Isolation and Characterization of a Temperature-sensitive Mutant of ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY

Val. 268, No. 22, Issue of August 5, pp. 16544-16550,1993 Printed in U.S.A.

Isolation and Characterizationof a Temperature-sensitive Mutant of Salmonella typhimurium Defective in Prolipoprotein Modification* (Received for publication, March 31, 1993, and in revised form, April 22, 1993)

Keda Gan,Sita D. Gupta, Krishnan Sankaran, Molly B. SchmidS, and Henry C. Wup From the Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799 and the $Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544

A temperature-sensitive ( t s ) mutant of Salmonella an in vitro system using unmodified Braun's prolipoprotein typhimurium that accumulated unmodified murein of Escherichia coli as. the substrate (3) and was found to be prolipoprotein at 42 O C but not at 30 "C was identified. associated with the inner membrane and detergent-sensitive In vivo and in vitro studies of the biosynthesis of (6). Lack of an efficient and simple assay system prevented Braun's lipoprotein revealed that this mutant further purification and characterization of the enzyme. (SE6221)was defectivein the glycerylmodification of In order to study the structure andfunction of prolipoproprolipoprotein. The ts mutation was mapped to 60.6 tein glyceryl transferase, we sought a mutant defective in the min of the S. typhimurium chromosome and was linked to argA and cysH. A clone with a 1.4-kilobase S. ty- first step of prolipoprotein modification. In the absence of a phimurium DNA insert that complemented the ts mu- predictable phenotype useful for the selection or screening of tation and restored the prolipoprotein modification ac- such amutant, we resorted to brute-force screening of a tivity both in vivo and in vitro was isolated. DNA collection of temperature-sensitive (ts)' mutants of Salmosequencing of the complementing region revealed an nella typhimuriurn ( 7 ) for the identification of mutants accuopen reading frame encoding a protein with 291 amino mulating unmodified murein prolipoprotein (UPLP) at the acids lacking NHz-terminal signal sequence. This open non-permissive temperature (42 "C). This paper describes the reading frame is immediately 5' to the thyA gene and isoIation and characterization of a ts mutant of S. typhimuis allelic to umpA of Escherichia coli.Wild-type strains rium (SE5221) that accumulates unmodified prolipoprotein harboring the cloned gene exhibited elevated levels of due to a defect in the activity of prolipoprotein glyceryl prolipoprotein modification activity. At the non-per- transferase at 42 "C,the mapping of the ts mutation on the missive temperature, the mutation affected both S. typhimurium chromosome, and the cloning and sequence growth and viability, and the mutant cells exhibited determination of the wild-type allele of the putative prolipoanomalouscell morphology. The ts phenotype was sup- protein glyceryl transferase (kt)gene. pressed by the introduction of a 1pp::TnlO mutation. These results suggest that the cloned gene encodes EXPERIMENTALPROCEDURES prolipoprotein glyceryl transferase ( l g t ) , and in the Materials-[%]Methionine (specific activity 1190 Ci/mmol) was wild-type background, this prolipoprotein modificafrom ICN Biomedicals, Inc. [3H]Palmitate (specific activtion enzyme is essential for the growth and viability of purchased ity 60 Ci/mmol) and \2-3H]glycerol (specific activity 11.5 Ci/mmol) S. typhimurium. were from Du Pont-New England Nuclear, and a-35S-dATPwas from

The known eubacterial lipoproteins have a common NH2terminal amino acid, N-acyl diglyceride-cysteine (1, 2). The modification and processing of the precursor prolipoproteins involve at least four enzyme activities according to the proposed pathway of lipoprotein maturation (Fig. 1, Ref. 3). The modification of prolipoprotein with glycerol and fatty acids precedes processing and is required for cleavage of signal peptide by prolipoprotein signal peptidase or signal peptidase I1 (3, 4).In vivo studies showed that thetransfer of the nonacylated glyceryl moiety from phosphatidylglycerol to the sulfhydryl group of the cysteine residue of prolipoproteins, catalyzed by phosphatidylglycerol-prolipoprotein glyceryl transferase, is the first committed step in the modification of prolipoprotein (5). This enzyme activity was demonstrated in

* This work was supported by National Institutes of Health Grant GM-28811. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s)reported in thispaper has been submitted to the GenBankTM/EMBLData Bank with accession numberfs) L23259. 3 To whom correspondence should be addressed.

Amersham Corp. Globomycin was a gift from Dr. M. Arai (Sankyo Co., Tokyo, Japan). Restriction enzymes and T4 DNA ligase were purchased from Life Technology Inc. DNA sequencing kit was purchased from U. S. Biochemical Corp. Bacterial Strains, Plasmids, and Culture Media-Strains used in the presentstudy included S. typhimurium wild-type strain LT2, strains TT146 (argA1832:TnZO) and NB145(cysHZJ1574::MudA) obtained from Dr. N. R. Benson (University of Utah, Salt Lake City, UT), and E. coli strain DH5a as the host strain in the cloning experiments. pBluescript I1 KS(+) and SK(+) were used as vectors for subcloning. Culture media included Lbrothand M9 minimal medium supplemented with glucose (0.4%) andrequired amino acids (20 pg/ml). Antibiotics were added to final concentrations of 50 gg/ ml ampicillin, 20 pg/ml chloramphenicol, or 10 pg/ml tetracycline. Labeling and Anulysis of Lipoprotein-Cells grown in M9 medium to ODm = 0.5 at 30 "C were pulse-labeled with 10 pCiof ["SI methionine/ml of culture for 2 min at 30 "C; for labeling at the nonpermissive temperature, cultures were shifted to 42 "C for 1 h and then labeled for 2 min a t 42 " C . To detect the formation of lipidmodified prolipoprotein, cells were preincubated with 50 gg/ml globomycin for 15 min before labeling. Labeling was terminated with the addition of trichloroacetic acid (10% final concentration), and lipoprotein species were immunoprecipitated and separated on Tricine-SDS gel (8). Rapid Mapping of the ts Mutation-The ts mutation was mapped The abbreviations used are: ts, temperature sensitive; UPLP, unmodified prolipoprotein; Tricine, N-tris(hydroxymethy1)methylglycine; kb, kilobase (s); ORF, open reading frame.

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press method (12, 13) and suspended in 20mM Tris-HC1, pH 8.0, containing 5 mM EDTA and 1 mM dithiothreitol. The assay was carried out by incubating inverted membrane vesicles (5-20pgof Met Leu-X-X-Cy'"prolipoprotein Unmodified protein) with denatured UPLP (20,000 counts/min) in 20 mM TrisPhosphaiidylglycerol HCl, pH 8.0, containing 5 mM EDTA, 1mM dithiothreitol, and 0.1% Glyceryl Transferase octyl glucoside in a final volume of 40 p1 at 37 "C for 1 h. ConcentraPhosphatidic acid tion of guanidinium chloride was kept below 0.1 M in the assay. The reaction was terminated by the addition of 10 pl of 5 X SDS-sample l?i20H buffer, heated at 100 "C for 5 min, and loaded on Tricine-SDSgel (8) to separate the intermediates in the lipoprotein maturation. Radioactive bands onthe SDSgel were revealed by either autoradiography or PhosphorImager analysis. Protein was estimated by the dye bindMet L~U-X-X-~-+ prolipoproteinGlyceryl ing method (14). Phospholipid(PE, PG,CL) Isolation of a Clone Thut Complemented the ts Mutation-6 pools O-acyl Transferase@) of pRSL libraries (each containing about 2000 transformants) carrying 8-12-kb fragments of S. typhimurium genomic DNAin pBR328 Lysophospholipid were obtained from Miller's laboratory at the University of Illinois (Urbana, IL). The P22 lysates made on this pRSL library were used FH20COR1 to transduce mutant SE5221. Transductants were selected on LB (X4OCOR2 plate containing 20 pg/ml chloramphenicol at 30 "C, replica-plated, EH2 and incubated a t 42 "C. Temperature-resistant ( t r ) colonies on each ? Diglycerlde Met plate were pooled, and the plasmids isolated from each pool were Leu-x-x-cYs " prolipoprotein once again transformed intothe mutant straina t 30 "Cand screened I for tr phenotype. The tr transformants were analyzed for the restoSignal PeptidaseII ration of prolipoprotein modification enzyme activity. One of the clones conferring temperature resistance was further subcloned, and FH20COR1 the physical map was determined. Using pBR328 and pBluescript ?iOCOR2 (SK, KS) as vectors, the smallest subclone that complemented the ts mutation and restored prolipoprotein modification enzyme activity p 2 was obtained. 7 DNA Sequence Determination-Double-stranded DNA sequencing Signal peptide+ H2N -Cys" -, Apolipoprotein using the Sequenase kit (U. S. Biochemical Corp.) was performed by Phosphoiipid(PE, PG, CL) the method of Sanger et al. (15). The pBluescript (SK) plasmid that N-acyl Transferase contained the 1.4-kb insert was purified by CsCl gradient. Initially, Lysophospholipid T7 and T3 primers were used for sequencing; based on the emerged sequence data, appropriate oligonucleotides (16-mers) were synthesized as primers. The DNA wassequenced in both directions, and the CH20COR1 CHOCOR2 sequence data were analyzed using the Genetics Computer Group's bH9 Sequence Analysis Software Package. Recombinant DNA Techniques-Restriction endonucleases and DNA ligase wereused according to themanufacturer's specifications. Purification of DNA on CsCl gradients, plasmid preparations, and FIG. 1. Proposed biosynthetic pathway for the maturation transformation were performed using standard procedures (16). of bacterial lipoproteins. RESULTS using a Mud-P22 mapping set, a gift from Dr. K. Sanderson (UniverScreening for Mutants Defective in Prolipoprotein Modifisity of Calgary), according to theprocedure described by Benson and cation-440 t s mutants of S. typhimurium (7) generated by Goldman (9). The mapping set consisted of 51 strains with Mud-P22 insertions covering the entire genome of S. typhimurium. Phage chemical mutagenesis with diethyl sulfate were screened for lysates obtained from each strain were spotted separately on a lawn defects in prolipoprotein modification by pulse labeling with plates were incubated a t 42 "C [35S]methionineat 42 "C for 2 min, followed by analysis of of the ts mutant strain, and the overnight. Based on the strainswhich complemented the ts mutation, lipoprotein species synthesized at the non-permissive temperthe approximate map position of the ts mutation was determined. ature by Tricine-SDS-polyacrylamidegel electrophoresis. One Construction of SE5221 Zpp::TnIO-TheZpp::TnIO allele from E. mutant strain, SE5221, was found to accumulate UPLP at coli strain E609 lpp::TnIO (10) was transduced into SA3858 mutH::Tn5 (11) of Salmonella by P1 transduction. P22 lysate on 42 "C. Fig. 2 shows the analysis of [35S]methionine-labeled SA3858 1pp::TnlO was used to transduce strain SE5221 at 30 "C; lipoprotein species in the wild-type and mutant cells at 30 lipoprotein-negative phenotype of the TetR transductants was con- and 42 "C in the presence or absence of globomycin,a specific firmed by Ouchterlony test using lipoprotein antiserum. inhibitor of signal peptidase I1 (17). Mutant strain SE5221 Growth Properties of SE5221 and Related Strains-Exponentially not only accumulated UPLP at 42 "C, but also showed a growing cultures of each strain were diluted in LB to an ODm nm of significant amount of unmodified prolipoprotein at 30 "C as 0.05 and incubated a t 30 and 42 "C. At regular time intervals, ODm nm was measured, and thenumber of viable cells was estimated by plating aliquots of serially diluted samples on LB media and MPLP~k MBW, "PLP incubated a t 30 "C overnight. Cell morphology was examined under L*c EUPLP UPLP= a phase contrast microscope. LP LP I n Vitro Assayof Prolipoprotein Glyceryl Transferase-In this assay we detected the glyceride-modified prolipoprotein formed after the 1 2 3 4 5 6 7 8 combined action of glyceryl transferase and 0-acyltransferase (2). [36S]Methionine-labeledUPLP was prepared by in vitro transcription FIG. 2. Tricine-SDS-polyacrylamidegel electrophoresis of and translation as described previously (12, 13). We have found that [S6S]methionine-labeled lipoprotein species in the wild-type treatment of UPLP with 1 M guanidinium chloride a t 100 "C for 5 and mutant strains. Cells were labeled in the absence (lanes 1-4) min just prior to theassay improved the efficiency of the modification or presence (lanes 5-8) of globomycin. Lanes 1,2,5, and6 represent reaction and obviated the need to prepare freshUPLP for the assay? wild-type strain LT2 grown at 30 "C (I and 5) or 42 "C (2 and 6 ) ; Inverted membrane vesicles were prepared from cells by the French lanes 3, 4, 7,and 8 represent mutant strain SE5221 grown at 30 "C ( 3 and 7) or 42 "C ( 4 and 8). MPLP, modified prolipoprotein; LP, * K. Sankaran andH. C. Wu, unpublished data. mature lipoprotein. -3 -2 4 + l

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compared to the wild-type cells. In the presence of globomycin, modified prolipoprotein accumulated in the wild-type strain at both 30 and 42 "C, and in mutantcells at 30 but not at 42 "C. These results show that mutant SE5221 is defective in a modification step prior to globomycin-sensitive signal peptidase I1 processing at 42 "C. Incorporation of [2-3H]glycerol or [3H]palmitate intolipoprotein was greatly reduced in strain SE5221 at 42 "C as compared to wild-type cells (Fig. 3), indicating that the defect lies in glyceryl modification of prolipoprotein. Analysis of total membrane phospholipids of the mutant cells at 42 "C indicated that SE5221 was not deficient in phosphatidylglycerol,an essential lipid precursor for prolipoprotein modification (data not shown) (5). It has been shown previouslythat theexport of lipoprotein in E. coli requires functional SecA, SecD, SecE, SecF, and SecY proteins, and conditionally lethal mutants defective in any of these sec genes accumulate UPLP at thenon-permissive temperature (18, 19). To determine whether the ts mutation inSE5221 represented an allele of the known essential sec genes in S. typhimurium, we examined the processing of proOmpA protein in wild-type and mutant cells both at 30 and at 42 "C.No accumulation of proOmpA was observedin the mutant cells at 42 "C (data not shown). We conclude, therefore, that the ts mutation in SE5221 does not represent a new allele of the known sec genes.The inverted membrane vesicles prepared from SE5221 grown at 30 "C (Fig. 6A, lane 1)or at 42 "C(data not shown) had negligible glyceryltransferase activity in vitro. Taken together, these results suggest that theaccumulation of UPLP in this mutantat 42 "Cis due to a defective glyceryltransferase. Mapping of the ts Mutation in SE5221"Using the mapping set (9) described under "Experimental Procedures," the ts mutation was located between 60-62.7 min of the S. typhimurium chromosome. Based on a linkage of the ts mutation to cysH and argA by P22 transduction at frequencies of 12 and 17%, respectively, the ts mutation was located at 60.6 min on the S. typhimurium (Fig. 4). Cloning and Sequence Determination of the Wild-type Allele of the ts Mutation in SE5221"To clone the gene comple-

menting the ts mutation in SE5221, we transformed strain SE5221 with the DNA prepared from pRSL libraries containing 8-12-kb inserts of s.typhimurium genomic DNA. Among 12 clones that complemented the mutantfor growthat 42 "C, four had the same 7.5-kb insert. The physical map of one of these clones (pGTOOl) is shown in Fig. 5. Subcloning revealed that a 2.7-kb SalI fragment (pGT004) complemented the ts defect in growth and restored the prolipoprotein modification activity (Figs. 5, and 6A, lane 5). The 2.7-kb fragment was further subcloned into pBluescript KS(+) and SK(+) vectors, and a clone (pSKOO4dP) containing a 1.4-kb fragment that complemented the defect in growth and restored prolipoprotein modification activity was obtained (Fig. 5). In fact, the prolipoprotein modification activity was found to be enhanced in the cell envelopeof the wild-type strain (DH5a)containing the cloned gene (Fig.6B). The sequence of the 1.4-kb fragment is shown in Fig. 7. It revealed a 873-base pair ORF encoding a protein of 291 amino acids. A putative promoter and ribosome-binding site are present upstream of this ORF. The 3' sequence of this fragment showed a very high homology to thyA sequence (20), and this ORF is immediately 5' to the thyA gene separated by only 6 base pairs. In E. coli, an umpA (unidentified membrane protein) gene immediately adjacent to 5' of thyA gene has been shown to be an essential gene (21). There is a very high homology(95%)between the partial deduced amino acid sequence ofumpAof E. coli available from the published sequence of the DNA fragment 5' to the thyA gene (20) and the corresponding region of the ORF of S. typhimurium. It is most likely that the umpA gene is allelic to this ORF of S. typhimurium. The deduced amino acid sequence shows that the protein does not contain a typical signal sequence at its NH2terminus but has significant stretches of hydrophobic sequencesinterrupted by charged hydrophilic segments (Fig. 8) (22). These hydrophobic segments may anchor this protein to the cell membrane. The deduced amino acidcompositionsuggests that this is a basic protein with a net charge of +6 at neutral pH and a PI of 10.58; the strong basic nature is due to the relative abundance of arginine residues (20/291). Interestingly, the enzyme lacks cysteine, but contains many Gly and Pro, helix-breaking amino acids. The Chou Fasman prediction suggests a predominantly @-sheetstructure, with a low a-helix LP -ill content. No significant homology to otherproteins in thegene banks including enzymes involvedin phospholipid synthesis, transacylases, and phospholipases was detected. 1 2 3 4 5 6 7 8 Suppression of the ts Mutation in SE5221 by1pp::TnlOSE5221 1pp::TnlO was constructed by transduction as deFIG.3. Incorporation of [2-3H]glycerol or['Hlpalmitate into lipoprotein. Lunes 1-4 represent [2-3H]glycerol-labeledlipo- scribed under "Experimental Procedures.'' The disruption of proteins, and lanes 5-8 [3H]palmitate-labeled lipoproteins. Lanes 1 the structural gene for Braun's lipoprotein suppressed the ts and 2 and 5 and 6 correspond to mutant strain SE55221 labeled a t 30 "C ( 1 and 5)or 42 "C (2 and 6), lanes 3 and 4 and 7 and 8 represent phenotype of SE5221, and the growth of SE5221 lpp::TnlO at wild-type strain LT2 labeled a t 30 "C ( 3 and 7) or 42 "C ( 4 and 8). 42 "C was similar to the wild-type LT2 (Fig. 9). At 30 "C, the growth rates for strains SE5221, LT2, and LP, mature lipoprotein. SE5221 lpp::TnlO were similar (data not shown). At 42 "C, the growth rate of SE5221 decreased, and OD, nm decreased CYM ts arsn after 1 h; the viability of mutant cells at 42 "C decreased I I 60 60.6 61 (min) immediately after the temperature shift to 42 "C (Fig. 9). In addition, the morphology of mutant cells at 42 "Cwas abnor12?h mal; cells became swollen and oval, and eventually lysed. The ts phenotype and itssuppression by 1pp::TnlO of the lgt allele 17% in strain 835221 are similar to those of the lnt mutant (strain FIG.4. Location of the t s mutation in SE6221 on S. typhi- 5312) described in the accompanying paper (28).

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murium chromosome. SE5221 cells were transduced with P22 phages grown on cysH::MudA or argA::TnlO strains as the donor a t 30 "C, and selected on Amp or Tet plates, respectively. The linkages of ts to these two marker were determined bv scoring the number of tr colonies on the platescontainingappropriate antibiotics.

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DISCUSSION

We have succeededin the isolation of a conditionally lethal mutant of S. typhimurium defective in thefirst step of proli-

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FIG.5. Identification of the Zgt gene by subcloning and complementation. The inserts in the recombinant plasmids and their physical maps are shown at the top. Dashed lines indicate deletions and the arrows denote the direction of transcription of lgt and thyA genes. Results of complementation of the ts phenotype are expressed as + or -.

of prolipoprotein glyceryl transferase enzyme in the cell envelope of wild-type strain harboring the cloned gene strongly suggests that thisgene encodes prolipoprotein glyceryl transferase. For this reason, we would name this gene Zgt. Verification of this designation awaits the purification of this enzyme from strains overexpressing the cloned gene. Since the assay for prolipoprotein modification used in the present 1 2 3 4 5 study is a coupled assay of both glyceryl transferase and 0acyltransferases, it is conceivable that the Zgt gene might encode a protein required for the overall reaction rather than B. the actual glyceryl transferase. Resolution of this issue would require a detailed study of the mechanism of this reaction using purified components. MPLP The ORF predicts a gene product of maximum length of UPLP291 amino acids with a calculated molecular mass of 34 kDa with three possible initiation codons; the actual size of the gene product remains to be determined. Based on SDS gel analysis, the UmpA protein of E. coli has been reported to be 1 2 3 4 5 6 25 kDa in size. Prolipoprotein glyceryl transferase of E. coli FIG.6. In vitro assay of prolipoprotein glyceryltransferase is an inner membrane protein (6) as is the case with UmpA in the inverted vesicles of mutant 835221, DH5a, and both (21); the absence of a typical signal sequence is consistent 935221 and DH5a containing different subclones. A: lane 1, SE5221; lanes 2-5,SE5221with subclones pGTOO2, pGT003, pGT001, with this observation. The high PI predicted for this protein and pGT004, respectively; 10 pg of cell envelope protein was used in and the presence of hydrophobic segments flanked by posieach assay. B: lane 1, DH5a; lanes 2-6, DH5a with subclones pSK004, tively charged residues suggest that thisprotein can interact pKS004dE, pKS004dP, pSK004dE, and pSK004dP, respectively; 5 with acidic phospholipids by ionic interactions as well as with pg of cell envelope protein was used in each assay. MPLP, modified lipid bilayer by hydrophobic interactions. prolipoprotein. The essentiality of the biosynthetic pathway for lipoproteins has been suggested by the fact that the inhibition of poprotein modification pathway. Mapping, cloning, and se- signal peptidase I1 activity by globomycin or by mutation quence determination of the wild-type allele of the ts mutation results in cell death (17,23). Sensitivity to globomycin can be indicate that the ts mutation resides in an ORF that corre- significantly reduced by a deletion of the lpp gene or by lpp sponds to theumpA gene of E. coli (21). The enhanced activity mutations which encode non-modifiable prolipoprotein (24).

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Lipid Modificationof E. coli Prolipoprotein 1 AGATCTTCGCTTGTGCGGCGAGATGGCGGGCGATCCGATGTGCGTGGCGATTCTTAT~ 61 TCTGGGATATCGCCATCTTTCGATGAA~CCGTTCGGTAGCGCGTGTG~TATCTGCT 121 G C G G C A T A T C G A T T T n ; A A G A C G C G ~ C C C T T G C C A G A A T 181 GGCGACCGAAGTGCGTCATCAGGTGGCGGCGTTTATGGAGCGCCGCGGGATGGGGGGATT 241

GATTCGAGGAGGGTTGTAACGACTGGAGCGGGAAAAGAGAGTGACTGTGGATTCACGGCG

301 CAGACAACACGCCTGCAAGATGAAAGACAATGCGTATACAATGCGTATACATATCTTTTAACGGTAATCGG

361 CATCTCGCTTTTAACCCTTGTGCTATTATTCGCACCTTTTGGAGCGTCTG~CGCCAGG -3s -10 421 C G C G C T T A T C A A T C G C T A T C T C T T C A G C G ~ T A A C A A G ~ T T G T ~ T G A C A G A T G A C C W T 2 481 AGTAGCTATCTGCATTTTCCGGACTZTCATCCGGTCATTGATCCGGTCATTTTCTC~TT~CCCGTCGCG S S Y L E F P D F D P V I F S I O P V A 22 541 CTTCACTGGTATGGCTTGATGTATCTGGT~TTCGTTTTCGCGATGTGGTTGGCGGTG L H W Y O L W Y L V G F V F A W W L A V 42 601 CGTCGCGCTAACCGTCCGGGAAGCGGTTGGACCAAAAACGAAGTTGAAAATTTACTCTAT R R A N R P O S O W T K N B V I N L L Y 62 661 GCAGGTTTCCTGGGGGTCTTCCTGGGGGGACGTATTGGCTATGTCCTGTTTTATAACTTC A G F L O V F L Q O R I G Y V L F Y N F 82 721 CCTCTGTTTCTGGATAACCCGCTCTATTTATTCCGCGTCTGGGACGGC~~TGTCCTTC P L F L D N P L Y L F R V W D O O W S F 102 781 CACGGCGGGCTTATCGGCGTGATACTGGTGATGATTATCTTCGC~GGCGCACGAAGCGC H O O L I O V I L V W I I F A R R T K R 122 841 TCGTTCTTTCAGGTGTCTGATTTTATTGCGCCGTTAATTCCGTTCGGCCTGGGCGCCGGG S F F Q V S D F I A P L I P F O L O A O 142 901 CGTCTGGGCAACTTTATCAACGGTGAATTGTGTG~CGCGTCGATCCTGACTTCCGGTTT R L O N F I N O B L W G R V D P D F R F 162 961 GCCATGCTCTTTCCTGGCTCACGCGCGGAAGATATTGCGCTGCTGCCGTCACATCCGC~ A W L F P O S R A B D I A L L P S E P Q 182 1021 TGGCAGCCTATTTTTGATACCTACGGCGTATTGCCACGCCACCCTTCC~GTTGTATGAG W Q P I F D T Y O V L P R H P S Q L Y B 202 1081 TTGGCATTAGAAGGCGTGGTGCTGTTTATCATCCTTAATCTCTTTATTCG~CCGCGT L A L S O V V L P I I L N L F I R K P R 222 1141 CCGATGGGCGCAGTCTCCGGATTATTCCTGATTGGCTATGGCGCGTTTCGTATCATTGTT P W O A V S O L P L I O Y O A P R I I V 242 1201 GAATTCTTCCGCCAGCCGGACGCGCAATTTACCGGCGCGTGGGTACAGTACATCAGCATG E F F R Q P D A Q F T O A W V Q Y I S W 262 1261 GGGCAGATTCTCTCTATCCCATGATTATCGCTGGCGCGATCATGATGGTTTGGGCATAT O Q I L S I P W I I A G A I W W V W A Y 282 1321 CGCCGCCGCCCGCAGCAACACGTTTCCTGAGGTTCCATG~CAGTATTTAGAACTGATG thyA 291 R R R P Q Q E V S * 1381 CAAAAAGTGCTGGATGAAGGCACACAG~CGACCGTACCGGCACCGGCACGCTTTCC 1441 ATTTTTGGCCATCAGATGCGTTTTAACCTGCAG FIG. 7. The nucleotide sequence of the 1.4-kb fragment and the deduced amino acid sequence of the putative lgi gene. Symbols: -, the putative promoter; =, the putative ribosome-binding site; *, stop codon; and -+, the startcodon of the thyA gene.

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10 h

FIG.9. Growth and cell viability of 836221, LT2, and SES221 1pp::TnlO at 42 "C. Symbok;:A and A, SE5221;0 and 0, LT2; ando, SE5221

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TIME (hr)

This has led to thesuggestion that thelethality of globomycin is related to the accumulation of lipid-modified outer membrane prolipoprotein in the inner membrane of globomycintreated cells. This explanation was somewhat weakened by the uncertain localization of lipid-modified prolipoprotein in globomycin-treated cells. Lipid-modified prolipoprotein was found in the inner membrane based on separation of inner and outer membrane fractions by sucrose density centrifugation (4), but was also reported to be Sarkosyl insoluble, suggestive of an outer membrane localization (25). Furthermore, lipid-modified prolipoprotein in globomycin-treated cells was found to be covalently linked to the peptidoglycan, suggesting that it is translocated to the outer membrane to form such a linkage (26, 27). It is worth noting that while E. coli strains deleted for or defective in lpp show increased resistance to globomycin, their growth is still inhibited by globomycin at 2100 pg/ml. Apparently, there may be essential lipoproteins in E. coli, and the maturation of these essential lipoproteins is inhibited by globomycinat high concentrations in cells lacking Braun's lipoprotein (24). The results reported in this paper and the accompanying manuscript (28) strongly supportthe notion that certain minor lipoproteins in bacteria are essential for the growth and viability of bacterial cells. Thus, among predicted genes involved in the lipoprotein maturation pathway (Fig. l),three have been identified, all of which appear to be essential for the cell growth, division, or viability in thepresence of Braun's lipoprotein as the major outer membrane protein (23, 28). When the biosynthetic pathway of bacterial lipoproteins is rendered suboptimal due to mutations or by antibiotic treatment such as globomycin, the presence of Braun's lipoprotein as the most abundant lipoproteins in E. coli further reduces the maturation of these essential lipoproteins to a rate insufficient €or its viability. A related observation is the sparing effect of lpp mutation or deletion in E. coli pgsA mutants severely defective in the synthesis of phosphatidylglycerol, an essential substrate inthe maturation of prolipoprotein (5,29). The high abundance of Braun's lipoprotein consumes limited biosynthetic capacity of mutants defective in phosphatidylglycerol synthesis or in prolipoprotein modification so that they cannotmeet the needs in the synthesis of essential acidic phospholipids or in the modification and processing of minor essential lipoprotein(s).Removal of the major competing sub-

strate for the limited supply of essential enzymes or substrates allows the formation of these essential proteins ormembrane lipids sufficient for cell growth. Both in E. coli and in S. typhimurium, it appears that Igt (or u m p A ) is 5' to thyA and shares a common promoter 5' to Igt (or u m p A ) . The physiological significance of this transcriptional linkage is not clear. Of three known or proposed genes involved in lipoprotein maturation pathway, none is linked to one another on the bacterial chromosome, nor linked to any structural gene of bacterial lipoproteins. Whether any of the genes encoding modification and processing enzymes are COregulated remains to be determined. The isolation of mutants defective in these enzymes and clones complementing these defects should facilitate future studieson the structures, functions, and regulation of these enzymes. Acknowledgments-We thank Drs. K. Sanderson, N. R. Benson, and C . G . Miller for the gifts of bacterial strains and genomic library, and Dr. M. Arai for the gift of globomycin. REFERENCES 1. Hantke, K., and Braun, V. (1973) Eur. J. Ewchem. 34, 284-296

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7. Schmid, M. B., Kapur, N., Isaacson, D. R., Lindroos, P., and Sharpe, C. (1989) Genetics 123,625-633 8. Schagger, H., and von Jagow, G. (1987) A m L Biochern. 166,368-379 9. Benson, N. R., and Goldman, B. S. (1992) J. Bacteriol. 174,1673-1681 10. Rotering, H., Fiedler, W., Rollinger, W., and Braun, V. (1984) FEMS Microbwl. Lett. 2 2 , 6 1 4 8 11. Shanabruch, W. G., Behlau, I., and Walker, G. C. (1981) J. Bacteriol. 1 4 7 , 827-835 12. Tian, G., Wu, H. C., Ray, P. H., and Tai, P. C. (1989) J. Bacteriol. 1 7 1 , 1987-1997 13. Hayashi, S., and Wu, H. C. (1992) in Li id Modificatiorw of Proteins: A Practrcal Approach (Hooper, N. M., ancfTurner, A. J., e&) pp. 261-285, Oxford University Press, Eynsham, Oxon., United Kingdom 14. Bradford, M. M. (1976) Anal. Eiochem. 7 2 , 248-254 15. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 7 4 , 5463-5467 16. Sambrook, J., Fritsch, E. F., and Maniatis, T.(1989) Mokcuhr Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, ColdSpring Harbor. NY

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E. coli Prolipoprotein 25. Inukai, M., and Inouye, M. (1983)Eor. J. Biochem. 130, 27-32 26. Inukai, M., Takeuchi, M., Shimizu, K., and Arai, M. (1979)J. Bocteriol. 140, 1098-1101 27. Ichihara, S., Hussain, M., and Mizushima, S. (1982)J. Biol. Chem. 267, 495-500 28. Gupta, S. D.,Can, K., Schmid, M. B., and Wu, H. C. (1993)J.Biol. Chern. 268, 16551-16556 29. Asai, Y.,, Katayose, Y., Hikita, C., Ohta, A,, andShibuya, I. (1989)J. Bacterwl. 171,6867-6869