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PubMed2003

A clonal lineage of VanA-type Enterococcus faecalis predominates in vancomycin-resistant Enterococci isolated in New Zealand.

Avoparcin was used as a feed additive in New Zealand broiler production from 1977 until June 2000. We report here on the effects of the usage and discontinuation of avoparcin on the prevalence of vancomycin-resistant enterococci (VRE) in broilers. Eighty-two VRE isolates were recovered from poultry fecal samples between 2000 and mid-2001. VRE isolates were only obtained from broiler farms that were using, or had previously used, avoparcin as a dietary supplement. Of these VRE isolates, 73 (89%) were VanA-type Enterococcus faecalis and nine (11%) were VanA-type Enterococcus faecium. All E. faecalis isolates were found to have an identical or closely related pulsed-field gel electrophoresis (PFGE) pattern of SmaI-digested DNA and were susceptible to both ampicillin and gentamicin. The PFGE patterns of the nine E. faecium isolates were heterogeneous. All VRE contained both the vanA and ermB genes, which, regardless of species or PFGE pattern, resided on the same plasmid. Eighty-seven percent of the VRE isolates also harbored the tet(M) gene, while for 63 and 100%, respectively, of these isolates, the avilamycin and bacitracin MICs were high (>or=256 microg/ml). Five of eight vancomycin-resistant E. faecalis isolates recovered from humans in New Zealand revealed a PFGE pattern identical or closely related to that of the E. faecalis poultry VRE isolates. Molecular characterization of Tn1546-like elements from the VRE showed that identical transposons were present in isolates from poultry and humans. Based on the findings presented here, a clonal lineage of VanA-type E. faecalis dominates in VRE isolated from poultry and humans in New Zealand.

Animals↗
PubMed2003

Impact of elements containing glycopeptide resistance genes on expression of virulence in Enterococcus faecalis peritonitis: a pilot study with rats.

The relationship between virulence and chromosomal elements containing glycopeptide resistance genes was experimentally assessed for two transconjugant strains of Enterococcus faecalis (VanA and VanB phenotypes) and compared to that for a susceptible wild-type strain. Microbiologic and inflammatory effects were assessed in a polymicrobial rat model of peritonitis. Mean peritoneal enterococcus concentrations +/- standard deviations at day 1 were 2.1 +/- 1.9, 1.3 +/- 1.1, and 1.7 +/- 2.0 log(10) CFU/ml for susceptible, VanA, and VanB strains, respectively (P < 0.05). At day 3 also there were lower concentrations of glycopeptide-resistant enterococcal strains in peritoneal fluid (3.2 +/- 3.4, 1.8 +/- 1.8, and 2.1 +/- 2.4 log(10) CFU/ml for susceptible, VanA, and VanB strains, respectively [P < 0.05]). Transconjugant glycopeptide-resistant strains were associated with increased peritoneal cell counts at the different evaluation times of the experiment (P < 0.001). Plasma alpha1-acid glycoprotein concentrations were lower in the presence of the susceptible strain (667 +/- 189 mg/liter) than in the presence of the VanA or VanB strain (1,193 +/- 419 or 1,210 +/- 404 mg/liter, respectively [P < 0.05]), while concentrations of tumor necrosis factor alpha and interleukin-6 in peritoneal fluid remained similar for the strains. These results suggest a trend toward variation of virulence of transconjugant strains compared to the wild-type strain in this peritonitis model.

Animals↗
PubMed2003

Presence of a vanA-carrying pheromone response plasmid (pBRG1) in a clinical isolate of Enterococcus faecium.

Sex pheromone plasmids, frequently found in Enterococcus faecalis, have rarely been detected in Enterococcus faecium. pBRG1 is an approximately 50-kb vanA-carrying conjugative plasmid of an E. faecium clinical isolate (LS10) that is transferable to E. faecalis laboratory strains. In cell infection experiments, E. faecium LS10 exhibited remarkably high invasion efficiency and produced cytopathogenic effects in Caco-2 cell monolayers. Growth in the presence of sex pheromones produced by E. faecalis JH2-2 was found to cause self-aggregation of both E. faecium LS10 and E. faecalis JH-RFV(pBRG1) (a transconjugant obtained by transfer of pBRG1 to E. faecalis JH2-2) and to increase the cell adhesion and invasion efficiencies of both E. faecium LS10 and E. faecalis JH-RFV(pBRG1). Sex pheromone cCF10 caused clumping of E. faecalis OG1RF(pBRG1) (a transconjugant obtained by transfer of pBRG1 to E. faecalis OG1RF) at a concentration approximately 100-fold higher than the one required for the control strain E. faecalis OG1RF(pCF10). PCR products of the expected sizes were obtained with primers internal to aggregation substance genes of E. faecalis pheromone response plasmids pAD1, pPD1, and pCF10 and primers internal to ash701 of E. faecium pheromone plasmid pHKK701. These findings suggest that pBRG1 of E. faecium LS10 is a sex pheromone response plasmid.

Bacterial Adhesion↗
PubMed2005

A novel engineered peptide, a narrow-spectrum antibiotic, is effective against vancomycin-resistant Enterococcus faecalis.

A novel antienterococcal peptide was prepared by fusing the enterococcal cCF10 pheromone to the channel-forming domain of colicin Ia, forming Enterococcus faecalis pheromonicin (PMC-EF). This peptide was bactericidal against vancomycin-resistant Enterococcus faecalis (VRE) organisms. Electron microscopy and vital dyes confirmed increased membrane permeability. All mice made bacteremic with VRE strains survived when they were treated with PMC-EF, while all controls died.

Anti-Bacterial Agents↗
PubMed2005

Transcriptional response of Enterococcus faecalis V583 to erythromycin.

A transcriptional profile of Enterococcus faecalis V583 (V583) treated with erythromycin is presented. This is the first study describing a complete transcriptional profile of Enterococcus. E. faecalis is a common and nonvirulent bacterium in many natural environments, but also an important cause of nosocomial infections. We have used a genome-wide microarray based on the genome sequence of V583 to study gene expression in cells exposed to erythromycin. V583 is resistant to relatively high concentrations of erythromycin, but growth is retarded by the treatment. The effect of erythromycin treatment on V583 was studied by a time course experiment; samples were extracted at five time points over a period of 90 min. A drastic change in gene transcription was seen with the erythromycin-treated cells compared to the untreated cells. Altogether, 260 genes were down-regulated at one or more time points, while 340 genes were up-regulated. Genes encoding hypothetical proteins and genes encoding transport and binding proteins were the two most dominating groups of differentially expressed genes. The gene encoding ermB (EFA0007) was expressed, but not differentially, which indicated that other genes are important for the survival and growth maintenance of V583 treated with erythromycin. One of these genes is a putative MsrC-like protein, which was up-regulated at all time points studied. Other specific genes that were found to be up-regulated were genes encoding ABC transporters and two-component regulatory systems, and these may be genes that are important for the specific response of V583 to erythromycin.

Anti-Bacterial Agents↗
PubMed2005

Mechanisms of resistance to imipenem and ampicillin in Enterococcus faecalis.

We found ampicillin- and imipenem-resistant isolates of vanA-possessing Enterococcus faecalis with MICs of 8 to 16 microg/ml and 4 to 32 microg/ml, respectively. There have been few reports about penicillin- and imipenem-resistant E. faecalis. Two mechanisms of beta-lactam resistance in E. faecalis, the production of beta-lactamase and the overproduction of penicillin-binding proteins (PBPs), have been reported. The resistant isolates in the current study did not produce any beta-lactamases and analysis of the PBPs showed no overproduction. However, the affinities of PBP4 for beta-lactams in the resistant strains were lower than those of susceptible strains but the affinities of other PBPs for beta-lactams did not change. Accordingly, whole pbp4 fragments from these resistant isolates were sequenced. Two amino acid substitutions at positions 520 and 605 were observed in the highly resistant strains compared to the susceptible ones, Pro520Ser and Tyr605His, and a single Tyr605His amino acid substitution was found in the low-resistance strains. These two point mutations exist in the region between the active-site-defining motifs SDN and KTG of the penicillin-binding domain, the main target of beta-lactams. A strong correlation was seen between these substitutions and decreasing affinities of PBP4 to beta-lactams. In E. faecalis, resistance due to mutations in PBPs has not been reported, though it has in Enterococcus faecium. Our results suggest that development of high-level resistance to penicillins and imipenem depends on point mutations of PBP4 at positions 520 and 605.

Amino Acid Substitution↗
PubMed2006

Impact of Enterococcus faecalis on the bactericidal activities of arbekacin, daptomycin, linezolid, and tigecycline against methicillin-resistant Staphylococcus aureus in a mixed-pathogen pharmacodynamic model.

We inoculated an in vitro pharmacodynamic model simultaneously with clinical isolates of methicillin-resistant Staphylococcus aureus and an enterocin-producing enterococcus (vancomycin-resistant Enterococcus faecalis, ampicillin susceptible) at 7 log10 CFU/ml to examine enterocin effects and antimicrobial activity on staphylococci. The investigated antimicrobial regimens were 100 mg arbekacin every 12 h (q12h), 6 mg daptomycin per kg of body weight/day, 600 mg linezolid q12h, and 100 mg tigecycline q24h alone and in combination (daptomycin, linezolid, and tigecycline) with arbekacin. Simulations were performed in triplicate; bacterial quantification occurred over 48 h, and development of resistance was evaluated throughout. When we evaluated the impact of antimicrobial activity against S. aureus alone, daptomycin demonstrated bactericidal activity (>or=3 log10 CFU/ml kill), whereas arbekacin, linezolid, and tigecycline displayed bacteriostatic activities (<3 log10 CFU/ml kill). In the mixed-pathogen model, early and distinctive stunting of S. aureus growth was noted (1.5 log CFU/ml difference) in the presence of enterocin-producing E. faecalis compared to growth controls run individually (P=0.02). Most noteworthy was that in the presence of enterocin-producing E. faecalis, bactericidal activity was observed with arbekacin and tigecycline and with the addition of arbekacin to linezolid. Antagonism was noted for the combination of tigecycline and arbekacin against S. aureus in the presence of enterocin-producing E. faecalis. Our research demonstrates that the inhibitory effect of E. faecalis contributed significantly to its overall antimicrobial impact on S. aureus. This contribution was enhanced or improved compared to the activity of each antimicrobial alone. Further research is warranted to determine the impact of polymicrobial infections on antimicrobial activity.

Acetamides↗
PubMed2006

Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information between human and animal (chicken) VRE reservoirs and suggest the potential for horizontal transmission of multiple drug resistance, including vancomycin resistance, between farm animals and humans via a pheromone-responsive conjugative plasmid.

Animals↗
PubMed2006

Complete sequence of the enterocin Q-encoding plasmid pCIZ2 from the multiple bacteriocin producer Enterococcus faecium L50 and genetic characterization of enterocin Q production and immunity.

The locations of the genetic determinants for enterocin L50 (EntL50A and EntL50B), enterocin Q (EntQ), and enterocin P (EntP) in the multiple bacteriocin producer Enterococcus faecium strain L50 were determined. These bacteriocin genes occur at different locations; entL50AB (encoding EntL50A and EntL50B) are on the 50-kb plasmid pCIZ1, entqA (encoding EntQ) is on the 7.4-kb plasmid pCIZ2, and entP (encoding EntP) is on the chromosome. The complete nucleotide sequence of pCIZ2 was determined to be 7,383 bp long and contains 10 putative open reading frames (ORFs) organized in three distinct regions. The first region contains three ORFs: entqA preceded by two divergently oriented genes, entqB and entqC. EntqB shows high levels of similarity to bacterial ATP-binding cassette (ABC) transporters, while EntqC displays no significant similarity to any known protein. The second region encompasses four ORFs (orf4 to orf7), and ORF4 and ORF5 display high levels of similarity to mobilization proteins from E. faecium and Enterococcus faecalis. In addition, features resembling a transfer origin region (oriT) were found in the promoter area of orf4. The third region contains three ORFs (orf8 to orf10), and ORF8 and ORF9 exhibit similarity to the replication initiator protein RepE from E. faecalis and to RepB proteins, respectively. To clarify the minimum requirement for EntQ synthesis, we subcloned and heterologously expressed a 2,371-bp fragment from pCIZ2 that encompasses only the entqA, entqB, and entqC genes in Lactobacillus sakei, and we demonstrated that this fragment is sufficient for EntQ production. Moreover, we also obtained experimental results indicating that EntqB is involved in ABC transporter-mediated EntQ secretion, while EntqC confers immunity to this bacteriocin.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4.

A simple two-step procedure was developed to obtain pure enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Chemical and genetic characterization revealed that the primary structure of enterocin 4 is identical to that of peptide antibiotic AS-48 from Enterococcus faecalis S-48. In contrast to the reported inhibitory spectrum of AS-48, enterocin 4 displayed no activity against gram-negative bacteria.

Amino Acid Sequence↗
PubMed1999

Characterization and heterologous expression of the genes encoding enterocin a production, immunity, and regulation in Enterococcus faecium DPC1146.

Enterocin A is a small, heat-stable, antilisterial bacteriocin produced by Enterococcus faecium DPC1146. The sequence of a 10, 879-bp chromosomal region containing at least 12 open reading frames (ORFs), 7 of which are predicted to play a role in enterocin biosynthesis, is presented. The genes entA, entI, and entF encode the enterocin A prepeptide, the putative immunity protein, and the induction factor prepeptide, respectively. The deduced proteins EntK and EntR resemble the histidine kinase and response regulator proteins of two-component signal transducing systems of the AgrC-AgrA type. The predicted proteins EntT and EntD are homologous to ABC (ATP-binding cassette) transporters and accessory factors, respectively, of several other bacteriocin systems and to proteins implicated in the signal-sequence-independent export of Escherichia coli hemolysin A. Immediately downstream of the entT and entD genes are two ORFs, the product of one of which, ORF4, is very similar to the product of the yteI gene of Bacillus subtilis and to E. coli protease IV, a signal peptide peptidase known to be involved in outer membrane lipoprotein export. Another potential bacteriocin is encoded in the opposite direction to the other genes in the enterocin cluster. This putative bacteriocin-like peptide is similar to LafX, one of the components of the lactacin F complex. A deletion which included one of two direct repeats upstream of the entA gene abolished enterocin A activity, immunity, and ability to induce bacteriocin production. Transposon insertion upstream of the entF gene also had the same effect, but this mutant could be complemented by exogenously supplied induction factor. The putative EntI peptide was shown to be involved in the immunity to enterocin A. Cloning of a 10.5-kb amplicon comprising all predicted ORFs and regulatory regions resulted in heterologous production of enterocin A and induction factor in Enterococcus faecalis, while a four-gene construct (entAITD) under the control of a constitutive promoter resulted in heterologous enterocin A production in both E. faecalis and Lactococcus lactis.

Amino Acid Sequence↗
PubMed1999

Atypical genetic locus associated with constitutive production of enterocin B by Enterococcus faecium BFE 900.

A purified bacteriocin produced by Enterococcus faecium BFE 900 isolated from black olives was shown by Edman degradation and mass spectrometric analyses to be identical to enterocin B produced by E. faecium T136 from meat (P. Casaus, T. Nilsen, L. M. Cintas, I. F. Nes, P. E. Hernández, and H. Holo, Microbiology 143:2287-2294, 1997). The structural gene was located on a 2.2-kb HindIII fragment and a 12.0-kb EcoRI chromosomal fragment. The genetic characteristics and production of EntB by E. faecium BFE 900 differed from that described so far by the presence of a conserved sequence like a regulatory box upstream of the EntB gene, and its production was constitutive and not regulated. The 2.2-kb chromosomal fragment contained the hitherto undetected immunity gene for EntB in an atypical orientation that is the reverse of that of the structural gene. Typical transport and other genes associated with bacteriocin production were not detected on the 12.0-kb chromosomal fragment containing the EntB structural gene. This makes the EntB genetic system different from most other bacteriocin systems, where transport and possible regulatory genes are clustered. EntB was subcloned and expressed by the dedicated secretion machinery of Carnobacterium piscicola LV17A. The structural gene was amplified by PCR, fused to the divergicin A signal peptide, and expressed by the general secretory pathway in Enterococcus faecalis ATCC 19433.

Amino Acid Sequence↗
PubMed2000

Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071.

The pH-neutral cell supernatant of Enterococcus faecalis BFE 1071, isolated from the feces of minipigs in Göttingen, inhibited the growth of Enterococcus spp. and a few other gram-positive bacteria. Ammonium sulfate precipitation and cation-exchange chromatography of the cell supernatant, followed by mass spectrometry analysis, yielded two bacteriocin-like peptides of similar molecular mass: enterocin 1071A (4.285 kDa) and enterocin 1071B (3.899 kDa). Both peptides are always isolated together. The peptides are heat resistant (100 degrees C, 60 min; 50% of activity remained after 15 min at 121 degrees C), remain active after 30 min of incubation at pH 3 to 12, and are sensitive to treatment with proteolytic enzymes. Curing experiments indicated that the genes encoding enterocins 1071A and 1071B are located on a 50-kbp plasmid (pEF1071). Conjugation of plasmid pEF1071 to E. faecalis strains FA2-2 and OGX1 resulted in the expression of two active peptides with sizes identical to those of enterocins 1071A and 1071B. Sequencing of a DNA insert of 9 to 10 kbp revealed two open reading frames, ent1071A and ent1071B, which coded for 39- and 34-amino-acid peptides, respectively. The deduced amino acid sequence of the mature Ent1071A and Ent1071B peptides showed 64 and 61% homology with the alpha and beta peptides of lactococcin G, respectively. This is the first report of two new antimicrobial peptides representative of a fourth type of E. faecalis bacteriocin.

Amino Acid Sequence↗
PubMed2003

Purification and characterization of a novel bacteriocin produced by Enterococcus faecalis strain RJ-11.

Lactic acid bacteria exhibiting activity against the gram-positive bacterium Bacillus subtilis were isolated from rice bran. One of the isolates, identified as Enterococcus faecalis RJ-11, exhibited a wide spectrum of growth inhibition with various gram-positive bacteria. A bacteriocin purified from culture fluid, designated enterocin RJ-11, was heat stable and was not sensitive to acid and alkaline conditions, but it was sensitive to several proteolytic enzymes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that enterocin RJ-11 had a molecular weight of 5,000 in its monomeric form. The amino acid sequence determined for purified enterocin RJ-11 exhibited high levels of similarity to the sequences of enterocins produced by Enterococcus faecium.

Amino Acid Sequence↗
PubMed1991

Nucleotide sequence of the gelatinase gene (gelE) from Enterococcus faecalis subsp. liquefaciens.

The gene coding for gelatinase (also called metalloendopeptidase II; microbial proteinase, EC 3.4.24.4) of Enterococcus faecalis subsp. liquefaciens strain OG1-10 was cloned in an Escherichia coli-Enterococcus shuttle vector, and its nucleotide sequence was determined. The DNA sequence encodes one large open reading frame (ORF) with 509 amino acid residues. The ORF contains a signal sequence in its N-terminal region, whereas the N-terminal amino acid sequence determined from the purified extracellular proteinase starts at residue 192 deduced from the ORF. This implies that the gelatinase is synthesized as a prepropolypeptide or prezymogen. The mature gelatinase contains 318 amino acid residues (molecular weight, 34,582) and has significant homology with neutral proteinases from Bacillus species and elastase from Pseudomonas aeruginosa.

Amino Acid Sequence↗
PubMed2000

Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence.

Three agr-like genes (fsrA, fsrB, and fsrC, for Enterococcus faecalis regulator) were found upstream of the previously reported gelatinase gene (gelE) and a downstream putative serine protease gene (sprE; accession number Z12296) of Enterococcus faecalis OG1RF. The deduced amino acid sequence of fsrA shows 26% identity and 38% similarity to Staphylococcus aureus AgrA (the response regulator of the accessory gene regulator system in the agr locus), FsrB shows 23% identity and 41% similarity to S. aureus AgrB, and FsrC shows 23% identity and 36% similarity to S. aureus AgrC (the sensor transducer of Agr system). Northern blot analysis suggested that gelE and sprE are cotranscribed and that fsrB and fsrC are also cotranscribed in OG1RF. Northern blot analysis of fsrA, fsrB, fsrC, gelE, and sprE insertion mutants showed that fsrB, fsrC, gelE, and sprE are not expressed in fsrA, fsrB, and fsrC mutants, while insertion in an open reading frame further upstream of fsrA did not effect the expression of these genes, suggesting that agr-like genes may be autoregulated and that they regulate gelE and sprE expression, as further confirmed by complementation of fsr gene mutations with a 6-kb fragment which contains all three fsr genes in the shuttle vector, pAT18. Testing of 95 other isolates of E. faecalis showed that 62% produced gelatinase (Gel(+)), while 91% (including all Gel(+) strains) hybridized to a gelE probe; 71% (including all Gel(+) strains) hybridized to an fsr probe, corroborating the conclusion that both gelE and fsr are necessary for gelatinase production. Testing of fsrA, fsrB, and sprE mutants in a mouse peritonitis model showed that sprE and agr-like gene mutants resulted in highly significantly prolonged survival compared to the parent strain OG1RF, a finding similar to what we had previously shown for a gelE mutant. These results suggest that sprE and agr-like genes contribute to the virulence of E. faecalis OG1RF in this model.

Amino Acid Sequence↗
PubMed2025

The adjacent ATP-binding protein-encoding genes of the Enterococcus faecalis phosphate-specific transport (pst) locus have non-overlapping cellular functions.

UNLABELLED: The widely conserved pst-phoU operon encodes a low-velocity, high-affinity, ATP-dependent importer for inorganic phosphate (Pi). The pstB gene encodes the ATPase that powers the import of Pi into the cell. In some Firmicutes, including the gastrointestinal commensal and opportunistic pathogen Enterococcus faecalis, the pst-phoU locus contains adjacent pstB genes. In this work, we compared the functionality of E. faecalis pstB1 and pstB2. E. faecalis pstB1 and pstB2 share sequence similarities with verified PstB ATPases from Escherichia coli and Streptococcus pneumoniae and only share ~60% amino acid identity with each other. Deletion of pstB1 was associated with a growth defect in low Pi-containing chemically defined medium (CDM), reduced Pi uptake, and a moderate increase in alkaline phosphatase (AP) activity. Deletion of pstB2 fully inhibited growth in CDM regardless of inorganic phosphorus source but did not hinder growth in rich, undefined medium. The &#x394;pstB2 mutant also exhibited a significant increase in AP activity that was associated with extracellular Pi accumulation. Overexpression of pstB2 in the pstB1 mutant was sufficient to restore growth in low-Pi CDM, Pi uptake, and AP activity, but this was not recapitulated with overexpression of pstB1 in the &#x394;pstB2 mutant. Deletion of either pstB paralog increased expression of the tandem paralog, and overexpression of pstB2 in &#x394;pstB2 reduced pstB1 expression. These results suggest that the E. faecalis pstB2-encoded ATPase is required for Pi import, while the pstB1-encoded ATPase has an accessory role in Pi import that can be duplicated by the presence of excess PstB2. IMPORTANCE: Phosphate is critical for all microbial life. In many bacteria, inorganic phosphate (Pi) is imported by the high-affinity, low-velocity Pst-PhoU system. The pstB gene encodes the ATPase that powers Pi import. The pst-phoU operon in many Firmicutes, including the human commensal and opportunistic pathogen Enterococcus faecalis, contains adjacent pstB genes, pstB1 and pstB2. No studies on the relative biological contributions of tandem pstB paralogs in any microbe have been published. This genetic study indicates that E. faecalis pstB1 and pstB2 do not have equivalent functions. The pstB2 gene encodes an ATPase that is required for Pi import, while the ATPase encoded by pstB1 has an accessory role in Pi import that can be duplicated by the presence of excess PstB2.

Enterococcus faecalis↗
PubMed2006

Characterization of the ends and target site of a novel tetracycline resistance-encoding conjugative transposon from Enterococcus faecium 664.1H1.

Enterococcus faecium 664.1H1 is multiply antibiotic resistant and mercury resistant. In this study, the genetic support for the tetracycline resistance of E. faecium 664.1H1 was characterized. The tet(S) gene is responsible for tetracycline resistance, and this gene is located on the chromosome of E. faecium 664.1H1, on a novel conjugative transposon. The element is transferable to Enterococcus faecalis, where it integrates into a specific site. The element was designated EfcTn1. The integrase of EfcTn1 is related to the integrase proteins found on staphylococcal pathogenicity islands. We show that the transposon is flanked by an 18-bp direct repeat, a copy of which is also present at the target site and at the joint of a circular form, and we propose a mechanism of insertion and excision.

Amino Acid Sequence↗