Vol. 170, No. 7

Expression of the Divergent Tricaryboxylate Transport Operon (tctl)of Salmonella typhimurium

K. A. WIDENHORN, J. M. SOMERS, AND W. W. KAY*

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada V8W 2Y2

Received 17 December 1987/Accepted 8 April 1988

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult todetect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, wascloned in both orientations behind the T7 phage promoter +10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was

shown to encode the periplasmic (29000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, anda 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000Mr. By TnS mutagenesis, subcloning of TnS insertions, and subcloning of various deletion mutants it was shownthat the tctl system is divergently transcribed. The periplasmic binding protein (C protein) is the first productof one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the oppositestrand only the 29,000-Mr protein was essential for tetI function, and it was found to be weakly attached to theinner membrane. Thus tetI encodes four proteins, one periplasmic, two integral, and one peripheral to thecytoplasmic membrane, with the genes arranged as c M

Bacterial transport systems are loosely classified as eithershock sensitive or shock insensitive. These two operationalgroups are distinguished on the basis of the functionalnecessity of a periplasmic component and by differing en-ergy requirements (2, 8). The tricarboxylate transport op-eron, tctI, of Salmonella typhimurium is an example of theformer as it deploys a 29,000-Mr substrate binding protein asan essential periplasmic transport component (14). Previousfine-structure genetic mapping suggested the presence of atotal of four genes (13). Subsequent cloning restricted thetctl system to a 4.5-kilobase (kb) DNA fragment (17) inwhich the periplasmic binding protein was centrally mappedto approximately 1 kb of DNA. Unfortunately, until now theother gene products have steadfastly resisted elucidation.

Bacterial shock-sensitive transport operons normally ex-hibit a high degree of polarity after transcription of the firstgene, invariably that encoding the periplasmic binding pro-tein (2). The other gene products, usually integral or periph-eral membrane proteins, often have been difficult to identify,and frequently their existence has only been deduced fromDNA sequence analysis (2).Using the T7 RNA polymerase-promoter system (15),

which allows the amplified exclusive expression of clonedgenes, we were able to identify all the gene products of thetctI system. The system is compared herein with otherwell-characterized shock-sensitive systems.

MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and

plasmids used are listed in Table 1. Strains harboring plas-mids were grown in the presence of the appropriate antibi-otic (6).

Genetic techniques. Plasmids were isolated according tothe alkaline lysis method described by Maniatis et al. (10).DNA was digested with restriction enzymes as describedbefore (17) and ligated in the presence of polyethylene glycol

* Corresponding author.

(R. W. Blakesley and P. V. King, Focus 8:1-3, 1986). Dele-tions were created using the procedure described by Dale etal. (5).A TnS mutagenesis. Escherichia coli DL291(pKW107) was

infected with X467. Plasmids carrying TnS insertions wereisolated as previously described (7, 17), and clones unable togrow on citrate were selected as white colonies on citrate-peptone tetrazolium agar (3, 13). The position of the TnSinsertion was located after restriction with endonucleaseBamHI and subsequent agarose-gel electrophoresis.

Exclusive labeling of plasmid proteins by the T7 RNApolymerase-promoter system. Cells containing pGP1-2 and apT7 recombinant plasmid were grown in LB at 30°C to anoptical density of 0.25 at 590 nm. A sample of 1.5 ml washarvested and washed once with M9 medium, and the pelletwas suspended in 3 ml of M9 medium to which 0.3 ml ofmethionine assay medium (Difco) was added. Cells weregrown at 30°C for 1 h before the temperature was shifted to42°C. After 30 min a 42°C, rifampin was added to a finalconcentration of 600 ,ugIml. A 1-ml sample of rifampin-treated cells was incubated for an additional 45 min at 30°C,before cells were pulse-labeled for 5 min with 10 ,uCi of[35S]methionine (New England Nuclear Corp.).

Cell fractionation. Six 1-ml samples of strain KS1070(pGP1-2, pKW108) were labelled with [35S]methionineas described. Cells were converted into spheroplasts andsubsequently broken by six consecutive freeze-thaw cyclesaccording to the method of Boeke and Model (4). Unbrokencells were removed by low-speed centrifugation. The super-natant was applied to an 11-ml discontinuous sucrose gradi-ent (1.5 ml of 70%, 2.5 ml of 53%, and 7.0 ml of 15% sucrose)and spun for 4 h at 130,000 x g, which routinely separatesouter and inner membrane fractions. Outer membrane wasconfirmed by the presence of 2-keto-3-deoxyoctulosonicacid in the lipopolysaccharide component, and the innermembrane was confirmed by the presence of succinic dehy-drogenase. Cross-contamination was always less than 5%.Fractions from the gradient were collected, and the radioac-

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3224 WIDENHORN ET AL.

TABLE 1. Bacterial strains, phages, and plasmids

Designation Known genotype and phenotypes Source or reference

BacteriaS. typhimurium KS1070 S. typhimurium hisFI009 trpB2 metA22 rpsL201 xyl-J galE Atctl This laboratoryE. coli LE392 F- supF supE hsdR galK trpR mutB lacY CSHaE. coli DL291 araDJ39 A(argF-lac)U169 relAl rpsLJSOflbB5301 deoCI ptsF25 glpR D. Ludtke

A(glpT-glpA)593 gyrA recABacteriophage

X467 A b221 rex::TnS X c1857 Oam29 Pam8O N. KlecknerPlasmids

pT7-5 Ampr S. TaborpT7-6 Ampr S. TaborpGP1-2 Kanr S. Tabor (15)pKW107 Ampr tctA+B+C+D+b (8 kb) This studypKW108 Ampr tctA+B+C+D' (4.5 kb) This studypKW109 Ampr tctA'B+C+D+c This studypKW110 Ampr tctB+C+D+ This studypKW111 Ampr tctD' This studypKW112 Ampr tctC'D+ This studypKW119 Ampr tctA+B+C+D+ (4.5 kb; insert in reverse orientation compared to pKW108) This study

a CSH, Cold Spring Harbor Laboratory genetics course.b The genotype tctA+B+C+D' indicates all genes are present and functional on the plasmid.I The genotype tctA' indicates the C-terminal end of the protein is truncated.

tivity of aliquots was assayed by scintillation counting inPCS solubilizer (Amersham).

Strain KS1070(pGP1-2, pKW119) labeled with [35S]me-thionine either was fractionated by the NaOH lysis methoddescribed by Russel and Model (12) or, alternatively,spheroplasts were prepared and broken by freeze-thaw asdescribed by Boeke and Model (4).

Analytical techniques. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE) separations weredone in 12% gels by the method of Ames (1). Immunoblot-ting using anti-C-protein antiserum was performed as de-scribed by Towbin et al. (16).

RESULTS

In vivo expression of tctl present on an 8-kb tetI insert. An8-kb EcoRI-BamHI restriction fragment containing the tctIsystem had been cloned previously in both E. coli and a AtctIS. typhimurium mutant (KS1070) (17). This fragment wasexcised by restriction endonuclease digestion and ligatedinto pT7-6 (15). This placed the 8-kb fragment containing thetctl insert behind the 410 promoter (pKW107) (Fig. 1).KS1070(pKW107) was then transformed with pGP1-2 (15), aplasmid which allows the expression of T7 RNA polymerase

0

UJI__,

I ECE o

C-Protein m010

(pT7-6)

EC0o C-Protein

0o01O(pT7-6)

_00I w .4 -

C-Protein (oTl) j

C- Protein (p75

pKW107

pKW18

pKW119

FIG. 1. Cloning of tctI behind the 4i10 promoter. pKW1O7 and

pKW108 represent 8- and 4.5-kb inserts into pT7-6, respectively.pKW119 represents the 4.5-kb insert in the reverse orientation.

upon temperature induction. The two-plasmid system(pKW107, pGP1-2) should permit the specific expression ofthe cloned genes. After a temperature shift to 42°C and theaddition of rifampin, only message transcribed from the T7promoter by the phage polymerase was expressed. Virtuallyall host background could be suppressed by rifampin inhibi-tion of bacterial RNA polymerase, allowing exclusiveexpression of cloned genes as well as radiolabeling of thegene products in vivo with radioactive amino acids. Figure 2demonstrates the results of such an expression experimentwith the 8-kb tctI insert with KS1070 as host. Lanes 1through 6 of Fig. 2 are controls, and lanes 3 and 6 show theabsence of host background in cells transformed with pGP1-2 (lane 3) or pGP1-2 and pT7-6 (lane 6) after temperatureinduction in the presence of rifampin. Lane 9 shows that in

pit4-. 4.

4dw 4.im

*0mw.. As s1F2 3 4 5 6 7 8 91

1 234 56 78 9

Mr 3x 10

4131 i

-~29-19

10FIG. 2. Expression of a 8.0-kb tctI+ and 4.5-kb tctI+ insert in

pT7-6. S. typhimurium strains KS1070(pGP1-2) (lanes 1 to 3),KS1070(pGP1-2, pT7-6) (lanes 4 to 6), KS1070(pGP1-2, pKW107)(lanes 7 to 9), and KS1070(pGP1-2, pKW108) (lane 10) are shown.All samples were grown in M9 medium supplemented with methio-nine assay medium at 30°C. The T7 polymerase remained uninducedin lanes 1, 4, and 7. The T7 polymerase was induced in lanes 2, 5,and 8, and in addition rifampin was added in lines 3, 6, 9, and 10 toinhibit E. coli RNA polymerase. Cells were labeled with [35S]methionine, and labeled proteins were separated by SDS-PAGE andsubjected to autoradiography. The molecular size of proteins en-coded by the 8-kb as well as the 4.5-kb fragment is indicated.

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tVtI EXPRESSION IN S. TYPHIMURIUM 3225

oo0 T- -V, I- Y- Y- W-3333 3Xe Y.Y

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4131 a29 ""sam

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4 3 2 1 0Kb a_LJ

E

H

HI

hPKW10O

Ba HpKWIO3

BE H.J pKWllO

H B 010 H- j pKWfl2

H B. HI L. .j pKWI11

FIG. 3. Subcloning and expression of tctI. pKW108 codes for all the components necessary for citrate transport, while the smallersubclones have a tctI-negative phenotype. The autoradiogram shows the SDS-PAGE profile of proteins expressed by the different subclones.Cells were induced for the production of T7 polymerase and incubated with rifampin before [35S]methionine labeling.

cells transformed with pGP1-2 and pKW107 at least sixmajor proteins were expressed. Since T7 polymerase oftendoes not efficiently recognize bacterial termination signals(11), it was evident that more than just the tctl genes was

being transcribed from the 8-kb DNA insert.Expression of tctI as a 4.5-kb insert. Since we previously

restricted the genes coding for a tctl-positive phenotype to a

4.5-kb DNA fragment (17), we cloned and expressed thisfragment in the T7 expression system in a manner analogousto that described above (Fig. 1, pKW108). In addition to theC protein (29,000 Mr) and its precursor (31,000 Mr), the4.5-kb fragment encoded three other products: a diffuse bandof approximately 41,000 Mr as well as 19,000-Mr and 21,000-Mr bands (Fig. 2, lane 10), which together represent theentire potential tctI gene products from the sense DNAstrand. The appearance of a 21,000-Mr band expressed fromthe 4.5-kb insert (lane 10) but not from the 8.0-kb insert (lane9) suggests it is a truncated downstream protein. The 41,000-Mr protein expressed from the 8-kb fragment was partiallyhidden by a nearby band (Fig. 2). The comparison inexpression patterns from the 8-kb and 4.5-kb fragmentsillustrates the need for subcloning to reduce the complexitiesarising from overexpression with T7.

Ordering of the gene products by A TnS mutagenesis. In anattempt to order these gene products, we submitted the 8-kbDNA fragment harboring the tctl insert in pKW107 to k Tn5mutagenesis (17). After mutagenesis, kanamycin-resistantclones were pooled, and the plasmid pool was used totransform E. coli LE392. The location of the TnS insert incitrate-negative transformants was subsequently mapped byrestriction endonuclease digestion and electrophoresis (17).T7 expression of these insertion mutations revealed thateither the 41,000-Mr diffuse band had disappeared, or the Cprotein had been truncated as evidenced by the cross-reacting material in an immunoblot, or by the same evidenceC protein was completely absent (data not shown). Thisresult ordered the 41,000-Mr protein distal to the C protein.Absence or reduction of the 19,000-Mr band suggested thatthis protein was immediately distal to the C protein. How-ever, it also pointed out a real problem of combining TnSmutagenesis with such overexpression systems, since theTnS termination signal was frequently not recognized, mak-ing the results equivocal.

Confirmation of the gene order by deletion cloning. Toovercome the problems described above, we decided to take

advantage of the HindIll restriction site in TnS to subclonevarious fragments of the tctI system (Fig. 3). The sizes of thecloned fragments and the corresponding gene products aredemonstrated (Fig. 3). pKW108 represents the 4.5-kb frag-ment and expresses all of the expected gene productsincluding the diffuse band. pKW109 has lost the external21,000-Mr protein and has a truncated, originally 41,000-Mr,diffuse protein which now migrates at approximately 27,000Mr. In pKW110 this protein is now completely lost. pKW112has lost the 19,000-Mr protein and also shows a truncated Cprotein whose identity was again confirmed by immunoblot-ting (data not shown). Finally, pKW111, the smallest clone,is completely devoid of all gene products including the Cprotein. These deletions are summarized in the diagram-matic insert of Fig. 3. Thus the order of gene products, distalto proximal, in the tctI operon was deduced to be 41,000 Mr(diffuse band), 19,000 Mr, and finally the 29,000 Mr periplas-mic C protein. No protein was apparently expressed fromthe first 1.3 kb of the operon even when expression was

carried out in the presence of [14C]leucine instead of[35S]methionine (data not shown). This then presented uswith either a three-protein periplasmic transport operon or a

more complex divergent operon with an unidentified area ofapproximately 1.3 kb preceding the operon.

Cloning and expression of tetI in the reverse orientation.The Tabor and Richardson expression system is convenientsince the polylinkers are arranged in both orientations. Wetherefore cloned the 4.5-kb tct-containing fragment in thereverse orientation compared to pKW108 and expressed it inthe usual way (Fig. 1, pKW119). Interestingly, from thereverse orientation two proteins, 29,000 and 14,000 Mr, were

expressed from the 1.3-kb region between the PstI site andthe beginning of tetC (Fig. 4, lane 1). To determine whetherthese two gene products were essential to the function of thetct operon, we created two deletions in this area. A plasmidwhich had the first 0.8 kb of the insert deleted still renderedE. coli citrate positive on citrate-tetrazolium agar, althoughexpression of the 14,000-Mr protein was abolished (Fig. 4,lane 2). When the deletion was extended to approximately1.0 kb, cells tested negative on citrate indicator plates,indicating that only the 29,000-Mr protein is an essentialcomponent of the tctI system. Therefore the operon consistsof four gene products, of which three are encoded on oneDNA strand and a fourth is read off the opposite strand in a

divergent manner.

Mr3xlO

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Mrx103 A B

Mr 3x 0

29. _

'4

FIG. 4. Proteins encoded upstream of tctC (pKW119). Lane 1,Autoradiogram of the SDS-PAGE protein profile of S. typhimuriumKS1070(pGP1-2, pKW119). Cells were induced for T7 RNA poly-merase and treated with rifampin before pulse-labeling with[35S]methionine as described. The molecular sizes of the twoproteins encoded by pKW119 are indicated. The arrowhead pointsto a repeatedly observed background band. Lane 2, Protein profileof a phenotypically tctl-positive subclone, which had been deletedfor the 14,000-Mr protein.

Cellular disposition of expressed tcti gene products. The Cprotein and the presumed two downstream genes were firstexpressed in the presence of [35S]methionine. Membranepreparations were then made by freeze-thaw lysis of sphero-plasts (4), and the inner and outer membranes were sepa-rated on a discontinuous sucrose gradient. The 41,000- to45,000-Mr diffuse protein and the 19,000-Mr protein werelocalized to the inner membrane (Fig. 5). In this experimentthe 29,000-Mr periplasmic C protein was not actually seensince it was released into the soluble fraction. Nevertheless,the 31,000-Mr precursor to the C protein was found lodgedsecurely in both the inner and outer membrane fractions.The 29,000-Mr protein coded for by the other DNA strand

was also expressed in a similar manner, but was not clearlydisposed to either membrane or soluble fraction. We triedseveral methods of demonstrating the cytological localiza-tion of this protein. The protein was found to be solublewhen the cells were fractioned by the fractionation method

A

+

lo

'

31K-aS &t.

N 40

2-..

20? *i

s

B

31 K

!19 K

FractionFIG. 5. Cellular disposition of tctA and tctB gene products. S.

typhimurium KS1070(pGP1-2, pKW108) was induced for T7 RNApolymerase, treated with rifampin, and labeled with [35S]methio-nine. Labeled cells were converted to spheroplasts and lysed. Thetotal membrane fraction was separated by sucrose gradient densitycentrifugation. The two peaks represent inner (B) and outer (A)membrane proteins. The two inserted autoradiograms indicate theSDS-PAGE protein profile of the peak fractions.

1 2 3 1 2 3FIG. 6. Cellular disposition of the tctD gene product. S. typhi-

murium KS1070(pGP1-2, pKW119) was induced for the T7 RNApolymerase in the presence of rifampin and radiolabeled with[35S]methionine. An autoradiogram of the SDS-PAGE of solubleand membrane proteins fractionated according to different methodsis shown. (A) Cells were converted into spheroplasts and lysed.Lane 1 shows the absence of labeled proteins in the periplasmicfraction. Lane 2, Soluble cytoplasmic proteins; lane 3, membranefraction. (B) Cells in lanes 2 and 3 were treated with 0.1 N NaOH asdescribed (12). Lane 1, Protein profile of unfractionated labeledcells; lane 2, soluble proteins; lane 3, labeled membrane proteins.

of Russell and Model (12) (Fig. 6B). However, by the cellfractionation method of Boeke and Model (4), which uses amore gentle lysis of prepared spheroplasts, this gene productappeared equally fractionated with both soluble and mem-brane fractions (Fig. 6A). This suggested to us that thisprotein was only weakly associated with the inner mem-brane.

DISCUSSION

The results of this study demonstrate that exclusiveexpression by the T7 system of Tabor et al. (15) is highlyeffective in identifying the gene products of the tctI operon.Using a variety of other methods such as minicells, maxi-cells, and in vitro transcription-translation systems, we havebeen unable to reproducibly reveal products of this operonother than the C protein and its precursor (unpublishedobservations). However, some problems were encounteredusing the T7 system. Since T7 RNA polymerase does notrecognize bacterial termination signals, we could not distin-guish tctI component from unrelated proteins by simplyexpressing the 8-kb DNA insert under T7 control. The sameproblem was encountered when TnS insertions abolishingthe tctl-positive phenotype were expressed in vivo. Again,unambiguous ordering of proteins to certain DNA fragmentswas not possible. Therefore we resorted to subcloning andexpressing distinct fragments of various sizes, which led tothe results summarized in Fig. 3. A protein of 21,000 Mrexpressed by the 4.5-kb insert (pKW108 but not pKW107)was an unidentified protein of higher molecular weight whichwas truncated by the subcloning step. Since pKW108 wasstill phenotypically tricarboxylate positive, we could ex-clude any essential function of this protein in citrate trans-port. Phenotypic testing of various subclones and deletionmutants, combined with in vivo expression studies, in bothE. coli and S. typhimurium as hosts led to the unambiguousidentification of four proteins encoded by tctI. Three of thesewere transcribed from one DNA strand with the periplasmicprotein (C protein) closest to the promoter. The 19,000-Mr

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Kb lI l

tctA tctE totC

totO

FIG. 7. Gene order and direction of transcription of the tctIoperon.

and 41,000-Mr proteins could be localized to the innermembrane. The fact that no cytoplasmic precursors withuncleaved signal sequences could be identified also sup-ported this finding. A protein of Mr 29,000 was the fourthcomponent of the tctl system, transcribed divergently fromtctCBA and weakly associated with the inner membrane.The gene order and direction of transcription are depicted inFig. 7.

Binding protein-dependent transport systems normallycomprise a periplasmic protein and at least two inner mem-brane proteins. A third membrane protein, either integral orperipheral, contains a nucleotide-binding site and is thoughtto be involved in energy coupling. At the outset tctl wouldappear to contain the appropriate number of components inagreement with other systems. The number and organizationof components also agree quite favorably with the fine-structure genetic map of tctl (13). However, mutants whichmapped upstream of tctC, that is, into tctD, do not behave asessential structural genes for transport but rather as regula-tory mutants with either polar or down-regulatory effects(13), thereby suggesting tctD to be a regulatory region. Inaddition, other mutations in tctD have been more recentlyisolated and mapped which either exhibit conditional-lethalregulatory effects or are constitutive (unpublished results).This reaffirms our suspicion that tctD encodes a regulatoryprotein. If this is subsequently confirmed, then the tctIoperon may differ from most shock-sensitive systems by theconspicuous absence of a fourth component. From theremaining DNA downstream of tctC (17) there is no geneticroom for another component, unless in the unlikely eventthat overlapping genes are encountered. Recently, the high-affinity L-arabinose transport operon was also shown tocomprise only three genes (9). It is possible that the hypo-thetical missing component is the product of another operon,since some amino acid-binding proteins have been shown toshare common membrane components (2). However, thisseems less likely since the tctI operon functions perfectly inE. coli (17), which harbors no native periplasmic tricarboxy-late transport system, although it is possible that E. colipossesses a related membrane component which comple-ments this system. A DNA sequence analysis of this regionshould resolve this difficulty, since the presumed ATP-binding proteins from six other operons for which sequenceinformation is available reveal clear homologies (2).

ACKNOWLEDGMENTS

This research was supported by a grant to W.W.K. from theNatural Sciences and Engineering Research Council of Canada.K.A.W. was supported by a University of Victoria Graduate Fel-lowship.

tctI EXPRESSION IN S. TYPHIMURIUM 3227

We are particularly indebted to Stanley Tabor for the T7 plasmidsand advice on their use.

LITERATURE CITED

1. Ames, G. F.-L. 1974. Resolution of bacterial proteins by poly-acrylamide gel electrophoresis on slabs. Membrane, soluble,and periplasmic fractions. J. Biol. Chem. 249:634-644.

2. Ames, G. F.-L. 1986. Bacterial periplasmic transport systems:structure, mechanism, and evolution. Annu. Rev. Biochem. 55:397-425.

3. Bochner, B. R., and M. A. Savageau. 1977. Generalized indica-tor plate for genetic, metabolic, and taxonomic studies withmicroorganisms. Appl. Environ. Microbiol. 33:434-443.

4. Boeke, J. D., and P. Model. 1982. A prokaryotic membraneanchor sequence: carboxyl terminus of bacteriophage fl geneIII protein retains it in the membrane. Proc. Natl. Acad. Sci.USA 79:5200-5204.

5. Dale, R. M. K., B. A. McClure, and J. P. Houchins. 1985. Arapid single-stranded cloning strategy for producing a sequentialseries of overlapping clones for use in DNA sequencing: appli-cation to sequencing the corn mitochondrial 18S rDNA. Plasmid13:31-40.

6. Davis, R. W., D. Botstein, and J. D. Roth. 1980. Advancedbacterial genetics. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

7. de Bruijn, F. J., and J. R. Lupski. 1984. The use of transposonTn5 mutagenesis in the rapid generation of correlated physicaland genetic maps of DNA segments cloned into multicopyplasmids-a review. Gene 27:131-149.

8. Furlong, C. E. 1986. Osmotic-shock-sensitive transport sys-tems, p. 768-796. In F. C. Neidhardt, J. L. Ingraham, K. B.Low, B. Magasanik, M. Schaechter, and H. E. Umbarger (ed.),Escherichia coli and Salmonella typhimurium: cellular andmolecular biology, vol. 1. American Society for Microbiology,Washington, D.C.

9. Horazdovsky, B. F., and R. W. Hogg. 1987. High affinityL-arabinose transport operon. Gene products expression andmRNAs. J. Mol. Biol. 197:27-35.

10. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

11. McAllister, W. T., C. Morris, A. H. Rosenberg, and F. W.Studier. 1981. Utilization of bacteriophage T7 later promoters inrecombinant plasmids during infection. J. Mol. Biol. 153:527-544.

12. Russel, M., and P. Model. 1982. Filamentous phage pre-coat isan integral membrane protein: analysis by a new method ofmembrane preparation. Cell 28:177-184.

13. Somers, J. M., and W. W. Kay. 1983. Genetic fine structure ofthe tricarboxylate transport (tet) locus of Salmonella typhimu-rium. Mol. Gen. Genet. 190:20-26.

14. Sweet, G. D., C. M. Kay, and W. W. Kay. 1984. Tricarboxylatebinding proteins of Salmonella typhimurium. Purification, crys-tallization and physical properties. J. Biol. Chem. 259:1586-1592.

15. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7RNA polymerase promoter system for controlled exclusiveexpression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078.

16. Towbin, H., T. Staeblin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets; procedures and some applications. Proc. Natl. Acad.Sci. USA 76:4350-4354.

17. Widenhorn, K. A., W. Boos, J. M. Somers, and W. W. Kay.1988. Cloning and properties of the Salmonella typhimuriumtricarboxylate transport operon in Escherichia coli. J. Bacteriol.170:883-888.

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