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Antimicrobial Agents and Chemotherapy, February 2009, p. 735-747, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00754-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac){triangledown}

Bo Zheng,1,3 Haruyoshi Tomita,1* Takako Inoue,1 and Yasuyoshi Ike1,2

Department of Bacteriology,1 Laboratory of Bacterial Drug Resistance, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan,2 Institute of Clinical Pharmacology, Peking University First Hospital, Beijing 10034, China3

Received 9 June 2008/ Returned for modification 18 July 2008/ Accepted 18 November 2008


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ABSTRACT
 
Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10–4 per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOBMG family, which is found in pheromone-independent plasmid pHTβ of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.


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INTRODUCTION
 
Multiple-drug-resistant enterococci and vancomycin-resistant enterococci (VRE), in particular, are opportunistic pathogens and major causes of nosocomial infections in immunocompromised patients (4, 7, 44). The isolation of VRE (VanA type) was first reported in 1988 in the United Kingdom (67) and France (41), and shortly thereafter it was reported in the United States (55). Since then, VRE have been identified in many countries. VRE have caused an increasing number of treatment-related problems, especially in the United States (4, 43), where they are estimated to account for about 15% of nosocomial enterococcal isolates (15). In Asia, VRE have been isolated from hospitalized patients or food animals in China, Japan, South Korea, Taiwan, and Thailand (49, 74). In particular, they have frequently been isolated in South Korea (72) and Taiwan (40). Since the first report of the isolation of VanA-type VRE from a patient in Japan, VRE have been isolated from both sporadic individual cases and outbreaks of nosocomial infections in several hospitals (29, 49). However, an outbreak of VRE nosocomial infection is still a very rare event in university teaching hospitals in Japan.

VRE isolates of the VanA and VanB types are the most commonly identified VRE isolates to be acquired. Their genomes are composed of operon gene clusters, and isolates of the VanA and VanB types have the same basic mechanism of resistance (12). The VanA-type determinant is encoded on the Tn1546 transposon or a Tn1546-like transposon (2), which frequently resides on a conjugative plasmid in VanA-type Enterococcus faecium (41). The vanB gene has been divided into three subtypes, vanB1, vanB2, and vanB3, on the basis of differences in the sequence of the vanB ligase (13, 22, 50). The vanB2 determinant is encoded on conjugative transposon Tn1549 (34 kb) (30) and the closely related transposon Tn5382 (27 kb) (3), which have similarities with the Tn916 family of conjugative transposons (10, 26). The transposable elements can be located on a conjugative or a nonconjugative plasmid or on the chromosome (3, 30, 53, 54). To our knowledge, there has been no report of a vanB determinant located on the pheromone-responsive highly conjugative plasmid.

The first outbreak of a VRE nosocomial infection in Japan was caused by a VanB-type Enterococcus faecalis strain in a hospital setting in July 1999. Twenty VanB-type E. faecalis isolates were obtained from three clinical specimens, nine rectal swab specimens from asymptomatic carriers, and eight swab specimens from the hospital environment and were examined for drug resistance by pulsed-field gel electrophoresis (PFGE) (47). Southern blot analysis of the PFGE gel with a vanB probe implied that the VanB-type determinants resided on a 110-kbp plasmid in 19 strains obtained from among the 20 isolates (47). As described in this report, we examined the plasmids carried by the VanB-type VRE and identified two pheromone-responsive plasmids: one plasmid encoding vancomycin resistance and a bacteriocin and the other plasmid encoding erythromycin resistance and cytolysin.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and media. The bacterial strains and plasmids used in this study are listed in Table 1 and Table 2. Of the 18 isolates studied, the results of PFGE and Southern hybridization analysis with the vanB probe for 11 isolates (i.e., isolates NKH1 to NKH7 and NKH15 to NKH18) have been described in a previous study (47). The E. faecalis strains were grown in brain heart infusion broth and agar (Difco Laboratories) or Todd-Hewitt broth (Difco Laboratories) at 37°C. Escherichia coli strains were grown in Luria-Bertani medium (GIBCO BRL, Life Technologies). The following antibiotics were used at the indicated concentrations for the selection of E. faecalis: erythromycin, 12.5 µg ml–1; streptomycin, 250 µg ml–1; spectinomycin, 250 µg ml–1; chloramphenicol, 20 µg ml–1; rifampin (rifampicin), 25 µg ml–1; and fusidic acid, 25 µg ml–1. The following antibiotics were used at the indicated concentrations for the selection of E. coli: ampicillin, 100 µg ml–1, and chloramphenicol, 50 µg ml–1. All antibiotics were obtained from Sigma Chemical Co. 5-Bromo-4-chloro-3 indolyl-β-D-galactopyranoside was used at 40 µg ml–1.


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  		TABLE 1. Bacterial strains and plasmids used in this study


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  		TABLE 2. VanB-type vancomycin-resistant first outbreak E. faecalis strains isolated from a Japanese hospital

Antimicrobial susceptibility testing. The MICs of the antibiotics were determined by the agar dilution method. An overnight pure culture of each strain grown in Mueller-Hinton broth (Nissui, Tokyo, Japan) was diluted 100-fold with fresh broth. An inoculum of approximately 5 x 105 cells was plated on a series of Mueller-Hinton broth (Eiken, Tokyo, Japan) cultures containing a range of concentrations of the test drug. The plates were incubated at 37°C, and the susceptibility results were finalized at 24 h of incubation. Susceptibility testing and interpretation of the results were in compliance with standards recommended by Clinical and Laboratory Standards Institutes (formerly NCCLS). E. faecium ATCC 9790 was used as a control strain.

Soft agar assay for bacteriocin production and immunity. The bacteriocin production assay was performed as described previously (36, 62). The test for immunity to the bacteriocin was performed essentially as described previously (36).

Plasmid and DNA methodology. Recombinant DNA techniques, analyses of plasmid DNA with restriction enzymes, and agarose gel electrophoresis were carried out by standard methods (56). The introduction of plasmid DNA into bacterial cells was carried out by electrotransformation, as described previously (27). Plasmid DNA was purified from E. faecalis as described previously (68). Restriction enzymes were purchased from New England Biolabs and Roche Co. PCR was performed with a Perkin-Elmer Cetus apparatus. Taq DNA polymerase was obtained from Takara.

DNA sequence analysis. Sequence analysis was performed with a Dye primer and a Dye Terminator cycle sequencing kit (Applied Biosystems) and with a 377 DNA sequencer and 310 gene analyzer (ABI Prism). To determine the DNA sequence of plasmid pMG2200, a shotgun cloning method was used (56). To determine the DNA sequences in the gap regions, PCR amplification was performed to obtain PCR products covering the gaps. The PCR products were sequenced directly by using custom primers. Open reading frames (ORFs) were identified and initially analyzed with Genetyx (version 5.1) computer software and the BLAST database to search for putative genes (1).

Conjugation experiments. Filter mating was performed as described previously (16, 37). Broth mating was carried out for 4 h. Transfer frequencies were expressed as the number of transconjugants per donor cell (at the end of mating).

Pheromone response (clumping) assay. Pheromone response assays were performed as described previously (18). The synthetic enterococci pheromones cAD1, cCF10, cPD1, cOB1, and cAM373 were prepared by Sawaday Technology Co., Ltd. (Tokyo, Japan).

Identification and genetic analyses of the oriT region of the pMG2200 plasmid. The amplified DNAs were cloned into the pAM401 vector plasmid. The oligonucleotides used as PCR primers were V43622F and V43943R, respectively (Table 3). Each of the pAM401 derivatives carrying pMG2200 segments to be tested for oriT activity was introduced by electrotransformation into E. faecalis UV202, which is defective in homologous recombination (63, 71). Conjugative plasmid pMG2200 was then introduced into each of the transformants carrying the pAM401 derivative (Cmr) by conjugation. Both broth matings and filter matings were performed with the transconjugants carrying the two plasmids as donor strains and JH2SS as the recipient strain.


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  		TABLE 3. Sequences of oligonucleotides used in the study

Southern hybridization analysis. Southern hybridization was performed with the digoxigenin-based nonradioisotope system of Boehringer GmbH (Mannheim, Germany), and all procedures were based on the manufacturer's manual and standard protocols (56). Plasmid pMG326 was used as the probe, as it contains the regulatory region of pheromone plasmid pPD1 (58).

Detection of the cytolysin (Hly/Bac) genes in the VRE isolates. To detect the cytolysin (Hly/Bac) gene encoded on the pAD1-like plasmid (31, 32, 34, 35, 36), PCR amplification with primer sets specific for the cyl genes cylLL, cylLS, cylA, and cylB was performed as described in the literature (20, 57).

PFGE. PFGE was carried out in a 1% agarose gel with 0.5% Tris-borate-EDTA buffer; and the following settings were applied: 1 to 23 s, 6 V/cm, and 22 h (with the CHEF Mapper system [Bio-Rad]) (49).

Nucleotide sequence accession number. The nucleotide sequence data reported in this article are available from the DDBJ, EMBL, and GenBank nucleotide sequence databases under accession number AB374546.


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RESULTS AND DISCUSSION
 
Antimicrobial susceptibilities of the VRE isolates. The MICs of the various antimicrobial agents used to test the 18 VRE isolates are shown in Table 2. All of the VRE isolates showed high levels of resistance to vancomycin (MICs, 32 to 256 µg/ml) and susceptibility to teicoplanin (MIC, 0.125 to 0.25 µg/ml). There were high levels of resistance to the aminoglycosides gentamicin and kanamycin and to tetracycline. Of the 18 VRE isolates, 12 isolates had high levels of resistance to erythromycin, and the remaining 6 isolates were susceptible to erythromycin.

PFGE analysis of SmaI-digested total DNA from the 18 VRE isolates showed that there were two PFGE patterns which differed with regard to the positions of two bands in the lower portion of the gels (data not shown); these observations are indicative of differences in plasmid contents (Fig. 1). These data indicate that the strains were identical but that the identical host strains contained different plasmids.


Figure 1
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  		FIG. 1. Agarose gel electrophoresis of restriction endonuclease-digested DNA of pMG2200 and pMG2201 and Southern hybridization with genes specific from the pheromone-responsive plasmid. Agarose gel electrophoresis of EcoRI-digested plasmid DNA (A) and Southern hybridization with pMG326 (58) (B). Lanes: 1, HindIII-digested bacteriophage lambda DNA; 2, wild-type strain NKH15; 3, pMG2200; 4; pMG2201; 5, pAD1.

Bacteriocin production. All of the 18 isolates showed bacteriocin activity against the E. faecalis strain among the indicator strains examined (Table 2). Of the 18 isolates, the 12 isolates that were resistant to erythromycin also showed cytolysin activity.

Conjugative experiments with drug resistance. The conjugative transfer of each of the vancomycin, erythromycin, gentamicin, and tetracycline resistance determinants from each of the VRE isolates to E. faecalis FA2-2 or E. faecium BM4105RF was examined by broth mating for 4 h or filter mating for 18 h at 37°C. The vancomycin resistance of the 18 isolates and the erythromycin resistance of the 12 isolates transferred to E. faecalis FA2-2 at frequencies of about 10–3 to 10–5 per donor cell in the broth mating experiments. Resistance to the other drugs was not transferred to E. faecalis FA2-2 at a detectable frequency (less than 10–8 per donor cell), even by filter mating, suggesting that resistance to the other drugs might be encoded on a nonconjugative plasmid(s) or the chromosome. The transconjugants of each strain were examined for their drug resistance and bacteriocin production. The vancomycin- and erythromycin-resistant transconjugants showed both bacteriocin and cytolysin activities, the vancomycin-resistant transconjugants showed only bacteriocin activity, and the erythromycin-resistant transconjugant showed only cytolysin activity.

The EcoRI restriction profiles of the plasmids found in the vancomycin-resistant transconjugants and the erythromycin-resistant transconjugants indicated that the plasmid found in each particular group was identical. Further plasmid analysis showed that the vancomycin-resistant transconjugants carried a 106.5-kbp plasmid and the erythromycin resistant transconjugants carried a 65.7-kbp plasmid (Fig. 1A).

The 106.5-kbp plasmid harbored by the vancomycin-resistant transconjugant and the 65.7-kbp plasmid harbored by the erythromycin-resistant transconjugant derived from the representative E. faecalis NKH15 strain were designated pMG2200 and pMG2201, respectively (Fig. 1A). pMG2200 conferred vancomycin resistance and bacteriocin production on the strain, and the bacteriocin was active only against E. faecalis strains. Bacteriocin 41 (Bac41), which was encoded on pheromone-responsive plasmid pYI14 isolated from bacteriocingenic strain E. faecalis YI714, was also active only against E. faecalis (61, 64). Strain OG1S carrying pMG2200 did not exhibit bacteriocin activity against E. faecalis FA2-2 carrying pYI14, and vice versa. These results indicated that the bacteriocin encoded on pMG2200 was identical to Bac41 with respect to its immunity characteristics. pMG2201 conferred erythromycin resistance and cytolysin activity (β-hemolysin/bacteriocin). Plasmid pMG2201 gave rise to the expected PCR product for the cytolysin genes (cylLL, cylLS, cylA, and cylB) of pAD1 by PCR analysis with primers specific for the genes, indicating that pMG2201 encoded the cytolysin (data not shown) (20, 36, 57).

Pheromone responses of pMG2200 and pMG2201. Donor cells of E. faecalis JH2SS carrying pMG2200 or pMG2201 were exposed for 2 h to either FA2-2 culture filtrate (i.e., pheromone) or synthetic pheromone cAD1, cPD1, cCF10, cOB1, or cAM373 (at a concentration of 100 ng/ml) for pheromone-responsive plasmids pAD1 (59.3 kb), pPD1 (58.9 kb), pCF10 (67.7 kb), pOB1 (64.7 kb), and pAM373 (36.7 kb), respectively, to induce the aggregation-mating function before a short (10-min) mating period (9). The short mating was carried out between the induced or noninduced donor cells and the plasmid-free recipient E. faecalis FA2-2. Plasmids pMG2200 and pMG2201 both responded to the FA2-2 filtrate and they responded to cCF10 and cAD1, respectively, indicating that pMG2200 and pMG2201 are identical to the pheromone-responsive plasmids pCF10 and pAD1, respectively, with respect to their pheromone responses (data not shown). Southern hybridization analysis with the 7-kbp probe specific for the conserved pheromone-responding genes of pPD1 (i.e., traC, traB, traA, ipd, traE, traF, orfY, sea1, and a part of asp1) indicated that both pMG2200 and pMG2201 contained the homologous genes shown in Fig. 1B (28, 58).

DNA sequence and genetic organization of plasmid pMG2200. The complete nucleotide sequence of pMG2200 was determined, and its molecular size was confirmed to be 106,527 bp (Table 4 and Fig. 2). The first G residue within an EcoRI site (GAATTC) was chosen as the first nucleotide of pMG2200, as shown in Fig. 2. All ORFs listed in Table 4 are numbered in relation to this nucleotide. The pMG2200 plasmid carries the conjugative transposon Tn1549-like element (33,812 bp) encoding VanB2-type vancomycin resistance, and it is located between 8,580 bp and 42,391 bp in the clockwise orientation of the plasmid map (Fig. 2). The Tn1549-like element of pMG2200 contained 29 ORFs which were almost identical to the 29 ORFs of Tn1549 (34 kbp) in pIP834, which is found in E. faecalis E93/268 (10, 30). The 18 ORFs from ORF13 to ORF30 that are located at the left-end extremity and that are aligned in the order identified in pMG2200 were completely identical to the 18 ORFs of Tn1549 (30), with the exception of ORF16 (99% amino acids identity). The Tn1549-like element contained the vanRB, vanSB, vanYB, vanWB, vanHB, vanB2, and vanXB genes, which correspond to the seven equivalent genes in the VanB2 operon of Tn1549 (30). The deduced amid acid sequence of VanB2 of the Tn1549-like element was almost identical to the deduced amid acid sequence of VanB2 of Tn1549 at a level of 99% amino acid identity. The ORFs from ORF46 to ORF57 in an approximately 6.2-kbp region running from 48,656 bp to 54,841 bp of the map and ORF79 to ORF108 in the approximately 30.55-kbp region running from 72,694 bp to 103,247 bp of the map showed a level of homology of 80 to 100% amino acid identity with the genes or the ORFs found in the pheromone-responding plasmids (pAD1, pCF10, pPD1, pAM373, and pTEF2) (5, 9, 14, 24, 28, 51). These regions contained ORFs that correspond to the ORFs pcfJ, pcfK, pcfL, pcfM, pcfN, pcfP, pcfQ, pcfR, pcfS, pcfT, pcfU, pcfY, and pcfZ of pCF10; the UV resistance genes uvrC, uvaB, uvrB, uvrA, uvaE, and uvaF (orfB, orfC) of pCF10 or pAD1; the plasmid maintenance genes (plasmid partition and replication) par and rep of pTEF2; and prgN, prgZ, prgY, prgX, and prgQ of pCF10 (33, 48, 51). Like the ORFs or genes in pCF10, these ORFs align in this order in pMG2200. ORF94 to ORF97 were identical to prgZ, prgY, prgX, and prgQ, respectively, which are the pheromone-responding regulatory genes that allow the cell surface receptor to take up exogenous pheromone, that shut down pheromone production or reduce endogenous pheromone levels, that act as the pheromone receptor and negative regulator for the downstream genes of prgQ, and that act as the pheromone inhibitor, respectively (19). The ORFs downstream of the regulatory genes, ORF98 to ORF108, were similar to those found in other pheromone-responsive plasmids, such as pAD1 and pPD1. ORFs 98, 99, 100, 102, and 103 were highly homologous to TraE1, OrfY, Sea1, Orf1, and Asa1 of pAD1, respectively. The deduced ORF98 protein showed 100% amino acid identity with TraE1 of plasmid pAD1, which is a key positive regulator for the pheromone-responsive plasmid. In contrast, the other regulatory genes corresponding to prgZ, prgY, prgX, and prgQ of plasmid pCF10 showed a high level of homology with the equivalent genes in plasmid pCF10. Like the pheromone-responsive plasmids, there were two inverted repeat sequences (IRS1 and IRS2) in the noncoding region between ORF97 (prgQ) and ORF98 (traE1) that stopped the transcript from the promoter region of the pheromone inhibitor of prgQ in the case of pCF10 (60). The sequence of the upstream region of IRS2 was identical to the sequence in pCF10, and the sequence of the downstream region of IRS2 was identical to the sequence in pAD1. Ten ORFs from ORF59 to ORF68 showed a high level of homology with genes in the region of the Bac41 determinant, which is active against E. faecalis, and consisted of bacL1, bacL2, bacA, and bacI, which are encoded on pheromone-responsive plasmid pYI14, which has been reported to be a novel bacteriocin for cell wall lysis found in E. faecalis YI714 (64). E. faecalis OG1S harboring pMG2200 showed resistance (immunity) to Bac41, indicating that pMG2200 encodes a Bac41-like bacteriocin.


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  		TABLE 4. ORFs identified in pMG2200


Figure 2
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  		FIG. 2. Genetic map of pMG2200. The open arrows show the ORFs and the direction of transcription. Each color indicates significant homology with a reported plasmid or mobile element. Representative homologous genes are indicated on the ORFs (Table 2). The first G residue within an EcoRI site (GAATTC) on the map indicates the first nucleotide of pMG2200, as shown in Table 4.

Cloning and genetic analysis of oriT region of pMG2200. The transfer origin (oriT) is thought to be characteristic of the conjugative plasmid and is essential for the transfer of the transferable or mobile element (25). Known oriT regions are classified into several groups on the basis of sequence similarities (73). No sequence that was identical or similar to the known oriT regions of conjugative plasmids in gram-positive bacteria was found in the pMG2200 sequences. It is characteristic of the oriT region that direct repeat sequences flank the oriT site and that the oriT sites are present within inverted repeat sequences (25, 73). In the noncoding region between ORF41 and ORF42, multiple direct repeats and two inverted repeats were found (Fig. 3). The direct repeats were composed of 14 copies of 5-bp TGCTA sequences. The 14 direct repeats (DR-1 to DR-14) were located several base pairs away (from 5 to 7 bp) from each other. The inverted repeats were composed of GCCTTGCA/TGCAAGGC (IR-1) and GGGTCAG/CTGACCC (IR-2).


Figure 3
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  		FIG. 3. Nucleotide sequence of the oriT region of plasmid pMG2200. The 332-bp noncoding DNA region between ORF41 and ORF42 is shown. The horizontal arrows under the sequences indicated the direct repeats TGCTA (DR-1 to DR-14) and inverted repeats (IR-1 and IR-2) in the oriT region. The names and the locations of the oligonucleotide primers used for the analysis of the oriT region are shown on the sequence with the right-angled arrows. The complementary sequence corresponding to 3'-GTCGAA-5' shows the possible nick site. The italicized characters in the 178-bp segment mapped between 43,733 bp and 43,910 bp indicate the sequences identical to the sequence found in plasmid pAM{alpha}1 (from positions 3618 to 3795 bp on the plasmid).

To identify the functional oriT region of pMG2200, a 322-bp segment containing a potential candidate for the oriT region was cloned into pAM401 (Cmr), and the resultant plasmid was designated pMG2210 (Fig. 3). pMG2210 (Cmr) containing the 322-bp region was mobilized by pMG2200 (Vmr) and transferred at the same frequency as the parent plasmid (data not shown). The results showed that the noncoding region between ORF41 and ORF42 is the functional oriT region of pMG2200.

The internal 178-bp segment within the oriT region located between 43,733 bp and 43,911 bp of the map showed significant homology (more than 80%) with the region from 3618 to 3795 bp of plasmid pAM{alpha}1 and the region from 2078 to 1901 bp of plasmid pS86; however, these regions were not related to the oriT regions of these plasmids (Fig. 3) (23, 42). Both pAM{alpha}1 (9.8 kb) and pS86 (5.2 kb) are nonconjugative but mobilizable plasmids found in E. faecalis (17, 42). Plasmids pMG2200, pAM{alpha}1, and pS86 each had seven copies of the 5-bp direct repeat sequences (TGCTA) and two inverted repeat sequences in the homologous regions.

Putative DNA relaxase/nickase gene ORF44. In addition to the oriT sequence, the relaxase/nickase is an important feature of conjugative plasmids that is essential for the initiation of DNA transfer (25, 73). ORF44, which encoded a 686-amino-acid protein, showed a significant level of similarity to the predicted relaxase/nickase gene traI (ORF34) of plasmid pHTβ isolated from a vancomycin-resistant E. faecium strain (63, 66). The three conserved motifs (motifs I to III) of the DNA relaxase were found in the N-terminal portion (i.e., about 377 amino acids) of the deduced ORF44 protein (Fig. 4). Motif I contained the catalytic Tyr residue involved in DNA cleavage-joining activity. Motif II was reported to be involved in DNA-protein contacts through the 3' end of the nick region, and a Ser residue is usually present. Motif III contains three conserved His residues and is known as the His3 motif. It has been suggested that the His residues aid with the nucleophilic activity of the Tyr residue in motif I, coordinate the required Mg2+ ions, and direct the activation of the active Tyr. These three motifs are thought to form part of the catalytic center of the relaxase (25, 72).


Figure 4
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  		FIG. 4. Comparison of the N-terminal region of the deduced ORF44 protein of pMG2200 with putative relaxases found in sequence databases. The boldface letters indicated the amino acid residues conserved in each protein. The asterisks on the sequences show the key residues, Tyr, Ser, and His3 (3His), in motifs I, II, and III, respectively. There are two motif III candidates (motifs Ia and Ib) in the most of the proteins. The GenBank accession numbers of the putative relaxases are as follows: for pXO2, NZ_ABJC01000063.1; for pCP13, NC_003042.1; for ATCC 12228, NC_004461.1; for NEM316, NC_004368.1; for pLI100, NC_003383.1; for pHTβ, NC_007594.1.

The conserved His3 sequence in motif III of the ORF44 of pMG2200 belonged to the MOBMG family [W(x4)H(x2)T(x3)HxH(x4)E(x4)R, where uppercase letters represent conserved amino acids and "x" indicates the variable residues] found in TraI of pHTβ (Fig. 4) (63).

Pheromone-specific plasmid transfers. The specific pheromone induces transfer of the corresponding plasmid. The transfer of pMG2200 was induced by the pheromone cCF10. Nucleotide sequence analysis of pMG2200 revealed that the deduced pheromone receptor and negative regulatory gene (i.e., ORF99 [prgX]) was identical to the prgX gene, a key negative regulator of pCF10, which is responsive to cCF10. pMG2200 encoded the deduced positive regulatory gene (i.e., ORF101 [traE1]) that is identical to traE1 of pAD1, which is derepressed by cAD1. The traE1 gene product, the E-region product(s), positively regulates the structural transfer genes, including the aggregation substance gene (asa1), downstream of traE1. The molecular mechanism of the regulation by traE1 has not been elucidated. There are reports that traE1 acts in trans (8, 45). Another report shows that traE1 acts as a cis element in gene regulation (46). All of the analyses were performed under artificial conditions with cloned elements of the regions on multicopy vector plasmids; thus, these activities might differ from the activity in the wild-type plasmid. pMG2200 was a naturally occurring chimeric plasmid, as described above. Its pheromone receptor was identical to PrgX of pCF10, and its positive regulator was identical to TraE1 of pAD1. Using plasmid pMG2200, we examined whether the traE1 gene regulated the plasmid transfer in a trans or a cis manner. We constructed a donor strain harboring two plasmids, pMG2200 (Vamr) and pAM714 (pAD1::Tn917 [Emr], a derivative of pAD1 showing the wild-type pheromone-response and transfer). Plasmids pMG2200 and pAM714 had different pheromone receptors, and the pheromones were PrgX for cCF10 and TraA for cAD1, but both plasmids encoded an identical positive regulator, the traE1 gene (E region). If the traE1 gene product regulates the expression of structural genes in a trans manner, either cCF10 or cAD1 would induce the expression of the traE1 gene encoded on a plasmid, and TraE1 would then positively regulate the expression of both structural genes encoded on the two plasmids, which would result in the transfer of both pMG2200 and pAM714 in the cell.

After pheromone induction by either cCF10 or cAD1, a short mating experiment between JH2SS(pMG2200, pAM714) and FA2-2 was performed, as described in Materials and Methods. After incubation with the pheromones, cell aggregates (clumping) were observed, indicating that the aggregation substance gene(s) was expressed in the cell. cCF10 induced only the transfer of pMG2200 (2.6 x 10–6 per donor cell) and did not induce the transfer of pAM714 (<1.1 x 10–8 per donor cell). cAD1 induced only the transfer of pAM714 (2.4 x 10–5 per donor cell) and not that of pMG2200(<1.1 x 10–8 per donor cell). This result implies that TraE1 acted in a cis manner for plasmid transfer. In the case of pCF10, it is considered that the small gene products (RNA molecules; i.e., PrgR and PrgS) of pCF10, which are located on the equivalent region of traE1 in pAD1, regulate the downstream structural genes in a cis manner by an unknown mechanism (6). A similar mechanism might exist in pAD1 and pMG2200, which both contain the traE1 gene. It is notable that the previous data showed that Tn917-lac insertion mutants in the noncoding downstream region of traE1 of pAD1 resulted in a defect in plasmid transfer and mating aggregates and that this region was genetically determined and mapped as the positive regulatory E region on pAD1 prior to the determination of the traE1 gene by nucleotide sequence analysis (21, 52, 69). There is no current explanation for the transfer-deficient Tn917-lac insertion mutants (i.e., pAM7314, pAM7330, and pAM2125) of the 3' noncoding region in the E region (52). The 3' terminal border of the E region is mapped by the Tn917-lac insertion of pAM2125 and is located 371 bp from the stop codon of traE1 (unpublished data). Further analyses of pMG2200 might provide clues that will allow elucidation of the regulation of the pheromone-responsive plasmids.

Concluding comments. The two pheromone-responsive conjugative plasmids pMG2200 (106.5 kbp) and pMG2201 (65.7 kbp) were isolated from VanB2-type E. faecalis isolates. This report describes the first case of the isolation and characterization of pheromone-responsive conjugative plasmid pMG2200 encoding the vanB resistance determinant. pMG2200 encoded vancomycin resistance and bacteriocin and responded to pheromone cCF10, and pMG2201 encoded erythromycin resistance and cytolysin (Hly/Bac) and responded to pheromone cAD1. Our results show that an E. faecalis strain can acquire these characteristics and that these characteristics provide a selective advantage for the organism by allowing it to obtain the pheromone-responsive plasmids encoding drug resistance or bacteriocins by conjugation with plasmid-bearing bacteria in patients in the clinical setting. The plasmid also conferred the cytolysin (Hly/Bac) function for pathogenesis (38, 39). The complete nucleotide sequence of pMG2200 showed that pMG2200 consists of five major segments: (i) conjugative transposon Tn1549-like elements (33,812 bp) encoding the vanB2-type determinant, (ii) genes that regulate the pheromone response of pheromone-responsive plasmids, (iii) genes for UV resistance, (iv) the bacteriocin determinant, and (v) the origin of plasmid transfer. The genes corresponding to the pheromone-responsive regulatory genes, with the exception of the positive regulator traE1 of plasmid pAD1, showed high levels of homology (100% amino acid identity) to those of pCF10. The data indicated that pMG2200 is a new type of pheromone-responsive plasmid which is a naturally occurring chimeric plasmid with regard to the negative regulatory gene prgX (prgQ) of pCF10 and the positive regulatory gene traE1 of pAD1, resulting in a prgX-prgQ-traE1 genetic organization. Using the chimeric plasmid, we showed that traE1 is cis acting. The nucleotide sequence of the plasmid origin showed a high level of homology to a region within plasmid pAM{alpha}1 of E. faecalis that is unrelated to the oriT region of pAM{alpha}1, and the ORF corresponding to the putative relaxase showed homology to that of E. faecium conjugative plasmid pHTβ (61, 64). These results indicate that the diversity within the genetic organization of housekeeping genes, such as the regulatory regions, origin of transfer, and plasmid replication in the pheromone-responsive plasmids, could result from genetic recombination between different pheromone-responsive plasmids or between a pheromone-responsive and a non-pheromone-responsive plasmid.

To our knowledge, there has been only one report on sequence analysis of the conjugative transposon Tn1549 encoding the VanB gene cluster (30). The conjugative transposon Tn1549-like element encodes a vanB2-type resistance determinant that is almost completely identical to the Tn1546 transposon residing on pIP834 of E. faecalis E93/268 (30). There has been no report to date of a pheromone-responsive highly conjugative plasmid carrying the Tn1549-like element encoding the VanB gene cluster. Our report is the first to describe a pheromone-responsive plasmid carrying the Tn1549-like element encoding the VanB2 gene cluster.


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ACKNOWLEDGMENTS
 
This work was supported by grants from the Japanese Ministry of Education, Culture, Sport, Science and Technology (Tokutei-ryoiki [Matrix of Infection Phenomena], Kiban [B], Kiban [C]) and the Japanese Ministry of Health, Labor and Welfare (H18-Shinko-11).

We thank E. Kamei for helpful advice on the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Bacteriology, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan. Phone: 81-27-220-7990. Fax: 81-27-220-7996. E-mail: tomitaha{at}med.gunma-u.ac.jp Back

{triangledown} Published ahead of print on 24 November 2008. Back


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Antimicrobial Agents and Chemotherapy, February 2009, p. 735-747, Vol. 53, No. 2
0066-4804/09/$08.00+0     doi:10.1128/AAC.00754-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.




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