Conjugative plasmid transfer is one of the most important mechanisms for the spread of antibiotic resistance genes and thereby the emergence of multiple resistant pathogenic bacteria. pIP501 is a 30,599-bp plasmid with the broadest known host range for a conjugative plasmid originating from grampositive (Gϩ) bacteria. pIP501 can self-transfer to a variety of Gϩ bacteria, including pathogens and nosocomial pathogens such as streptococci, staphylococci, enterococci, listeria, multicellular Streptomyces lividans, and also to gram-negative (GϪ) Escherichia coli (32). The pIP501 tra region is organized in an operon comprising almost half of the plasmid genome. It bears 15 genes, all of which are putatively involved in conjugative plasmid transfer. Cotranscription of all 15 tra genes was shown by reverse transcription-PCR in Enterococcus faecalis JH2-2 (33). The tra genes are transcribed throughout the growth cycle of E. faecalis, and their expression level remains constant independent of the growth phase. The pIP501 tra operon is negatively autoregulated at the transcriptional level by the first gene product of the operon, the TraA relaxase (33). The TraA relaxase was biochemically characterized as the enzyme attacking a specific dinucleotide within oriT, thereby initiating the directed transfer of the plasmid single strand to the recipient. The TraA relaxase and its amino-terminal relaxase domain TraAN 246 (the first 246 amino-terminal amino acids) were shown to bind to oriT and to the tra operon promoter, P tra , which partially overlaps with oriT (30, 33).

d pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraG⌬TMH) and shown to possess peptidoglycan-degrading activity. TraG⌬TMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.

The International Space Station (ISS) and the Antarctic Research Station Concordia are confined and isolated habitats in extreme and hostile environments. The human and habitat microflora can alter due to the special environmental conditions resulting in microbial contamination and health risk for the crew. In this study, 29 isolates from the ISS and 55 from the Antarctic Research Station Concordia belonging to the genera Staphylococcus and Enterococcus were investigated. Resistance to one or more antibiotics was detected in 75.8 % of the ISS and in 43.6 % of the Concordia strains. The corresponding resistance genes were identified by polymerase chain reaction in 86 % of the resistant ISS strains and in 18.2 % of the resistant Concordia strains. Plasmids are present in 86.2 % of the ISS and in 78.2 % of the Concordia strains. Eight Enterococcus faecalis strains (ISS) harbor plasmids of about 130 kb. Relaxase and/or transfer genes encoded on plasmids from gram-positive bacteria like pIP501, pRE25, pSK41, pGO1 and pT181 were detected in 86.2 % of the ISS and in 52.7 % of the Concordia strains. Most pSK41-homologous transfer genes were detected in ISS isolates belonging to coagulase-negative staphylococci. We demonstrated through mating experiments that Staphylococcus haemolyticus F2 (ISS) and the Concordia strain Staphylococcus hominis subsp. hominis G2 can transfer resistance genes to E. faecalis and Staphylococcus aureus, respectively. Biofilm formation was observed in 83 % of the ISS and in 92.7 % of the Concordia strains. In conclusion, the ISS isolates were shown to encode more resistance genes and possess a higher gene transfer capacity due to the presence of three vir signature genes, virB1, virB4 and virD4 than the Concordia isolates.

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On the basis of pIP501, a green fluorescent protein (GFP)-tagged monitoring tool was constructed for quantifying plasmid mobilization among Gram-positive bacteria and between Gram-positive Enterococcus faecalis and Gram-negative Escherichia coli. Furthermore, retromobilization of the GFP-tagged monitoring tool was shown from E. faecalis OG1X into the clinical isolate E. faecalis T9. The mechanisms of conjugative transfer in Gram-negative bacteria are fairly well understood (e.g., see references 2, 9, 18, 33, 36, and 41), whereas conjugation in Gram-positive bacteria has been studied in greater detail for only the last decade leading to the first model of a type IV secretion-like system in Gram-positive bacteria (1,2,16,17,41). The antibiotic resistance plasmid pIP501 from Streptococcus agalactiae has a very broad host range for conjugative plasmid transfer and mobilization. Its host range includes virtually all tested Gram-positive bacteria, including the multicellular filamentous streptomycetes and Gram-negative Escherichia coli (17,24).For Gram-positive systems, molecular tools for in situ detection of horizontal gene transfer by conjugation are still very limited in contrast to 7,8,29,37,38). Nieto and Espinosa (30) have constructed a green fluorescent protein (GFP)-tagged derivative of plasmid pMV158 from Streptococcus pneumoniae, pMV158GFP, that was shown to be mobilizable to different low-GC Gram-positive bacteria like Enterococcus faecalis and Lactococcus lactis. Lorenzo-Díaz and Espinosa applied pMV158GFP to intra-and interspecies mobilization between different Gram-positive bacteria in large-scale filter mating assays (26).Recently, Babic and coworkers (3) demonstrated conjugative transfer of the integrative and conjugative element ICEBs1 from Bacillus subtilis donor cells to B. subtilis recipient cells in real time using a lacO or LacI-GFP system for visualization of transfer events.Here, we report the construction and mobilization of a GFPtagged mobilizable plasmid based on the pIP501 tra region to monitor horizontal gene transfer between Gram-positive bacteria and between Gram-positive and Gram-negative bacteria by the formation of a green fluorescent phenotype in transconjugants. The mobilizable plasmid is based on a nisin-inducible expression system (NICE) and replicates in both Gram-positive and Gramnegative bacteria.Plasmid construction. The oriT region from the broad-hostrange plasmid pIP501 (oriT pIP50 ) was subcloned with primer pair oriT-HindIII-fw (fw stands for forward) and oriT-HindIII-re (re stands for reverse) (see Table S1 in supplemental material) via HindIII into plasmid pJP rel GFP encoding a gfp gene improved for expression in prokaryotes (28, 31). All bacterial strains and plasmids used in this work are described in Table 1. The gfp-oriT pIP501 cassette was inserted into the E. coli shuttle plasmid pMSP3535VA (6) via XmaI/XbaI with primer pair P rel -gfp-XmaI-fw and oriTXbaI-re under the control of a nisin-inducible nisA promoter. Then, the phage t 0 terminator was cloned downstrea...