Conjugal transfer of the vancomycin resistance determinant vanB between enterococci involves the movement of large genetic elements from chromosome to chromosome

Abstract Resistance to various levels of vancomycin and susceptibility to teicoplanin in enterococci (VanB phenotype) is mediated by the vanB gene cluster. VanB-type resistance was transferred by intra- and inter-specific conjugation between different strains of Enterococcus . Analysis of S⨍i I-digested genomic DNA by zero integrated-field electrophoresis followed by Southern hybridization revealed vanB -containing chromosomal insertions of approximately 90–250 kb in the transconjugants. Thus, transfer of VanB-type resistance is associated with the movement of large genetic elements from chromosome to chromosome.     

VanA-Type Vancomycin-Resistant Enterococci in Equine and Swine Rectal Swabs and in Human Clinical Samples

Vancomycin-resistant enterococci (VRE) in healthy people and in food-producing animals seems to be quite common in Europe. The existence of this community reservoir of VRE has been associated with the massive use of avoparcin in animal husbandry. Eight years after the avoparcin ban in Europe, we investigated the incidence of VanA enterococci, their resistance patterns, and the mobility of their glycopeptide-resistance determinants in a sampling of animal rectal swabs and clinical specimens. A total of 259 enterococci isolated from equine, swine, and clinical samples were subcultured on KF-streptococcus agar (Difco Laboratories, Detroit, MI) supplemented with vancomycin and teicoplanin; 7 (6.7%), 10 (16%), and 8 (8.6%) respectively were found to be glycopeptides resistant (VanA phenotype). Slight differences in antimicrobial resistance patterns resulted among VRE recovered from the different sources. Polymerase chain reaction amplification demonstrated the presence of the vanA gene cluster and its extrachromosomal location in VRE plasmid DNA. VanA resistance was transferred in 7 out of 25 mating experiments, 4 with clinical, 2 with swine, and only 1 with equine donors. The conjugative plasmids of animal strains showed a high homology in the restriction profiles, unlike plasmids of clinical microrganisms. Our observations confirmed the possible horizontal transfer of VanA plasmids across different strains and, consequently, the diffusion of the vancomycin-resistance determinants.     

Comparative study of vanA gene transfer from Enterococcus faecium to Enterococcus faecalis and to Enterococcus faecium in the intestine of mice

Vancomycin-resistant enterococci represent a large reservoir in animals because of the use of avoparcin as a growth promoter in Europe. These strains of animal origin enter the food chain and can either colonize the human gut or transfer their resistance genes to the human microbiota. In this study, we compared the transfer of vancomycin resistance from resistant animal Enterococcus faecium to sensitive human Enterococcus faecalis and E. faecium. We analysed these transfers in dibiotic mice and human faecal flora-associated mice. VanA transfer from animal E. faecium to human E. faecalis occurred in dibiotic mice. The transconjugants appeared rapidly and persisted at levels between 3.0 and 4.0 log10 colony-forming units g(-1) of faeces. In human faecal flora-associated mice, vanA gene transfer was not detected towards E. faecalis but was possible between E. faecium strains. Our experiments revealed the possibility of vanA transfer from animal E. faecium to human E. faecalis in vitro and in vivo in the intestine of dibiotic mice. However, intraspecies transfer of vanA gene seems more common than interspecies transfer among enterococci.     

Conjugative transfer of glycopeptide and macrolide resistant genes among Enterococci and from Enterococcus faecalis to Staphylococcus aureus

The resistance determinants were transferred from clinical strains of enterococci to Staphylococcus aureus strains. As recipients methicillin-resistant and methicillin-susceptible strains were used and the filter-mating procedure was performed. The transconjugants resistant to erythromycin were obtained in the case of all recipients, in one case the vanA determinant conferring the resistance to vancomycin was transferred together with erythromycin resistance. However the resistance was very unstable and the level was not as high as in the case of Enterococcus faecalis donor strain. The vanA determinant was easily transferred between enterococcal strains by the conjugation and a transfer occurred of vanA alone or together with erythromycin resistance.     

Intra- and interspecies genomic transfer of the Enterococcus faecalis pathogenicity island

Enterococci are the third leading cause of hospital associated infections and have gained increased importance due to their fast adaptation to the clinical environment by acquisition of antibiotic resistance and pathogenicity traits. Enterococcus faecalis harbours a pathogenicity island (PAI) of 153 kb containing several virulence factors including the enterococcal surface protein (esp). Until now only internal fragments of the PAI or larger chromosomal regions containing it have been transferred. Here we demonstrate precise excision, circularization and horizontal transfer of the entire PAI element from the chromosome of E. faecalis strain UW3114. This PAI (ca. 200 kb) contained some deletions and insertions as compared to the PAI of the reference strain MMH594, transferred precisely and integrated site-specifically into the chromosome of E. faecalis (intergenic region) and Enterococcus faecium (tRNAlys). The internal PAI structure was maintained after transfer. We assessed phenotypic changes accompanying acquisition of the PAI and expression of some of its determinants. The esp gene is expressed on the surface of donor and both transconjugants. Biofilm formation and cytolytic activity were enhanced in E. faecalis transconjugants after acquisition of the PAI. No differences in pathogenicity of E. faecalis were detected using a mouse bacteraemia and a mouse peritonitis models (tail vein and intraperitoneal injection). A 66 kb conjugative pheromone-responsive plasmid encoding erm(B) (pLG2) that was transferred in parallel with the PAI was sequenced. pLG2 is a pheromone responsive plasmid that probably promotes the PAI horizontal transfer, encodes antibiotic resistance features and contains complete replication and conjugation modules of enterococcal origin in a mosaic-like composition. The E. faecalis PAI can undergo precise intra- and interspecies transfer probably with the help of conjugative elements like conjugative resistance plasmids, supporting the role of horizontal gene transfer and antibiotic selective pressure in the successful establishment of certain enterococci as nosocomial pathogens.     

Chromosomal integration of tcb chlorocatechol degradation pathway genes as a means of expanding the growth substrate range of bacteria to include haloaromatics

The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.     

Plasmid donor affects host range of promiscuous IncP-1beta plasmid pB10 in an activated-sludge microbial community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI(q). The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the alpha- and gamma-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

Transfer of resistance plasmids from Staphylococcus epidermidis to Staphylococcus aureus: evidence for conjugative exchange of resistance.

The ability of Staphylococcus epidermidis to transfer antimicrobial resistance to Staphylococcus aureus was tested by mixed culture on filter membranes. Two of six clinical isolates examined were able to transfer resistance to S. aureus strains 879R4RF, RN450RF, and UM1385RF. Subsequent S.aureus transconjugants resulting from matings with S. epidermidis donors were able to serve as donors to other S. aureus strains at similar frequencies. Cell-free and mitomycin C-induced filtrates of donors and transconjugants showed no plaque-forming ability. Addition of DNase I, citrate, EDTA, calcium chloride, and human sera to mating mixes and agar showed no effect on transfer. Nonviable donor cells were unable to transfer resistance and transfer did not occur at 4 degrees C. Cell-to-cell contact was required since transfer did not occur in broth or when filters of donor and recipient, respectively, were placed back-to-back so cells were not in direct contact. Analysis of DNA from S. epidermidis isolate UM899, its subsequent S. aureus transconjugants, and cured derivatives demonstrated that all resistance markers which transferred resided on plasmids. Mating experiments suggested a central role for the gentamicin plasmid pAM899-1 in the transfer process. It is concluded that our results are consistent with a conjugative transfer of resistance from S. epidermidis to S. aureus analogous to plasmid transfer demonstrated in streptococcal species for plasmids such as pAM beta 1. This represents a novel mechanism for gene exchange among staphylococci.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.     

Comparison of enterococcal populations related to urban and hospital wastewater in various climatic and geographic European regions

AIMS: Scarce knowledge about the distribution of enterococci species in wastewaters limits any statement on their reliability as faecal indicators or the implications of antibiotic resistance transmission by these organisms through the water cycle. Enterococci have been involved in nosocomial infections and the spreading of antibiotic resistance through the food chain. The species distribution of enterococci and the presence of resistant strains to vancomycin and erythromycin were analysed in more than 400 raw and treated urban wastewaters, surface waters receiving these treated wastewaters and hospital wastewaters from three European countries. METHODS AND RESULTS: A total of 9296 strains were isolated and biochemically phenotyped. The species identification was based on the comparison of biochemical profiles with those of more than 20000 enterococci isolates from an international study. The prevalence of enterococcal isolates resistant to erythromycin (ERE) and vancomycin (VRE) was also analysed. ERE strains were present in a high proportion in all the studied samples. VRE strains were also isolated in all studied countries despite the time elapsed since the use of antimicrobial glycopeptides in animal production was banned in the European Union. CONCLUSIONS: Enterococcus faecalis and Ent. faecium were the most abundant species in all the studied wastewaters. All the studied wastewaters demonstrated high diversity and similar population structure and composition. ERE and VRE isolates were detected in most of the wastewaters. SIGNIFICANCE AND IMPACT OF THE STUDY: Urban and hospital wastewaters are useful targets for the evaluation of the prevalence of ERE and VRE isolates in the environment. It appears that these bacteria could pass through wastewater treatment plants and be transferred to surface waters.     

Chromosomal Integration of tcb Chlorocatechol Degradation Pathway Genes as a Means of Expanding the Growth Substrate Range of Bacteria To Include Haloaromatics

The tcbR-tcbCDEF gene cluster, coding for the chlorocatechol ortho-cleavage pathway in Pseudomonas sp. strain P51, has been cloned into a Tn5-based minitransposon. The minitransposon carrying the tcb gene cluster and a kanamycin resistance gene was transferred to Pseudomonas putida KT2442, and chromosomal integration was monitored by selection either for growth on 3-chlorobenzoate or for kanamycin resistance. Transconjugants able to utilize 3-chlorobenzoate as a sole carbon source were obtained, although at a >100-fold lower frequency than kanamycin-resistant transconjugants. The vast majority of kanamycin-resistant transconjugants were not capable of growth on 3-chlorobenzoate. Southern blot analysis revealed that many transconjugants selected directly on 3-chlorobenzoate contained multiple chromosomal copies of the tcb gene cluster, whereas those selected for kanamycin resistance possessed a single copy. Subsequent selection of kanamycin resistance-selected single-copy transconjugants for growth on 3-chlorobenzoate yielded colonies capable of utilizing this carbon source, but no amplification of the tcb gene cluster was apparent. Introduction of two copies of the tcb gene cluster without prior 3-chlorobenzoate selection resulted in transconjugants able to grow on this carbon source. Expression of the tcb chlorocatechol catabolic operon in P. putida thus represents a useful model system for analysis of the relationship among gene dosage, enzyme expression level, and growth on chloroaromatic substrates.     

Bacteriocin production, plasmid content and plasmid location of enterocin P structural gene in enterococci isolated from food sources

AIMS: To characterize bacteriocin production, antimicrobial spectrum and plasmid content in bacteriocinogenic enterococci from foods. METHODS AND RESULTS: Bacteriocinogenic Enterococcus faecium (14 isolates) and Enterococcus faecalis (three isolates) showed two different patterns of bacteriocin production in liquid broth: exponential-phase and stationary-phase production. Bacteriocin concentrates from all enterococci were inactivated by trypsin, but seldom by heat (100-117 degrees C), extremes of pH (2.0 to 9.0) or reducing agents (such as dithiothreitol). All bacteriocin concentrates were active against Listeria innocua and Listeria monocytogenes, and most were also active against many Ent. faecalis and Ent. faecium isolates. Enterococci clustered in three main groups according to their plasmid content (which included plasmids from 2.0 to 53 kb). Several isolates from different foods showed almost identical plasmid profiles. The enterocin P structural gene (entP) was detected by hybridization on plasmids of c. 19, 26 and/or 35-38 kb. CONCLUSIONS: Enterococci from food show different patterns of bacteriocin production and different plasmid content in spite of carrying similar bacteriocin-encoding genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on the diversity of bacteriocinogenic enterococci from food sources carrying apparently similar enterocin genes.     

肠球菌万古霉素耐药基因簇遗传特性

万古霉素耐药肠球菌自20世纪80年代后期被发现以来,已逐渐发展成为重要的医院感染病原菌。此类耐药肠球菌携带的万古霉素耐药基因簇编码产物可催化合成与万古霉素、替考拉宁等糖肽类抗生素亲和力极低的细胞壁前体导致耐药。目前已在肠球菌中发现的万古霉素耐药基因簇根据基因序列及构成不同分为9个型别;依据它们编码的连接酶合成产物不同又可分为D-Ala:D-Lac连接酶基因簇(VanA、VanB、VanD及VanM型)和D-Ala:D-Ser连接酶基因簇(VanC、VanE、VanG、VanL和VanN型)。这些耐药基因簇介导的耐药水平及其传播模式各有特点。文章综述了肠球菌中万古霉素耐药基因簇的类型、基因构成及传播特性。     

Plasmid Donor Affects Host Range of Promiscuous IncP-1β Plasmid pB10 in an Activated-Sludge Microbial Community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI^q. The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the α- and γ-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

Teicoplanin biosynthesis genes in Actinoplanes teichomyceticus

The genetic determinants for the biosynthesis of the glycopeptide antibiotic teicoplanin were identified. In order to isolate the corresponding gene cluster, oligonucleotides derived from highly conserved motifs in peptide synthetases were used. These synthetic probes, and gene fragments derived from the balhimycin gene cluster of Amycolatopsis mediterranei, led to the identification of the likely teicoplanin gene cluster centered on a region of ca. 110 kb from the genome of Actinoplanes teichomyceticus, the teicoplanin producer. Partial nucleotide sequences identified partial ORFs likely to encode two glycosyltransferases, three P-450 monooxygenases and one ABC transporter. The corresponding genes have been found in other glycopeptide gene clusters. Furthermore, upstream to the peptide synthetase region a segment was identified with a remarkable similarity to the vanHAX operon, conferring resistance to glycopeptides in enterococci. Thus, in contrast to the other glycopeptide producers thus far analyzed, in A. teichomyceticusthe genes for teicoplanin biosynthesis are closely linked to homologs of glycopeptide resistance commonly found in vancomycin-resistant enterococci.     

The composition and persistence of faecal coliforms and enterococcal populations in sewage treatment plants

AIMS: The changes in structure and composition of faecal coliforms and enterococcal populations in sewage from different treatment plants, and the elimination of vancomycin- and erythromycin-resistant enterococci (VRE and ERE, respectively) in these treatment plants was analysed to determine any selective reduction. METHODS AND RESULTS: Faecal coliforms, enterococci, VRE, ERE and spores of sulphite-reducing bacteria were enumerated using standard methods. Samples were enriched where necessary in order to isolate antibiotic resistant strains. The structure and composition of these bacterial populations were determined by biochemical fingerprinting and clustering analysis. High diversity and similarity indexes were detected among all the bacterial populations in raw and treated sewage, independently of their origin and the treatment processes employed. Antibiotic resistant strains were detected in all sewage tested and no selective reduction was observed. CONCLUSIONS: The faecal coliforms and enterococci populations did not differ in the sewage samples studied. The vancomycin and erythromycin resistances of the enterococcal populations were similar in the sewage samples. Resistance to both antibiotics persisted after the treatment process independently of raw sewage flow, faecal origin or size of the human population contributing to sewage. However, sewage of mixed origin (human and animal) presented a lower similarity index for the two bacterial populations compared with that of the other human sewage analysed. SIGNIFICANCE AND IMPACT OF THE STUDY: Although a significant reduction in bacterial populations was observed, the persistence of VRE and ERE strains in the same proportions in sewage suggests that there is no selective elimination of bacterial populations during the treatment processes. The ability of antibiotic resistance strains to survive sewage treatment systems should be considered in certain water reuse programmes.     

Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States

Aims: In this study, mechanisms of antimicrobial resistance and genetic relatedness among resistant enterococci from dogs and cats in the United States were determined. Methods and Results: Enterococci resistant to chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, streptomycin, lincomycin, quinupristin/dalfopristin and tetracycline were screened for the presence of 15 antimicrobial resistance genes. Five tetracycline resistance genes [tet(M), tet(O), tet(L), tet(S) and tet(U)] were detected with tet(M) accounting for approx. 60% (130/216) of tetracycline resistance; erm(B) was also widely distributed among 96% (43/45) of the erythromycin‐resistant enterococci. Five aminoglycoside resistance genes were also detected among the kanamycin‐resistant isolates with the majority of isolates (25/36; 69%) containing aph(3′)‐IIIa. The bifunctional aminoglycoside resistance gene, aac(6′)‐Ie‐aph(2″)‐Ia, was detected in gentamicin‐resistant isolates and ant(6)‐Ia in streptomycin‐resistant isolates. The most common gene combination among enterococci from dogs (n = 11) was erm(B), aac(6′)‐Ie‐aph(2″)‐Ia, aph(3′)‐IIIa, tet(M), while tet(O), tet(L) were most common among cats (n = 18). Using pulsed‐field gel electrophoresis (PFGE), isolates clustered according to enterococcal species, source and antimicrobial gene content and indistinguishable patterns were observed for some isolates from dogs and cats. Conclusion: Enterococci from dogs and cats may be a source of antimicrobial resistance genes. Significance and Impact of the Study: Dogs and cats may act as reservoirs of antimicrobial resistance genes that can be transferred from pets to people. Although host‐specific ecovars of enterococcal species have been described, identical PFGE patterns suggest that enterococcal strains may be exchanged between these two animal species.     

Antimicrobial resistance of Listeria spp. recovered from processed bison

AIMS: The current study examined the antimicrobial susceptibility of 86 Listeria spp. isolated from processed bison carcasses. MATERIALS AND METhods: Susceptibility to 25 antimicrobial agents was determined using E-test and National Antimicrobial Resistance Monitoring System (NARMS) panels. Most Listeria isolates (88-98%) exhibited resistance to bacitracin, oxacillin, cefotaxime, and fosfomycin. Resistance to tetracycline (18.6%) was also common. Of the 16 tetracycline-resistant Listeria isolates, 15 carried tetM and 2 contained integrase of Tn1545 transposons. Rifampicin and trimethoprim-sulfamethoxazole were the most active antimicrobial agents against Listeria spp., with a MIC(90) of 0.38 microg ml(-1). Ampicillin, erythromycin, penicillin, gentamicin, and tobramycin also exhibited good activity against Listeria spp., with MIC(90) not exceeding 1 microg ml(-1). Differences in resistance among Listeria spp. was displayed, as Listeria innocua strains were more resistant than other Listeria species. CONCLUSIONS: The study showed that Listeria monocytogenes strains from bison were susceptible to the antibiotics most commonly used to treat human listeriosis. However, the presence of antimicrobial resistance in L. innocua indicates the potential for transfer of resistance and a conjugative transposon to L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of our study will provide useful information for the development of public health policy in the use of antimicrobials in food animal production.     

A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica

The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism.