ICESluvan, a 94-Kilobase Mosaic Integrative Conjugative Element Conferring Interspecies Transfer of VanB-Type Glycopeptide Resistance, a Novel Bacitracin Resistance Locus, and a Toxin-Antitoxin Stabilization System

ABSTRACT A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis consists of a mosaic of genetic fragments from different Gram-positive bacteria. A variant of ICESluvan was confirmed in S. lutetiensis from a patient. A complete Tn5382/Tn1549 with a vanB2 operon is integrated into a streptococcal ICESde3396-like region harboring a putative bacteriophage exclusion system, a putative agglutinin receptor precursor, and key components of a type IV secretion system. Moreover, ICESluvan encodes a putative MobC family mobilization protein and a relaxase and, thus, in total has all genetic components essential for conjugative transfer. A 9-kb element within Tn5382/Tn1549 encodes, among others, putative proteins similar to the TnpX site-specific recombinase in Faecalibacterium and VanZ in Paenibacillus, which may contribute to the detected low-level teicoplanin resistance. Furthermore, ICESluvan encodes a novel bacitracin resistance locus that is associated with reduced susceptibility to bacitracin when transferred to E. faecium. The expression of a streptococcal pezAT toxin-antitoxin-encoding operon of ICESluvan in S. lutetiensis, E. faecium, and E. faecalis was confirmed by reverse transcription (RT)-PCR, indicating an active toxin-antitoxin system which may contribute to stabilizing ICESluvan within new hosts. Junction PCR and DNA sequencing confirmed that ICESluvan excised to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium. Transfer between E. faecalis cells was observed in the presence of helper plasmid pIP964. Sequence analysis of the original S. lutetiensis donor and enterococcal transconjugants showed that ICESluvan integrates in a site-specific manner into the C-terminal end of the chromosomal tRNA methyltransferase gene rumA.

H orizontal gene transfer is a key factor in bacterial evolution, and mobile genetic elements (MGEs) play an important role in the dissemination and persistence of antimicrobial resistance in enterococci. Genome sequence analysis and comparative genome hybridizations of seven Enterococcus faecium isolates from various sources have revealed large differences in genome size, mostly due to the variable presence of mobile genetic elements. The E. faecium pan-genome is considered to be unrestricted in size (1), implying that excess genes, such as those involved in environmental persistence, colonization, and virulence, can easily be incorporated into the E. faecium gene pool. Up to 38% of the E. faecium genome may be noncore, and differences in gene content indicate that gain and loss of genes as important in the evolution of E. faecium (1,2,3). These findings are consistent with the presence of more than 25% mobile or foreign DNA in Enterococcus faecalis V583 (3,4), suggesting extensive genome plasticity.
An approximately 100-kb transferable chromosomal element containing vanB2 has been described in a patient isolate of Streptococcus lutetiensis previously designated Streptococcus bovis biotype II with vanB3 (22,42) and in the plasmid-free S. lutetiensis strain 5-F9 from animal feces (20). The vanB2 cluster of the 5-F9 strain has previously been shown to be an integral part of a Tn5382/Tn1549 element (20) and was transferred to E. faecium and E. faecalis at relatively high frequencies (10 Ϫ7 to 10 Ϫ5 ). In contrast, the element was unable to transfer between E. faecium and E. faecalis strains (20,42), which suggests a coresident transfer system in Streptococcus (42). Retransfer of the ϳ100-kb element from the patient isolate between E. faecalis strains, including to a recombination-deficient recipient, was obtained in the presence of a conjugative helper plasmid (42), and the ϳ100-kb element appeared to integrate in a site-specific manner (20,42). Transfer of this ϳ100-kb chromosomal element from the S. lutetiensis strain that contains no visible plasmids into enterococci suggests self-encoded transfer functions consistent with an integrative and conjugative element (ICE) (20). The transfer mechanism has not been resolved.
Here, we describe the sequencing, annotation, and functional analysis of the 94-kb element designated ICESluvan, originally detected in S. lutetiensis from animal feces in the Netherlands. A variant of this element was confirmed in the S. lutetiensis patient isolate from France. The element encodes VanB-type vancomycin resistance on conjugative transposon Tn5382/Tn1549, a VanZ homologue which may contribute to the detected low-level resistance to teicoplanin, a novel bacitracin locus which seems to contribute to bacitracin resistance in S. lutetiensis and E. faecium, and a streptococcal toxin-antitoxin (TA) pezAT locus, as well as all essential components for conjugative transfer. ICESluvan excises to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium and integrates in a site-specific manner. However, transfer between enterococci required a helper plasmid. The expression of the pezAT operon indicates a functional toxin-antitoxin system.

MATERIALS AND METHODS
Bacterial strains and construction of BAC clones. The bacterial strains used in this study and their relevant characteristics are given in Table 1. Briefly, genomic DNA from the E. faecium transconjugant MM5-F9a was used to generate a bacterial artificial chromosome (BAC) library with an average insert size of 90 kb (MWG Biotech AG). MM5-F9a was obtained by transfer of the ICESluvan element from S. lutetiensis 5-F9, isolated from Selection and sequencing of BAC clones. Sequential dot blot and Southern hybridizations were used to identify BAC clones positive for both vanB2 and Tn5382/Tn1549. PCR-based probes were made (PCR digoxigenin [DIG] probe synthesis kit; Boehringer Mannheim) for vanB using the consensus primers 5=-CAAAGCTCCGCAGCTTGCATG-3= and 5=-TGCATCCAAGCACCCGATAATAC-3= (29) and for Tn5382 using primers 5=-GTTCTTATTCCGCAGGTGGTGATT-3= and 5=-ACGCC ATGCTATTTACTTCCGGC-3= (40). Hybridizations were detected using the DIG luminescent detection kit (Boehringer Mannheim). Plasmid DNA was isolated from BAC clones that scored positive for one or both probes (n ϭ 24). Clones were grown overnight in yeast extract-tryptone (YT) medium containing 12.5 g ml Ϫ1 chloramphenicol, plasmid replication was induced (Epicentre induction solution; Epicentre), and BAC DNA was purified using the E.Z.N.A BAC/PAC kit (Omega Bio-Tek). NotI (New England BioLabs)-digested plasmid DNA was separated on a 1.2% agarose gel, 5 to 15 s, 6 V/cm, 16 h, at 15°C on a CHEF-DR III (Bio-Rad) and analyzed by Southern hybridization. Five clones positive for both probes and with an average insert size of 90 kb were selected for sequencing.
Libraries of the BAC inserts of each selected clone were constructed with the use of multiplex identifiers (MIDs) and sequenced to an average depth of 91.5-times coverage using 454-GS-FLX technology. The BAC inserts were de novo assembled individually with Newbler (Roche).
End sequencing of the BAC inserts provided additional geographical information for contig ordering. End sequencing and extension of contigs were set up using extracted BAC DNA and BigDye 3.1 for cycle sequencing and an ABI prism 377 genetic analyzer (Applied Biosystems). The cycle sequencing program was as follows: initial denaturation at 96°C for 5 min followed by 99 cycles of 96°C for 30 s, 52°C for 10 s, and 60°C for 4 min (primers available on request).
DNA extraction and sequencing of transconjugants. Genomic DNA from transconjugants was obtained using a bacterial DNA kit (E.Z.N.A) with the following modifications: lysis was performed at 30°C for 40 min using 1.5 mg lysozyme and 100 U mutanolysin in a total volume of 220 l. Direct genome sequencing of the transconjugants was performed as for BAC end sequencing.
Verification of sequence assembly by PCRs. To verify the sequence assembly, over 40 PCRs with an average product size of 2.5 kb were performed in ICESluvan (see Table S1 in the supplemental material), using genomic DNA of transconjugants E. faecium MM5-F9a and OG5-F9a and E. faecalis JH5-F9a as templates. Some of these primers were used to confirm the presence of ICESluvan regions (see Table S1) in streptococci (S. lutetiensis 5-F9 and NEM760 and S. gallolyticus 4-C11 and 4-G10) and to sequence the bacitracin locus in the E. faecalis transconjugants OG5-F9a and JH5-F9a.
RNA extraction for expression analyses. The expression of the pezAT operon was analyzed by semiquantitative reverse transcription (RT)-PCR. Total RNA was extracted from stationary-phase cultures using an RNeasy minikit (Qiagen) with an extended lysis step of 1 h with 10 mg lysozyme and 10 U mutanolysin in a total volume of 200 l. DNA contamination was removed by DNase I treatment, and RNA quality and quantity examined by gel analyses and spectrophotometric measurements prior to cDNA synthesis. Reverse transcription of 100 ng total RNA was performed using SuperScript II (Invitrogen). For each sample, a control reaction (without reverse transcriptase) with no enzyme was included. PCR was performed using primer sets specific for each of the genes (pezAF [5=-TGACCTGGAATCGTCTTTCC-3=] and pezAR [5=-TGGCCGAATT GATTAACACA-3=] and pezTF [5=-TTTTTCCTGCAAATCCCTTG-3=] and pezTR [5=-GACAAAGCGGAGCAGGTAAG-3=]). In addition, PCR using primers pezTF and pezAR was performed. Amplicons were visualized on an agarose gel and confirmed by sequencing using BigDye 3.1 technology (Applied Biosystems).
Transfer of ICESluvan. Transfer of the ICESluvan element to E. faecalis strains BM4110 and JH2-2 with pIP964 and retransfer from these firstgeneration transconjugants to recipients (E. faecalis strains JH2-2, UV202, and BM4110) without this helper plasmid were performed by filter mating as previously described (33), with some modifications. Briefly, donor and recipient strains were grown to an A 600 of 0.6 and mixed in a ratio of 1:1 in 2 ml of culture. The suspension was pelleted, resuspended in 150 l brain heart infusion (BHI), and used for filter mating before application on selective agar plates using 8 mg liter Ϫ1 vancomycin, 1,000 mg liter Ϫ1 streptomycin, 20 mg liter Ϫ1 rifampin, and 10 mg liter Ϫ1 fusidic acid. A control experiment (E. faecalis JH5-F9a ϫ BM4110) with recipients containing no helper plasmid was also carried out. The presence of the vanB gene of ICESluvan was confirmed by PCR (29), and ICESluvan insertion in the transconjugant genome was confirmed by visual inspection of the SmaI pulsed-field gel electrophoresis (PFGE) pattern. The presence of the pheromone-responsive helper plasmid pIP964 (46) was determined by PCRs that detect the pCF10 replication initiation gene rep pCF10 as described by Jensen et al. (47), and the presence of the pheromone plasmid's conserved aggregation substance (agg) was detected using primers 5=-GC TCGTGGTGATGTTCTTTC-3= and 5=-CTTTTCTACCACTAATGGCT CTAC-3=. PFGE analyses. Total DNA was digested with SmaI (New England BioLabs) and analyzed by PFGE as previously described (48), with some modifications. Briefly, 5 l lysozyme (100 mg ml Ϫ1 ) and 2 l mutanolysin (10 U l Ϫ1 ) were added to the agarose plugs before molding, as well as to the lysis buffer.
Nucleotide sequence accession number. The whole sequence of ICESluvan is available in GenBank under the accession number HE963029.

RESULTS AND DISCUSSION
Gap closure and ICESluvan mosaic structure. The five selected BAC clones were sequenced to an average depth of 91.5-times coverage and assembled individually into an average of 28 contigs (Ͼ500 bp). Mapping of contigs against reference sequences and end sequencing of BAC inserts provided scaffold information for the gap closure that resulted in one large, 94-kb contig (GenBank accession number HE963029) containing 86 open reading frames (ORFs) ( Fig. 1; see also Table S2 in the supplemental material). Nine smaller contigs of Ͼ1 kb with BLAST similarities to unculturable organisms and transposases were discarded from further analysis. Extensive control PCRs (see Table S1) verified the assembly for E. faecium transconjugant MM5-F9a and confirmed the presence of the ICESluvan element in E. faecalis transconjugants OG5-F9a and JH5-F9a (Table 1). Some primer combinations used for MM5-F9a did not give PCR products with OG5-F9a and JH5-F9a. However, the presence of these regions was confirmed by altering the combination of the primers (denoted by plus signs in Table S1). Some of the PCRs worked at one time point and not at another, and when we sequenced the bacitracin locus of the E. faecalis transconjugants, we found that some of the primer combinations did not give a PCR product even though both primers matched the sequence 100%. Thus, we think there might be other reasons than nucleotide differences, possibly related to the primary or secondary DNA sequence, why PCR products were not achievable for all transconjugants in some specific regions.
A complete vanB2 Tn5382/Tn1549 (ORF18 to -30 and ORF39 to -59) putative conjugative transposon was integrated into the partial ICESde3396-like sequence in ORF17, similar to ICESde3396_54. The vanB2 Tn5382/Tn1549 sequence of ICESluvan was 99% iden-tical at the nucleotide level to the fully sequenced Tn1549 ( Fig. 1; see also Table S2 in the supplemental material) (30). Five more ORFs were identified in the Tn5382/Tn1549 element inserted in ICESluvan compared to those from previous reports (see Table  S2). Identical sequences were also present in Tn1549 (30) but have not been annotated previously. In addition, a 9-kb contig located within ORF30 in Tn5382/Tn1549 was flanked by directly repetitive sequences with 291 of 292 nucleotides identical. Annotation of the 9-kb contig revealed a TnpX site-specific recombinase (ORF31), a pseudotransposase of the IS30 family (ORF33), a putative VanZ family protein (ORF34), a putative mobilization protein (ORF36), and a DNA primase (ORF37), as well as hypothetical proteins (ORF32, -35, and -38) with 40 to 96% amino acid identity to putative proteins in Faecalibacterium, Lactobacillus, and Paenibacillus species, respectively (see Table S2).
The mosaic pattern of the ICESluvan element, which consists of genes from several different streptococcal species, Faecalibacterium, enterococci, and possibly other intestinal bacteria (see Table  S2 in the supplemental material), indicate that mobilization and recombination events have been key factors in the assembly of this element. A mosaic pattern was also observed for ICESde3396, which is a montage of genes derived from group A, B, and G Streptococcus organisms, in addition to genes acquired from nonstreptococcal Gram-positive bacteria, such as Listeria innocua and E. faecalis (49).
ICESluvan variant confirmed in an S. lutetiensis isolate from a patient in France. During examination of mixed fecal samples from 556 veal calf herds in the Netherlands, four vancomycinresistant Streptococcus isolates were found. In addition to S. lutetiensis 5-F9, S. gallolyticus isolates 4-C11 and 4-G10 were positive for the vanB gene and found to have a vanB2 cluster integrated in Tn5382/Tn1549 (20). Furthermore, S. lutetiensis NEM760, isolated from a stool swab of a patient in France, showed transfer of an approximately 100-kb element containing vanB2 (22,42) that inserted into the same SmaI PFGE fragment as ICESluvan in both E. faecium BM4105-RF-and E. faecalis JH2-derived recipients (20,22,42). Since 4-C11, 4-G10, and NEM760 are likely candidates to contain ICESluvan or variants of this element, we have examined these isolates by using primers selected to confirm the ICESluvan sequence assembly (see Table S1 in the supplemental material). The circa-100-kb element found in NEM760 was indeed very similar to ICESluvan, with some differences in the right end of the element, while 4-C11 and 4-G10 showed the presence of Tn5382/ Tn1549 and adjacent regions but not the left or right end of ICESluvan, which is not surprising since transfer of vanB was not achieved for these isolates (19,20). The presence of highly similar variants of ICESluvan capable of interspecies transfer in two S. lutetiensis isolates from completely different environments implies that ICESluvan is a successful element that may contribute to resistance in enterococci.
ICESluvan type IV secretion system components. A putative transfer system (ORF11 to -16 and ORF79 to -80) (see Table S2 in the supplemental material) was identified in the ICESluvan element, in addition to those encoded by Tn5382/Tn1549 (ORF21 to -23, ORF26, and ORF43 and -44) (see Table S2) (30). ORF11 to -15 were 94 to 99% identical to ICESde3396_59 to ICESde3396_55 (49). The ORFs display the same genetic order in the ICESluvan element and belong to a type IV secretion system. Three factors have been recognized as important for DNA transfer by type IV secretion system in Gram-positive bacteria. These are murein hydrolase (50), which is involved in controlled local degradation of the peptidoglycan, making space for the formation of a mating channel, the VirB4 ATPase, which provides energy for the translocation, and the VirD4 coupling protein, which links the DNA transfer intermediate to the mating channel (50,51). In the ICESluvan element, we identified two putative virB4 genes (ORF15 and OFR26) encoding ATPases, a putative virB6 gene (ORF13) encoding a putative membrane protein that has previously been shown to interact with several other Vir proteins mediating DNA substrate transfer through the cytoplasmic membrane channel (50), and two putative virD4 genes (ORF11 and ORF21) encoding coupling proteins of the TraG/TraD family. ORF11 is truncated by a frameshift after codon 465, but ORF21 encodes a putative coupling protein which belongs to the same coupling-protein family. Moreover, ICESluvan ORF16 encodes a putative N-acetylmuramoyl L-alanine amidase that may aid in local degradation of the peptidoglycan by hydrolyzing the amide bond between the N-acetylmuramic acid side chain and L-alanine of the short peptide (52). Genes encoding putative mobilization proteins of the MobC family (ORF79 and ORF44) and relaxases (ORF80 and ORF43) were also identified in ICESluvan. Taken together, this indicates that the ICESluvan element contains most genes necessary for mobilization and conjugative transfer. Furthermore, ICESluvan is transferable by conjugation from S. lutetiensis organisms with no visible plasmids, strongly suggesting that its transfer system is intact (20).
ICESluvan circularization, transfer, and integration. A putative site-specific serine recombinase (ORF86) (see Table S2 in the supplemental material) that can be involved in the genomic exit and integration of mobile genetic elements was found at the right flank of the ICESluvan. Although it lacks 1 or 20 amino acids in the C-terminal end compared to the sequences of its closest homologues, SsuiDRAFT_2393, identified in S. suis (98% amino acid identity), and SP70585_1107, identified in S. pneumoniae (95% amino acid identity), we hypothesize that the putative site-specific recombinase functions as normal in a transfer situation. This hypothesis was supported by circularization of the ICESluvan element both in the S. lutetiensis donor 5-F9 and the Enterococcus transconjugants, as shown by PCR and confirmed by sequence analyses (Fig. 2). Furthermore, we have transferred ICESluvan from S. lutetiensis 5-F9 to both E. faecium and E. faecalis (20). Retransfer was only possible when ICESluvan was transferred into recipients that contain the helper plasmid pIP964 (Tra ϩ ), which is restricted to E. faecalis. We did not test broad-host-range plasmids to achieve retransfer between E. faecium strains. Retransfer from E. faecalis BM4110 ICESluvan pIP964 to E. faecalis JH2-2 or UV202 (Fig. 3, lanes 4 to 6) and from JH2-2 ICESluvan pIP964 to BM4110 (data not shown) was confirmed by growth on selective plates, ICESluvan-and pIP964-specific PCRs, SmaI PFGE analysis (Fig. 3), and hybridization with vanB probe (data not shown) showing transconjugant patterns with an approximately 100-kb enlargement of one of the two 240-kb fragments compared to the recipient patterns (Fig. 3, lanes 4 to 6). However, transconjugant BM4110 ICESluvan pIP964 had gained an extra copy of ICESluvan, as shown by the presence of a fragment around 220 kb in Fig. 3, lane 4, which hybridized with vanB (data not shown). The presence of more than one copy of ICESluvan has been shown before in other JH-derived transconjugants (20). Both SmaI PFGE patterns and DNA sequencing show that the ICESluvan element is integrated in a site-specific manner into the recipient chromosome, although the E. faecium and E. faecalis integration sites within the C-terminal end of tRNA methyltransferase gene rumA showed some sequence differences (Fig. 4). The same integration site was identified within each species for all transconjugants tested (data not shown). The ICESluvan integration site in S. lutetiensis is also within the rumA gene. However, the right flank of ICESluvan in S. lutetiensis showed an extended 5.7-kb region identical to part of the E. faecalis G1-01247 vanG operon which was not transferred together with ICESluvan into the enterococci (see Fig.  S1 in the supplemental material). Interestingly, ICESluvan ORF1 is a truncated homolog identical to the 13-amino-acid C-terminal part of S. pneumoniae rumA (see Table S2). This C-terminal part is in the same translational frame as the N-terminal part of the enterococcal rumA, and together, they form a recombined fulllength rumA gene that is likely to be functional.
Since ICESluvan was shown to transfer from Streptococcus to Enterococcus but retransfer between enterococci required a helper plasmid, we hypothesize that a host factor is necessary for transfer. This has been shown for SXT elements in Vibrio cholerae, which require the host factors IHF and Fis for excision, recombination, and conjugation (53). Host factors may be chromosomally encoded and not on the MGE itself, leading to the inability of the MGE to conjugate in their absence. Transfer of the E. faecalis V583 pathogenicity island (PAI), containing the features of an ICE, by an ICE-independent mechanism has been reported. Characterization of V583 PAI transconjugants showed cotransfer of selectable markers representing virtually all regions of the chromosome, including a vancomycin resistance transposon, capsule genes, and alleles which are used for multilocus sequence typing. PAI transfer was dependent upon helper plasmid function in the donors (54).
Putative bacteriophage exclusion system. The ICESluvan ORF17 located upstream and ORF60 to -64 located downstream from Tn5382/Tn1549 are 81 to 97% identical to ICESde3396_54 to ICESde3396_48. ORF60 and ORF17 are homologous to IC-ESde3396_54 and ICESde3396_53, encoding the abortive infection proteins AbiGI and AbiGII, respectively. The Lactococcus lactis abiG resistance genes confer complete resistance to 712 phages (936 phage species) and partial resistance to c2 phages (2 species) (55). Most Abi systems are plasmid encoded and are widespread in bacteria (56). The abi genes are also known to be involved in increased stress tolerance of the host. These genes are commonly found in Streptococcus (57). Transposon Tn5382/ Tn1549 was inserted in the C-terminal end after codon 251 of abiGII in ICESluvan (Fig. 1), thereby disrupting this gene. Since this leads to an AbiGII protein lacking 30 amino acids compared to its closest homologue, it is not known whether these abi genes encode a functional system.
Putative virulence determinants. The ICESluvan ORF61 was 95% identical at the nucleotide level to ICESde3396_52, which encodes a putative agglutinin receptor precursor protein that harbors an LPXTG motif known to be common among several virulence factors in enterococci (58). Human salivary agglutinin has previously been shown to interact with streptococci in a calciumdependent reaction for oral bacterial aggregation (59,60). The ICESluvan putative agglutinin receptor precursor is located next to a putative Ca 2ϩ binding protein (ORF62). Studies have shown that transformation of nonaggregating E. faecalis with the streptococcal surface antigen SSP-5 confers an aggregation-positive phenotype in the presence of saliva agglutinin (61).
Resistance to glycopeptides and bacitracin. The ICESluvan element encodes vancomycin resistance mediated by a vanB2 cluster (ORF48 to -54) located in Tn5382/Tn1549. S. lutetiensis 5-F9 and the transconjugants, as well as S. lutetiensis NEM760 and S. gallolyticus 4-C11 and 4-G10, accordingly expressed resistance to vancomycin, whereas the recipient strains E. faecium BM4105-RF and E. faecalis OG1-RF, JH2-2, UV202, BM4110, and BM4110 pIP964 were susceptible to vancomycin according to EUCAST clinical breakpoints for enterococci (MIC, Յ4 mg liter Ϫ1 ) ( Table 1). In addition, ORF34 on the 9-kb element inserted into Tn5382/Tn1549 encoded a protein with 40% amino acid similarity to a putative VanZ family protein from Paenibacillus (see Table S2 in the supplemental material). The vanZ gene, contributing to teicoplanin resistance, is normally located within the vanA gene cluster, whereas vanB usually only confers resistance to vancomycin. The functionality of the VanZ homologue was supported by Etest results showing MICs of 3 or 4 mg liter Ϫ1 to teicoplanin (low-level resistance according to EU-CAST clinical breakpoints for enterococci) for S. lutetiensis NEM760 and 5-F9 and the 5-F9 transconjugants, which was at least 3-fold higher than the MICs of the corresponding susceptible recipient strains (BM4105-RF and UV202 MIC, 0.5 mg liter Ϫ1 ; JH2-2 MIC, 1 mg liter Ϫ1 ; BM4110 pIP964 MIC, 0.38 mg liter Ϫ1 ; and OG1-RF MIC, 0.25 mg liter Ϫ1 ) ( Table 1). The finding of this vanZ gene illustrates the dynamics and the potential of the Streptococcus and Enterococcus genomes to acquire and collect genes which may increase their ability for environmental adaptation.
The streptococcal ICESde3396 encodes resistance toward cadmium and arsenic (49). These resistance genes were not identified in ICESluvan. Rather, ORF81 to -84 encode an ABC transporter (ATPase and permease) and a putative two-component system (histidine kinase and response regulator) resembling proteins BceA, -B, -R, and -S (previously designated MbrABCD), which are involved in bacitracin resistance in S. mutans through active efflux of bacitracin (GenBank accession number AB078507) (62,63). ORF81 encodes 249 amino acids with 48% identity to the original S. mutans BceA (250 amino acids) and 82% identity to a putative BceA of S. mitis (GenBank accession number EFN94736) (245 amino acids), ORF82 encodes 672 amino acids with 29% identity to the original S. mutans BceB (667 amino acids) and 67% identity to a putative ABC transporter permease of S. mitis (GenBank accession number EFN94737) (671 amino acids), ORF83 encodes 524 amino acids with Ͼ20% identity to BceS (249 amino acids) and 65% identity to a putative membrane protein with a histidine kinase domain of S. mitis (GenBank accession number EFN94738) (524 amino acids), and ORF84 encodes 198 amino acids with 24% identity to BceR (223 amino acids) and 75% identity to a putative response regulator of S. mitis (GenBank accession number EFN94739) (198 amino acids). Compared to the bceABRS locus of S. mutans, ICESluvan holds a different gene order of the putative response regulator and histidine kinase. The putative bacitracin locus of ICESluvan shows about 20% amino acid identity to components encoded by a bcrRABD locus conferring highlevel bacitracin resistance in E. faecalis, which show yet another synteny with the regulator gene upstream from the ATPase, permease, and kinase genes (64). Phenotypic testing indeed shows at  (20). The presence of the pheromone-responsive plasmid pIP964 was verified by rep pCF10 and agg PCRs. least a 5-fold-increased MIC for bacitracin associated with transfer of ICESluvan from S. lutetiensis 5-F9 to E. faecium. The E. faecium transconjugant MM5-F9a expressed a bacitracin MIC of Ն256 mg liter Ϫ1 , while the MIC of the corresponding E. faecium recipient BM4105-RF was 48 mg liter Ϫ1 , implying functionality of the putative bacitracin locus of ICESluvan as a novel bacitracin resistance locus. On the other hand, the E. faecalis transconjugants and their corresponding recipients showed similar low or high MICs to bacitracin, except for the second-generation transconjugant JH2-2 ICESluvan pIP964, which after transfer from the highbacitracin-MIC background in E. faecalis BM4110 pIP964 showed at least a 16-fold-increased MIC compared to that of JH2-2 (Table  1). Undecaprenyl pyrophosphate phosphatase has recently been shown to account for the low-level resistance to bacitracin (MICs of 32 to 48 mg liter Ϫ1 ) in both laboratory (JH2-2) and clinical (V583) strains of E. faecalis (65), while variants of the transferable bcrRABD locus may be responsible for the high-level bacitracin resistance (MIC Ն128 mg liter Ϫ1 ) in enterococci (64,66). Alternative primer combinations had to be used to confirm the bacitracin locus region of ICESluvan (see Q38 in Table S1 in the supplemental material) in E. faecalis JH5-F9a and OG5-F9a compared to that in E. faecium MM5-F9a, indicating some sequence differences. However, sequencing E. faecalis JH5-F9a and OG5-F9a revealed identical nucleotides of the bacitracin locus (nt 85669 to 91794) compared to the sequence of MM5-F9a (GenBank accession number HE963029). Thus, host-related factors of E. faecalis and E. faecium may be a possible explanation for the lack of increased bacitracin MICs when ICESluvan was introduced directly into E. faecalis strains with low-level resistance to bacitracin.
ICESluvan expresses a pezAT TA system. ICESluvan ORF75 and ORF76 are 93 and 89% identical at the nucleotide level to the pezAT plasmid maintenance system previously described in S. pneumoniae (67), S. agalacticae (57,67), and S. suis (57). The pezAT gene cassette consists of two genes, encoding an epsilon antitoxin and a zeta toxin, respectively. Toxin-antitoxin systems are traditionally known as plasmid addiction systems that ensure stable maintenance of plasmids in a bacterial population (68). However, during recent years, these gene loci have been found on MGEs and shown to promote the maintenance of ICE elements in V. cholerae (69,70). Experimental evidence also indicated that TA systems are involved in the stress response, enabling the cell to survive hostile growth conditions (71,72). Analyses of the DNA sequence upstream from the pezAT locus showed the presence of a putative Ϫ10 promoter sequence (TATAAT) 34 bp upstream from an indicated start codon and a Ϫ35 promoter sequence (GT GCGTT) 19 bp from the putative Pribnow box. In addition, a putative ribosome-binding site (AGGAG) was shown 12 bp upstream from the putative start codon. All except the Ϫ35 sequence were identical to the S. pneumoniae sequences (67). RT-PCR analyses revealed the expression of both pezAT genes (data not shown) in all of the donor and transconjugant strains tested. Figure 5 shows the PCR products of both genes. Moreover, both genes were detected in a single transcript (Fig. 5), confirming that pezA and pezT constitute an operon, as in S. pneumoniae (67). PCR products were not detected in the absence of reverse transcriptase or in the recipient strain (BM4105-RF). All sequences were identical to the sequence found in our E. faecium transconjugant. These observations suggest that this is a functional toxin-antitoxin system that may contribute to the stabilization of ICESluvan after integration into new hosts.
In conclusion, our report describes the complete genetic structure of a 94-kb element, named ICESluvan, originally detected in S. lutetiensis from veal calf feces in the Netherlands. A variant of ICESluvan is confirmed for an S. lutetiensis isolate from a patient in France. ICESluvan encodes glycopeptide and bacitracin resistance loci, a putative bacteriophage exclusion system, a putative virulence gene, and components necessary for conjugative transfer, as well as a toxin-antitoxin stabilization system. This element is a novel ICE, since it forms a circular intermediate, is self-transferable from streptococci to enterococci, and integrates into the chromosome in a site-specific manner.