TraG encoded by the pIP501 type IV secretion system is a two-domain peptidoglycan-degrading enzyme essential for conjugative transfer.

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 (TraGΔTMH) and shown to possess peptidoglycan-degrading activity. TraGΔTMH 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.

Comparison of antibiotic resistance, biofilm formation and conjugative transfer of Staphylococcus and Enterococcus isolates from International Space Station and Antarctic Research Station Concordia.

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.

Green fluorescent protein-labeled monitoring tool to quantify conjugative plasmid transfer between Gram-positive and Gram-negative bacteria.

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.

Evaluation of plasmid content and tetracycline resistance conjugative transfer in Enterococcus italicus strains of dairy origin.

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.

A type IV-secretion-like system is required for conjugative DNA transport of broad-host-range plasmid pIP501 in gram-positive bacteria.

Plasmid pIP501 has a very broad host range for conjugative transfer among a wide variety of gram-positive bacteria and gram-negative Escherichia coli. Functionality of the pIP501 transfer (tra) genes in E. coli was proven by pIP501 retrotransfer to Enterococcus faecalis (B. Kurenbach, C. Bohn, J. Prabhu, M. Abudukerim, U. Szewzyk, and E. Grohmann, Plasmid 50:86-93, 2003). The 15 pIP501 tra genes are organized in a single operon (B. Kurenbach, J. Kopec, M. Mägdefrau, K. Andreas, W. Keller, C. Bohn, M. Y. Abajy, and E. Grohmann, Microbiology 152:637-645, 2006). The pIP501 tra operon is negatively autoregulated at the transcriptional level by the conjugative DNA relaxase TraA. Three of the 15 pIP501-encoded Tra proteins show significant sequence similarity to the Agrobacterium type IV secretion system proteins VirB1, VirB4, and VirD4. Here we report a comprehensive protein-protein interaction map of all of the pIP501-encoded Tra proteins determined by the yeast two-hybrid assay. Most of the interactions were verified in vitro by isolation of the protein complexes with pull-down assays. In conjunction with known or postulated functions of the pIP501-encoded Tra proteins and computer-assisted prediction of their cellular location, we propose a model for the first type IV-secretion-like system encoded by a conjugative plasmid from gram-positive bacteria.