JOURNAL OF BACTERIOLOGY,0021-9193/98/$04.0010

Mar. 1998, p. 1215–1223 Vol. 180, No. 5

Copyright © 1998, American Society for Microbiology

MppA, a Periplasmic Binding Protein Essential for Import of theBacterial Cell Wall Peptide L-Alanyl-g-D-Glutamyl-meso-Diaminopimelate

JAMES T. PARK,1* DEBABRATA RAYCHAUDHURI,1 HONGSHAN LI,1 STAFFAN NORMARK,2

AND DOMINIQUE MENGIN-LECREULX3

Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts 021111;Microbiology and Tumorbiology Center, Karolinska Institutet, S-17177 Stockholm, Sweden2; and

Unite de Recherche Associee 1131 du Centre National de la Recherche Scientifique,Biochimie Moleculaire et Cellulaire, Universite Paris-Sud, 91405 Orsay, France3

Received 7 July 1997/Accepted 30 December 1997

Mutants of a diaminopimelic acid (Dap)-requiring strain of Escherichia coli were isolated which failed togrow on media in which Dap was replaced by the cell wall murein tripeptide, L-alanyl-g-D-glutamyl-meso-diaminopimelate. In one such mutant, which is oligopeptide permease (Opp) positive, we have identified a newgene product, designated MppA (murein peptide permease A), that is about 46% identical to OppA, theperiplasmic binding protein for Opp. A plasmid carrying the wild-type mppA gene allows the mutant to growon tripeptide. Two other mutants that failed to grow on tripeptide were resistant to triornithine toxicity,indicating a defect in the opp operon. An E. coli strain whose entire opp operon was deleted but which carriedthe mppA locus was unable to grow on murein tripeptide unless it was provided with oppBCDF genes in trans.Our data suggest a model whereby the periplasmic MppA binds the murein tripeptide, which is then trans-ported into the cytoplasm via membrane-bound and cytoplasmic OppBCDF. In assessing the affinity of MppAfor non-cell wall peptides, we have found that proline auxotrophy can be satisfied with the peptide Pro-Phe-Lys,which utilizes either MppA or OppA in conjunction with OppBCDF for its uptake. Thus, MppA, OppA, andperhaps the third OppA paralog revealed by the E. coli genome sequence may each bind a particular family ofpeptides but interact with common membrane-associated components for transport of their bound ligands intothe cell. As to the physiological function of MppA, the possibility that it may be involved in signal transductionpathway(s) is discussed.

During growth, Escherichia coli breaks down over one-thirdof its cell wall each generation and efficiently reutilizes thetripeptide therefrom for synthesis of new murein in a sequenceof events termed the recycling pathway (9, 11, 32; see reference33 for a review). In this pathway, murein is degraded toN-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-g-D-glutamyl-meso-diaminopimelate (GlcNAc-anhMurNAc-tri-peptide) by the combined action of lytic transglycosylases, en-dopeptidases, and D,D- and L,D-carboxypeptidases which arepresent in the periplasm (39). The muropeptide, GlcNAc-anhMurNAc-tripeptide, presumably is transported into the cy-toplasm via the membrane-bound AmpG permease (20, 24).The tripeptide is then released from the muropeptide byAmpD anhydro-N-acetylmuramyl-L-alanine amidase (19, 21).Surprisingly, almost all murein tripeptide for recycling is trans-ported into the cell as GlcNAc-anhMurNAc-tripeptide via theAmpG permease and is then released by the cytoplasmicAmpD amidase (20, 32), rather than being transported as thefree tripeptide via the oligopeptide permease (Opp) as wasoriginally proposed (10). Direct utilization of the tripeptide forcell wall synthesis was assumed to depend on a hypotheticalligase which would attach tripeptide to UDP-MurNAc, therebyreintroducing it into the biosynthetic pathway for wall synthesis(9, 20, 33). In fact, the enzyme responsible for this activity hasrecently been identified, and the gene, mpl, was shown to bethe open reading frame (ORF) yifG at 96 min on the E. colimap (29). An mpl null mutant was completely devoid of ligase

activity, and cells of this mutant were viable and accumulatedtripeptide in their cytoplasm (29).

During a search for mutants lacking this murein peptideligase activity, four mutants were isolated from a pool of mu-tagenized diaminopimelic acid (Dap)-negative (dap) parentalcells in a screen that assayed the growth of cells on free tri-peptide as a source of Dap. In this report, we describe theisolation and initial characterization of one such mutant. Anew genetic locus, mppA, has been identified which codes fora periplasmic binding protein required for uptake of mureinpeptides. Two other mutants, one with a mutation in oppB andthe other with a mutation in groESL (unpublished), were foundto be defective in Opp function because of their resistance totriornithine toxicity. The oppB mutation indicates that mureintripeptide is transported from MppA into the cytoplasm viamembrane components of Opp, and the groE mutation sug-gests that the chaperonin is involved in the proper folding andassembly of the components of the peptide transport system.

MATERIALS AND METHODS

Bacterial strains and growth conditions. The E. coli strains used in this studyare listed in Table 1. Cells were grown at 37°C in L broth (38) or 23 YT (38)supplemented with 50 mg of Dap per ml where required. Antibiotic-resistantstrains were selected in the presence of 15 mg of chloramphenicol (Cm) per ml,30 mg of kanamycin (Kan) per ml, or 100 mg of ampicillin (Amp) per ml asneeded. Tests for triornithine resistance were done on M9-glucose agar supple-mented with 0.1% Casamino Acids, 1 mg of thiamine per ml, and 500 mMtriornithine (Bachem). Tests for growth of proline auxotrophs were done asdescribed in the footnote to Table 2.

Mutagenesis. E. coli TP981 was mutagenized by transposon mutagenesis withlNK1324, a lambda phage which carries the transposon miniTn10Cm that con-tains the Cm acetyltransferase (cat) gene in place of the tetracycline resistancedeterminant of Tn10 (16). lNK1324, with a titer of 1010 as determined on asuppressor strain (23), produced no plaques (,105) on TP981, which is suppres-sor negative. Eighty microliters of lNK1324 were mixed with 0.1 ml of a 10-fold

* Corresponding author. Mailing address: Department of MolecularBiology and Microbiology, Tufts University, 136 Harrison Ave., Bos-ton, MA 02111. Phone: (617) 636-6753. Fax: (617) 636-0337. E-mail:jpark@opal.tufts.edu.

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concentrated overnight culture of TP981. After adsorption for 15 min at roomtemperature and for 15 min at 37°C, 5 ml of L broth containing 50 mM sodiumcitrate was added; this was followed by centrifugation. The cells were resus-pended in 5 ml of L broth with citrate and shaken at 37°C for 45 min. Aliquotsof this suspension were plated on L agar-Cm-Dap agar plates (see Results).

Mapping, DNA, and transformation techniques. Mapping was done by P1transduction (30, 38), by Southern hybridization (31), and by DNA sequencing asindicated. Small- and large-scale plasmid isolations were carried out by thealkaline lysis method (38), and plasmids were further purified with cesium chlo-ride-ethidium bromide gradients or with a plasmid kit (Qiagen Inc., Chatsworth,Calif.). Standard procedures for restriction endonuclease digestions, ligation,and agarose gel electrophoresis were followed (38). E. coli cells were madecompetent and transformed with plasmid DNA either by the method of Dagertand Ehrlich (6) or by electroporation (38).

Recovery of the mutant gene on plasmids and strategy for sequencing. Torecover the transposon-disrupted gene on a plasmid, chromosomal DNA fromthe mutant was digested with PstI or HindIII, restriction enzymes which do notcleave the miniTn10Cm transposon, and the resultant fragments were ligated topBluescript SK1 (Stratagene) or pBR322 DNA digested with the correspondingrestriction enzyme and with calf intestinal alkaline phosphatase. The ligationmixtures were used to transform competent TOP10F9 cells, and the cells wereplated on L agar containing Dap and Cm. From CmR colonies, plasmids whichcarried the Cm marker and the flanking chromosomal DNA containing part orall of the gene of interest were recovered. The plasmid DNA served as a templatefor sequencing the chromosomal DNA flanking the transposon. The primersused for this purpose were complementary to the two ends of the cat genepresent in the transposon (see Results). Primer 59-CCTCCCAGAGCCTGATAA-39 is complementary to the 59 end of the noncoding strand of cat and was usedto determine the sequence downstream of the cat gene. The primer 59-AAGCACCGCCGGACATC-39, complementary to the promoter region of the codingstrand of cat, was used to sequence the DNA upstream of the transposon.

Construction of plasmids. The cloning vectors used were pTrc99A (Pharma-cia), pBluescript SK1, pACYC177, pACYC184, and pBR322. pB2 (AmpR) is apBR322 derivative that harbors the promoter distal region of the opp operonbeginning at the EcoRV site in oppA and extends through the end of the operon.Thus, it encodes oppB, oppC, oppD, and oppF. Transcription is driven by the Tetpromoter of pBR322 (39a). pBf30 (CmR) is a pACYC184 derivative that con-tains the opp regulatory region, the oppA gene, and part of oppB (39a). Expres-sion plasmids for overproduction of MppA were constructed by the followingprocedure. PCR primers were designed to incorporate a BspLU11I site (givenhere in bold) that included the initiation codon (underlined) of mppA (59-AATTTACATGTCGGTTAGAGGGAAAC-39) on the forward primer and a uniqueBglII site (in bold) on the reverse primer (59-CGCCAGATCTCATCACATCAATGCTTCAC-39). Since another potential initiation codon was found 21 basesdownstream of the first ATG in the mppA sequence, PCR amplification of thisshorter version was also done, using, in this case, 59-AAACTCATGAAGCACTCTGTTTCAG-39 as the forward primer that introduced a BspHI site (in bold)that included the initiation codon (underlined). These primers were used toamplify the two versions of the mppA gene from the chromosome of E. coli

JM83. The amplified DNA products were treated with BspLU11I or BspHI andBglII, and the resulting fragments were ligated into the compatible NcoI andBamHI sites of the expression vector pTrc99A. The ligation mixtures were usedto transform strain JM83, and the transformants were selected for Amp resis-tance at 37°C. Plasmid DNA from about 1,000 AmpR colonies was restricted byBamHI to linearize the cloning vector (there is no BamHI site in the desiredplasmids), and the restriction mixture was used to transform JM83. AmpR clonesselected this way all contained the expected plasmids. pMLD1285 (long form)and pMLD1493 (short form) allowing expression of the mppA gene under thecontrol of the isopropyl-b-D-1-thiogalactopyranoside (IPTG)-inducible Trc pro-moter were selected for further study. pHSL1 (KanR) is a pACYC177 derivativein which the 2.3-kb fragment from pMLD1285 cut with HpaI and HindIII andcarrying mppA was blunted with the Klenow fragment of DNA polymerase I andinserted into pACYC177 which had been cut with ScaI and blunted. Followingligation, a KanR AmpS transformant was shown to express mppA (Table 2,experiment 2).

Preparation of crude protein extracts. Cells (0.5-liter cultures) were grownfrom a 1% inoculum at 37°C in 23 YT medium with vigorous aeration. Theinoculum was grown overnight from a single colony. All cultures contained 100mg of Amp per ml. When required, 1 or 5 mM IPTG was added in early log phaseand growth was continued for approximately 4 h. In all cases, cells were harvestedin the cold when the optical density at 600 nm reached the range of 0.7 to 1 andwere washed with 40 ml of cold 20 mM potassium phosphate (pH 7.4) containing0.3 mM MgCl2 and 0.1% b-mercaptoethanol. The cell pellet was suspended in 5ml of the same buffer and disrupted by sonication (Sonicator 150; T. S. Ulltra-sons, Annemasse, France) for 10 min with cooling. The resulting suspension wascentrifuged at 4°C for 30 min at 200,000 3 g in a Beckman TL-100 ultracentri-fuge. The supernatant was dialyzed overnight at 4°C against 100 volumes of thephosphate buffer, and the resulting solution (5 ml; 10 to 12 mg of protein/ml),designated the crude extract, was stored at 220°C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of proteins was per-formed as previously described with 13% polyacrylamide gels (25). Protein con-centrations were determined by the method of Lowry, with bovine serumalbumin as a standard (26).

Isolation of periplasmic proteins. Cells (100-ml cultures) growing exponen-tially at 37°C in 23 YT medium were harvested and washed with 40 ml of 30 mMTris-HCl (pH 8.0). After centrifugation, the cell pellet was resuspended in 8 mlof the buffer containing 30% (wt/wt) sucrose, and 20 to 30 min later EDTA wasadded to a final concentration of 10 mM. The suspension was kept at roomtemperature for a further 15 min and then centrifuged. The supernatant con-taining the released periplasmic proteins was dialyzed against 100 volumes of 30mM Tris-HCl (pH 8.0) and concentrated on PM10 Amicon membranes. The cellpellet, following release of the periplasmic proteins, was resuspended in 2 ml ofTris buffer and sonicated, and the cytoplasmic proteins were recovered followinghigh-speed centrifugation.

Murein tripeptide isolation. E. coli TP73 (DampDE), which accumulates largequantities of anhMurNAc-tripeptide in its cytoplasm (20), was grown to earlystationary phase in 20 liters of 23 YT broth. The cells (150 g of cell paste) wereharvested, washed once in 10 mM potassium phosphate buffer (pH 7.0), and

TABLE 1. Bacterial strains

Strain Genotype Source or reference

AT980 dapD2 relA1 spoT1 thi-1 l2 Hfr (defective) E. coli Genetic Stock Center (YaleUniversity, New Haven, Conn.), no. 4545

TP72 F2 ampG::kan lysA opp araD139 rpsL150 relA1 deoC1ptsF25 flbB5301 rbsRD(argF-lac)

20

TP981 AT980 ampG::Kan P1(TP72) 3 AT980TP984 TP981 mppA::miniTn10Cm This workTP985 AT980 mppA::miniTn10Cm P1(TP984) 3 AT980AW1043 MC1000 D phoA (E-15) proC::Tn5 A. WrightTP987 AT980 proC::Tn5 P1(AW1043) 3 AT980TP988 TP985 proC::Tn5 P1(AW1043) 3 TP985CH483 D(trp-tonB-oppABCDF)467 thi lac pro galE 18VC6121 thi-1 relA1 spoT1 dapB17::Mu zaa1::Tn5 Hfr E. IshiguroTP986 CH483 dapB17::Mu zaa1::Tn5 P1(VC6121) 3 CH483SS320 F2 lacI22 lacZ pro-48 met-90 trpA trpR his-85 rpsL azi-9 gyrA l2 P1S 3SS5013 SS320 D(tdk-oppABCDF-tonB-trp) S. ShortTP989 SS5013 mppA::miniTn10Cm P1(TP985) 3 SS5013TP73 F2 DampDE lysA opp araD139 rpsL150 relA1 deoC1 ptsF25 flbB5301 rbsR

D(argF-lac)20

CH212 hsdS20 (rB2 mB

2) recA13 ara-14 proA2 lacY1 galK2 (SmR) xyl-5 mtl-1supE44 oppA462

16

JM83 ara D(lac-proAB) rpsL thi [f80 dlacD(lacZ)M15] 44TOP10F9 F9 [lacIq Tn10(Tetr)] mcrA D(mrr-hsdRMS-mcrBC) f80lacZDM15 DlacX74

deoR recA1 araD139 D(ara-leu)7697galU galK rpsL (StrR) endA1 nupGInvitrogen

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resuspended in 130 ml of cold 10% (wt/vol) trichloroacetic acid (TCA). After 10min, the extract was recovered by centrifugation and the pellet was extractedtwice with 100 ml of cold 2% TCA. The combined extracts were extracted threetimes with cold ethyl ether to remove the TCA. After removal of ether, theextract was neutralized with 1 ml of 10 N NaOH, lyophilized, and dissolved in 20ml of water. After removal of high-molecular-weight materials by precipitationwith 50% ethanol, anhMurNAc-tripeptide was isolated by molecular sieve chro-matography on a Toyopearl HW-40S column (1.6 by 78 cm; TosoHaas, Mont-gomeryville, Pa.) that was equilibrated and eluted with 20 mM potassium phos-phate buffer (pH 7.0). The fractions containing anhMurNAc-tripeptide werepooled, desalted on a Sephadex G-10 column, and then fractionated by high-pressure liquid chromatography on a C-18 reverse-phase column (LiChrospherRP-18; 250 by 4 mm; 3-mm particle size; E. Merck), employing a linear gradientof 0 to 20% acetonitrile in 0.05% trifluoroacetic acid (20). Because of the limitedcapacities of the chromatography columns, repeated runs were necessary, duringeach of which about 50,000 cpm of 3H-Dap-labeled TP73 extract was added tofacilitate monitoring of the column fractions. All the batches of high-pressureliquid chromatography-purified anhMurNAc-tripeptide were pooled, lyophi-lized, dissolved in 1 ml of 5 mM Tris-HCl (pH 7.5), and digested with 2 mg ofpurified AmpD amidase (21) for 16 h at 37°C. Tripeptide was isolated from thisdigest by chromatography on a QMA MemSep 1010HP anion exchange mem-brane (Millipore Corp., Bedford, Mass.), employing a gradient of 0 to 500 mMNaCl in 20 mM Tris-HCl (pH 8.0) over 20 min. Tripeptide eluted after about 11min.

Complementation of the mutant requiring murein tripeptide for growth.Growth of the mutant, containing various plasmids, on L agar-Dap-Amp plateswas compared with growth on L agar-tripeptide-Amp plates in which the con-centration of tripeptide was twice the amount required for growth (about 10mg/ml). IPTG (0.1 mM) was added when induction of the Trc promoter wasrequired.

Nucleotide sequence accession number. Our nucleotide sequence of the mppAgene has been deposited in the GenBank database under accession numberU88242.

RESULTS

Isolation of mutants that require murein tripeptide forgrowth. Prior evidence indicated that E. coli could utilizemurein tripeptide in place of Dap for synthesis of cell wallmurein (9). In order to search for mutants which could notutilize tripeptide, E. coli TP981 (opp1 ampG::kan lysA1

dapD2) was constructed. Opp was assumed to be required foruptake of the tripeptide, based on the report of Goodelland Higgins that an opp deletion strain and an oppA non-polar mutant did not utilize tripeptide (10). Since GlcNAc-anhMurNAc-tripeptide, transported into the cell via AmpG, isthe principal source of cytoplasmic tripeptide (20, 32),ampG::kan was introduced to prevent reutilization of tripep-tide originating from this source. Dap decarboxylase (LysA)was present to destroy free Dap that might be released fromtripeptide so that it could not be used for growth. It was shown

that TP981 required Dap for growth and that murein tripep-tide could replace the required Dap.

Aliquots (10 ml) of the mutagenized TP981 culture (seeMaterials and Methods) plated on L agar-Cm-Dap agar platesyielded about 300 CmR colonies. These master plates werereplicated onto media containing murein tripeptide in place ofDap. From about 90 such master plates, four mutants wereobtained which did not grow on tripeptide-containing medium.

Test of the murein tripeptide-requiring mutants for Oppfunction. Since oligopeptides are normally transported into thecytoplasm by Opp, one class of mutants isolatable by the pro-cedure employed would be opp mutants. Therefore, the mu-tants were tested for resistance to triornithine, a toxic tripep-tide dependent on Opp for transport into the cell (4). Two ofthe mutants proved to be resistant to triornithine, a fact indi-cating a possible defect in the Opp pathway. The other twomutants remained sensitive to triornithine, a fact indicatingmutations in loci other than opp, and sequencing of the DNAflanking the transposon insertions revealed that the two trior-nithine-sensitive mutants were identical.

Identification of the mppA locus in the triornithine-sensitivemutant TP984. P1vir grown on selected strains from the col-lection described by Singer et al. (40) were used for mappingby transduction. The recipient was TP984 CmR opp1. Whentransduced with P1vir grown on zci-3117::Tn10kan, which isvery close to opp at 28.0 min, all KanR transductants remainedCm resistant, indicating that the mutation was unlinked to opp.Transduction with P1vir grown on zda-3061::Tn10 (30.4 min)converted 24 out of 40 TetR transductants to Cm sensitivity,indicating that the mutation is about 2 min clockwise from theopp operon.

Sequencing with primers complementary to each end of thecat gene present in the transposon revealed an ORF with thetransposon and the characteristic 9-base pair repeat sequence(GTTAAAGCG) located 171 nucleotides upstream from thetermination codon. The cloned HindIII fragment (.10 kb)contained the complete gene, while the cloned PstI fragment(4.5 kb) lacked a short N-terminal region because of the pres-ence of a PstI site.

Comparison of the amino acid sequence of the ORF (acces-sion no. U88242) with sequences in the database by means ofthe BLAST algorithm (2) revealed that the mutation was in apreviously unidentified gene whose product is about 46% iden-tical to the amino acid sequence of OppA, the periplasmic

TABLE 2. Growth responses of strains to murein tripeptide and Pro-Phe-Lys

Experiment no.a Strain Relevant genotype Plasmid genotype Growthb

1 TP986 DoppABCDFdapB17

2

TP986/pB2 DoppABCDFdapB17

oppBCDF 1

2 TP985 dapD2 mppA 2TP985/pHSl1 dapD2 mppA mppA 1

3 SS5013/pB2 pro-48 DoppA 1TP989/pB2 pro-48 DoppA mppA 2TP989/pB2, pHSL1 pro-48 DoppA mppA mppA 1SS5013/pB2, pBf30 pro-48 DoppA oppA 111

4 TP987 proC 1TP988 proC mppA 1

a Experiments 1 and 2 were performed in L broth with murein tripeptide as the sole source of Dap. Experiments 3 and 4 were carried out in M9-glucose-B1 mediumwith required amino acids except for Pro, and Pro-Phe-Lys was used at 30 mg/ml. Pro was shown to be required for growth. Growth was observed after 1-ml standingcultures were incubated at 37°C for 18 to 20 h.

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binding protein of the opp operon (Fig. 1). The gene wasnamed murein peptide permease A (mppA) because of itsstructural and functional similarity to oppA and because, asshown below, it is a periplasmic protein required for the uptakeof murein tripeptide. Hybridization of a 32P-end-labeled, sin-gle-stranded 18-mer mppA probe to a membrane containing anordered set of Kohara phage clones with overlapping segmentsof the complete E. coli genome (29) showed that mppA waspresent in both l260 and l261. Comparison with the currentphysical map (5) places the gene at 29.95 min. Our mppAsequence agrees perfectly with the sequence of ORF_IDo260#13 (accession no. D90771) from the E. coli genomesequence (1).

Complementation of mppA::miniTn10Cm in TP984. Expres-sion vectors carrying the wild-type mppA gene or an alternateform starting at an ATG 21 bases from the presumed startcodon (see Materials and Methods) were tested for their abil-ities to complement TP984. As illustrated in Fig. 2, both formscomplemented the mutant for growth on tripeptide in thepresence of IPTG and had no effect on sensitivity to triorni-thine. Actually, the basal level of expression from the Trcpromoter was sufficient for complementation by pMLD1285,whereas pMLD1493 did not complement unless IPTG wasadded (data not shown).

Overproduction of MppA and demonstration of its presencein the periplasm. Since MppA is expected to be a periplasmicbinding protein, it is presumably first made in a precursor form

and processed to the mature periplasmic form in a mannersimilar to that of OppA. Because OppA is a major protein inthe periplasm (15), to test for overproduction of MppA, whichis predicted to be of similar size, the expression plasmids wereintroduced into E. coli CH483, a strain in which the opp operonis deleted (18). As shown in Fig. 3, induction with IPTG causedoverproduction of a protein of approximately 58 kDa that ispresent predominantly in the periplasm. For unknown reasons,the plasmid with the longer signal peptide (pMLD1285) over-expresses MppA significantly better than pMLD1493 (Fig. 3).MppA produced from pMLD1285 has an arginine in place oflysine at residue 49 of the precursor protein, presumably amutation introduced during PCR, but this is unlikely to be thereason for the superior production of MppA from pMLD1285.

Periplasmic MppA is processed by signal peptide cleavage.To determine if periplasmic MppA lacks the putative signalsequence, the N-terminal amino acid sequence of the highlyoverproduced 58-kDa protein expressed from pMLD1285 wasdetermined from the corresponding band transferred to anImmobilon P membrane following SDS-PAGE. The N-termi-nal sequence found, AEVPSGTVLA, is identical to aminoacid residues 30 to 39 of MppA (Fig. 1). This confirms that theoverproduced protein is the processed form of MppA. Process-ing of the MppA precursor occurs between the two alanines atpositions 29 and 30; the corresponding signal peptide cleavagesite in OppA is between two alanines located at positions 26and 27 (Fig. 1). However, the signal sequences of the two

FIG. 1. Comparison of the amino acid sequences of MppA and OppA from E. coli. Identical amino acids are identified by their single-letter codes, conservativeamino acids are indicated by 1. Amino acid numbering for both precursor proteins is shown on the right. Aligned MppA and OppA protein sequences are derivedfrom the nucleotide sequences with accession numbers U88242 and P23843, respectively. The shaded area represents the signal sequences of MppA and OppA, andthe arrow shows the site of signal peptide cleavage.

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proteins differ remarkably. MppA has nine hydroxyl- or thiol-containing amino acids in the hydrophobic region, whereasOppA has only a serine (Fig. 1). The mature protein has apredicted molecular weight of 57,618 that agrees with the sizededuced from its mobility on SDS-polyacrylamide gels. It has apI of 8.32.

Uptake of murein tripeptide requires components of Opp.In E. coli CH483, the entire opp operon is deleted (18). How-ever, the CH483 genome carries the mppA locus because aDNA fragment of the expected size was obtained by PCR (withTaq polymerase) on CH483 chromosomal DNA with 59-TATGTGCTTTACCGCATTTTG-39, which begins 153 nucleo-

tides upstream of the start codon on the mppA coding strand,and 59-ATTTGAAGATTATCGATA-39, which begins 154 nu-cleotides downstream of the stop codon on the antisensestrand, as primers. The PCR product was cloned into pTrc99A(cut with EcoRV and a T-overhang added with Taq polymerasein the presence of dTTP as described in reference 13), and theresulting plasmid complemented the mppA mutant, TP984, forgrowth on tripeptide (data not shown). To test if Opp compo-nents might be involved in supporting growth on tripeptideas the source of Dap, dap was introduced into CH483 bycotransduction with zaa1::Tn5 from E. coli VC6121 to yieldstrain TP986 (DoppABCDF, dap). TP986 failed to grow on Lagar plus murein tripeptide, whereas TP986 containing pB2(oppBCDF1) grew well on L agar plus murein tripeptide (Ta-ble 2, experiment 1). This indicates that some or all of themembrane and cytoplasmic components of Opp are necessaryand sufficient for transfer of murein tripeptide from MppAinto the cytoplasm. OppA is clearly not required.

Is MppA specific for murein tripeptide? To investigatewhether MppA could facilitate transport of non-cell wall pep-tides via OppB, OppC, OppD, and OppF, we determined theability of the tripeptide Pro-Phe-Lys to provide proline forgrowth of proline auxotrophs. As shown in Table 2, experiment3, E. coli SS5013 (pro his trp met DoppABCDF) did not growin minimal medium when Pro-Phe-Lys was used as the sourceof proline. However, E. coli SS5013 carrying plasmid pB2(oppBCDF) did grow in the same medium (although growthwas poor), indicating that MppA (or the third OppA paralog,ORF-f535, shown in Fig. 4) could facilitate transport of theproline-containing tripeptide when OppB, OppC, OppD, andOppF were present and that OppA was not essential for itsuptake. E. coli TP989, an SS5013 derivative carrying themppA::Cm mutation, could no longer grow on Pro-Phe-Lys inthe presence of pB2, strongly suggesting that MppA was in-volved in peptide uptake. Introduction into TP989/pB2 of asecond plasmid, pHSL1, compatible with pB2 and carrying themppA locus, restored growth, confirming that MppA directlyparticipated in peptide transport.

To determine if Pro-Phe-Lys could also be transported viaOppA, proC derivatives of TP981 (opp1) and TP984 (opp1

mppA::Cm) were tested. Pro-Phe-Lys supported growth ofboth strains (Table 2, experiment 4). Thus, OppA, in the ab-sence of MppA, can also allow growth of a proline auxotrophon Pro-Phe-Lys. Though TP989 (mppA::Cm DoppABCDF)/pB2 (oppBCDF)/pHSL1 (mppA) grew poorly during 18 h onPro-Phe-Lys, when the compatible plasmid, pBf30, expressingoppA, replaced pHSL1, significantly more rapid growth oc-curred (Table 2, experiment 3). Since both pHSL1 and pBf30are pACYC derivatives, higher oppA gene dosage cannot ex-plain this result. A more likely explanation is that Pro-Phe-Lysbinds preferentially to OppA rather than MppA. We haverecently observed that a 15-fold excess of Pro-Phe-Lys does notcompete with murein tripeptide for binding to MppA (;95%purity) in vitro (unpublished data). This is consistent with ourobservation that MppA transports Pro-Phe-Lys poorly and thatMppA transports L-Ala-g-D-Glu-meso-Dap efficiently.

Genomics of mppA. Comparison of the amino acid sequenceof MppA with the sequences in the GenBank protein databaserevealed that, in addition to a 46% identity with OppA from E.coli and Salmonella typhimurium, MppA is about 39% identicalto E. coli ORF-f535 (accession no. U28377), 28% identical toBacillus subtilis Spo0KA/OppA (36, 37), 25% identical to B.subtilis DPPE (28), and 39 and 24% identical to two ORFs inHaemophilus influenzae (7). A multiple sequence alignment ofthese seven paralogs and orthologs of MppA is shown in Fig. 4.

A comparison with the two ORFs in H. influenzae is inter-

FIG. 2. Complementation of mppA by pMLD1285 or pMLD1493. The plateon the left shows growth of the wild-type and mppA cultures on L mediumcontaining Dap, Amp, and 0.1 mM IPTG. The plate in the center shows growthsof the same cultures on L medium containing murein tripeptide, Amp, and 0.1mM IPTG. The plate on the right demonstrates that none of the strains can growon M9 medium containing 0.1% Casamino Acids, 1 mg of thiamine per ml, Dap,and 500 mM triornithine. All grew on the M9 medium in the absence of trior-nithine (data not shown). Sector 1, AT980/pTrc99A (the parent); sector 2,TP985/pTrc99A (mppA); sector 3, TP985/pMLD1285 (pTrcmppA1); sector 4,TP985/pMLD1493 (pTrcmppA1).

FIG. 3. Overproduction and cellular localization of MppA in E. coli cells.Crude protein extracts (total soluble proteins) and periplasmic extracts preparedfrom strain JM83 or CH483 carrying either the pTrc99A plasmid vector, thepMLD1285 plasmid, or the pMLD1493 plasmid were analyzed by SDS-PAGE.Shown are crude extract from JM83 (lane A); crude extracts from CH483 cellscarrying either pTrc99A (lane B), pMLD1285 (lane C), or pMLD1493 (lane D)after growth for 4 h in the presence of 5 mM IPTG; and periplasmic extractsfrom the strains listed for lanes A to D (lanes E to H). Molecular mass (mw)standards indicated on the left are as follows: pyrophosphorylase b (94 kDa),bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase(30 kDa).

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esting. ORF HI1124 (accession no. U32792), although mostclosely related to MppA, is clearly the ortholog of OppA (50%identity) because it is part of the opp operon of H. influenzae(6). Examination of the alignments in Fig. 4 reveals that the

presence of two cysteines is not critical for OppA, even thoughthe crystal structure of S. typhimurium OppA (43) shows thetwo cysteines (Cys296 and Cys442) forming a disulfide bridgethat straddles the peptide ligand.

FIG. 4. Alignment of E. coli MppA with its paralogs and selected orthologs. Positions with identical residues for at least five of the eight sequences are solid black,and gaps are indicated by dashes. The sequences were aligned by the Clustal method (MegAlign; DNASTAR). The sequences shown are as follows: Sty OppA, S.typhimurium OppA (17); Eco OppA, E. coli OppA (22); Eco ORF-f535, E. coli ORF-f535 (GenBank accession no. U28377); Eco MppA, E. coli MppA (GenBankaccession no. U88242); Hin HI1124, H. influenzae OppA ortholog (7); Hin HI0213, H. influenzae OppA ortholog (7); Bsu SpoOKA, B. subtilis OppA (36, 37); and BsuDPPE, B. subtilis DPPE (28). The putative hexapeptide motif in MppA and HI0213 and the two cysteines in Sty OppA, Eco OppA, and Hin HI0213 are highlighted in lightgrey.

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ORF HI0213 contains a hexapeptide sequence (179RIELDK 184) which is tantalizingly similar to a motif (174KIQLDK 179) in the aligned sequence of MppA (Fig. 4). Itwill be interesting to see if this hexapeptide motif (K/RIQ/ELDK) can serve as a signature for functional MppA orthologsin other gram-negative bacteria. At present, there are insuffi-cient data to determine whether MppA is found in differentbacterial species or is restricted to E. coli.

Examination of the E. coli genome sequence surroundingmppA at 29.95 min shows that there are no genes with partialidentity to opp genes, as might be expected if an entirely inde-pendent Mpp transport operon were present. Immediately up-stream of mppA, reading clockwise as with mppA, we find xapR(pndR) (accession no. P23841), a putative xanthosine operonregulatory protein, and immediately downstream is yggB (ac-cession no. P11666) that is present in the opposite orientation(1). Furthermore, mppA has a putative transcription termina-tion signal consisting of a 30-nucleotide stem and loop begin-ning 11 nucleotides after the stop codon, which also suggeststhat mppA is not at the beginning of a special transport operon.

DISCUSSION

Recycling of cell wall peptides required the presence of anenzyme that would link the tripeptide L-Ala-g-D-Glu-meso-Dap to UDP-N-acetylmuramate, thereby directly reintroduc-ing it into the biosynthetic pathway for wall synthesis (9, 20). Inseeking this hypothetical murein tripeptide-adding enzyme, weundertook a search for mutations in the putative gene. Wereasoned that a mutant of a Dap-requiring strain which lackedthe tripeptide-adding enzyme would not be able to grow onmedia in which murein tripeptide replaced Dap. A mutantisolation procedure was designed which relied on the uptake ofthe exogenously added murein tripeptide for growth. The mu-tant sought should grow on Dap but not on tripeptide. Con-currently with our mutant search, we identified the tripeptide-adding enzyme, or murein peptide ligase gene, mpl, by itshomology to murC and demonstrated that an mpl-null mutantcould not grow when Dap was replaced by murein tripeptide(29). Thus, our genetic screen for mutant isolation was sound.

However, to our surprise, instead of finding a mutant of mpl,as was suggested earlier (34), we discovered that one of themutations was in a gene required for the uptake of mureintripeptide, namely, mppA. The other two mutants we isolatedwere in genes required for a functional Opp system becausethese mutants were resistant to triornithine, which requiresOpp for uptake.

The new gene, mppA, mapping at 29.95 min, codes for theprecursor of a periplasmic binding protein that is required foruptake of murein tripeptide. We have cloned and overpro-duced MppA and shown that the mature protein is present inthe periplasm (Fig. 3). Plasmids carrying the mppA gene undercontrol of the IPTG-inducible Trc promoter complement themppA mutant when induced with IPTG (Fig. 2).

MppA and OppA are similar in size, and their amino acidsequences are about 46% identical (Fig. 1). OppA is an abun-dant periplasmic protein, whereas E. coli CH212 oppA462,which lacks OppA, contains only a trace protein of similar size,suggesting that MppA is a minor component in the periplasm(16). CH212 does not transport murein tripeptide (10), andthis led us to suspect that, contrary to the published report(10), oppA462 is a polar mutation because, as we have shown,the absence of OppA does not prevent the utilization oftripeptide via MppA and the membrane and cytoplasmic Oppcomponents. In fact, we have now demonstrated that oppA462

is a polar mutation because CH212 (proA2) will grow on Pro-Phe-Lys as the source of proline only when carrying plasmidpB2 (oppBCDF). Thus, a nonpolar oppA mutation is not pres-ently available in E. coli.

Two of the tripeptide-requiring mutants are resistant to tri-ornithine, which is usually diagnostic for a defect in Opp func-tion (4). We have recently found that one of these mutantshas its transposon located in oppB and is complementedby pB2, a plasmid expressing OppBCDF (unpublished data).This is consistent with our result demonstrating that TP986(DoppABCDF, dap)/pB2 will grow on murein tripeptide andstrongly suggests that MppA uses some or all of the membraneand cytoplasmic Opp components for transfer of murein trip-eptide into the cytoplasm.

The other triornithine-resistant mutant was found to have itstransposon in the promoter region of groESL, and this resultsin greatly reduced levels of the encoded proteins in the mutant(25a). This is, to our knowledge, the first example indicatingthat GroESL chaperonin function is essential for formation ofa functional ABC transporter.

Since the periplasmic binding proteins, MppA and OppA,are about 46% identical and perform similar functions, they, aswell as their orthologs, presumably evolved from a commonancestor following gene duplications. For MppA to use theOpp pathway, one would expect that MppA and OppA have acommon surface that interacts with one or both of the mem-brane-bound components of the Opp pathway. This would becomparable to the case with the hisJ and argT gene products ofS. typhimurium, another example of apparent gene duplicationthat gave rise to the periplasmic binding proteins required fortransport of histidine and arginine (15). Both proteins dock onshared membrane-bound components in order to dischargetheir cargo into the cytoplasm (15).

OppA is a remarkably nonspecific binding protein believedcapable of binding most tri-, tetra-, and pentapeptides linkedtogether by normal a-peptide bonds. Tame et al. (43) haverecently explained the structural basis for this sequence-inde-pendent peptide binding. The peptide is completely enclosedin a voluminous hydrated cavity that can accommodate thevarious amino acid side chains, and all bonding is directed tothe peptide bonds between the a-amino acids. In contrast, thesecond bond in murein tripeptide is an amide bond betweenthe g-carboxyl of D-Glu and the L-amino group of meso-Dap. Itis thus conceivable that the binding sites in OppA are notpositioned correctly to accommodate peptides containing thisnovel g-D-glutamyl amide bond.

While MppA is essential for the transport of murein tripep-tide, MppA can also transport ordinary a-linked tripeptidessuch as Pro-Phe-Lys, although, judging by the growth response,MppA is much poorer than OppA for the transport of Pro-Phe-Lys. The binding constants of different tripeptides forOppA vary over a wide range; e.g., triornithine is at least600-fold less able to bind than Ala-Phe-Gly (12). Presumably,a similar variation in the abilities of various tripeptides to bindto MppA (and the other OppA paralog in E. coli) will befound, but, judging by the example of Pro-Phe-Lys, it seemslikely that a-linked tripeptides will prove to be poor ligands forMppA compared to peptides that contain the g-D-glutamylbond.

Further complicating the transport of peptides in E. coli, aprotein called OppE is required for the uptake of certaintripeptides and is independent of Opp (3). This is similar to thesituation with S. typhimurium, in which TppB is a proteinessential for the uptake of some tripeptides. However, thesetransporters apparently do not depend on periplasmic binding

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proteins for their function, since only a single mutation site isknown for each.

MppA binds murein tripeptide precisely because it may haveevolved for this purpose. Following gene duplications, oneparalog, OppA, evolved to relative nonspecificity; another,MppA, evolved to high affinity for the unique murein tripep-tide; and the properties of the third OppA paralog in E. coli(ORF-f535 [Fig. 4]) are completely unknown at present. E. coliperiplasm contains lytic transglycosylases (39) which degradeat least 30% of the murein sacculus to GlcNAc-anhMurNAc-tri- and -tetrapeptide each generation (8, 32) as well as aperiplasmic MurNAc-L-alanine amidase which can release thepeptide from the anhydromuropeptide, although this activity isvery poor (35). The principal pathway for uptake and reuti-lization of tripeptide from the cell wall is indirect, as it requiresthe transport of GlcNAc-anhMurNAc-tripeptide into the cellvia the AmpG permease, followed by cleavage by AmpD ami-dase to release tripeptide (19, 20). Therefore, the question ofwhy E. coli has this capacity to bind and import free mureintripeptide remains unanswered. It would seem that MppAserves some purpose other than recycling.

Goodell (9) estimated a Km of 2 mM tripeptide for theincorporation of exogenously added tripeptide into mureinsacculi by intact cells, suggesting that the true Km may be evenlower. Goodell and Schwarz (11) have demonstrated that E.coli accumulates murein tri- and tetrapeptides as well as thedipeptide Dap-D-Ala in the medium during growth, and it hasrecently been shown that medium conditioned by E. coligrowth augments transcription from the two E. coli promotersupstream of the ftsQAZ gene cluster required for cell division(41). One of the promoters (P1) is RpoS-stimulated, and theother (P2) is regulated by SdiA, which is a member of the LuxRsubfamily of transcriptional activators involved in quorumsensing (autoinduction) (8, 41). The nature of the factors in E.coli-conditioned medium that stimulate transcription from theP1 and P2 promoters is unknown, but it is unlikely that thesefactors include an N-acyl-L-homoserine lactone (41), especiallysince E. coli lacks an ortholog of the Vibrio fischeri LuxI re-quired for production of N-acyl-L-homoserine lactone (8).

Since the recycling pathway presumably supplies a constantlevel of tripeptide in the cytoplasm, it is difficult to imaginehow entry of a smaller amount through the MppA pathwaywould have any significant effect thereon. It is tempting tospeculate that upon E. coli growth to a quorum (8), the accu-mulated murein peptides (11) may bind to MppA and, uponcontact with the required Opp components or another specificmembrane receptor, may stimulate expression from selectedpromoters through a signal transduction pathway. Experimentsto test these possibilities and that may illuminate the physio-logical function of MppA are under way.

An example of periplasmic binding proteins mediating signaltransduction is the chemotactic response (42). The periplasmicbinding proteins for ribose, galactose, and glucose, whenloaded with their specific sugars, trigger the chemotactic re-sponse by binding to the Trg receptor (42). Likewise, theperiplasmic binding protein of E. coli dipeptide permease ac-tivates the chemotactic response through the Tap receptorwhen loaded with dipeptide (27, 42). In a similar vein, a sig-naling pathway dependent on liganded MppA may exist forchemotaxis or may affect entry of E. coli into the stationaryphase by regulating expression of rpoS and/or the RpoS-de-pendent promoters (14).

ACKNOWLEDGMENTS

We thank Marten Hammar for providing bacteriophage lNK1324and for the gift of primers KS4508 and KS4509, which were used to

sequence the cat markers; Stephen A. Short for E. coli SS5013 andplasmids pB2 and pBf30; Christopher Higgins for E. coli CH212; EdIshiguro for E. coli VC6121; Andrew Wright for E. coli AW1043; andChristine Jacobs for the gift of purified AmpD amidase. We thankKeith Merdek for excellent technical assistance and the DigestiveDisease Center (NIDDK, P30 DK34928) for production of E. coliTP73 cells.

This work was supported in part by the Swedish Medical ResearchCouncil, by JT.P., and by Public Health Service grant GM51610 fromthe National Institute of General Medical Sciences.

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