucleic acid interactions. Techniques in molecular biology including isolation and preparation of DNA, polymerase chain reaction and DNA sequencing.Graduate Assistant 1989-93. Department of Biochemistry, James H. Quillen College of Medicine, East Tennessee State university.Sigma XiAmerican society for MicrobiologySecond Place Graduate Division 2 1993,9th Annual College of Medicine Research Forum, East Tennessee State universitySecond Place Graduate Division 2 1992,8th Annual College of Medicine Research Forum, East Tennessee State universityRecipient of Biomedical Research Support Grant (BSRG), 1990-91

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committee Service at ETSU:

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Biochemistry Chairperson Search Committee, 1993Secretary/Treasurer, Graduate Student Association, 1992-1993Departmental Representative, Graduate StudentAssociation, 1991-1992Learning Resources Committee, 1989-91Chittum, H. and W.S. Champney. Erythromycin Inhibits Assembly of the Large Ribosomal Subunit in Escherichia coll. (Submitted 1993)Chittum, H. and W.S. Champney. Ribosomal Protein Sequence Changes in Erythromycin Resistant Mutants of Escherichia coli. (in Preparation 1993)Chittum, H. and W.S. Chairpney. Molecular Basis for Erythronycin Sensitivity and Resistance in Escherichia coli. American Society for Microbiology, 93rd General Meeting, Atlanta Georgia, (May, 1993)Chanqpney, W.S,, H. Chittum, and R. Samuels. Ribosomes from Trichomonad Protozoa have Prokaryotic Characteristics, int. J.Biochem. 24(7):1125-1133 (1992)Chanipney, w.s., H. chittum and R. Samuels. Ribosomes from TrichomonadB have Prokaryotic Characteristics. Society of Protozoologists, 44th Annual Meeting,Bozeman, MT (July, 1991)Chittum, H., R. Samuels and w.s. Champney. Characterization of the Ribosomes of Four Trlchomonads. Society of Protozoologists,40th Annual Meeting, Champaign-Urbana, IL (July, 1987)

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