Biofilm-Forming Clinical Staphylococcus Isolates Harbor Horizontal Transfer and Antibiotic Resistance Genes.

Infections caused by staphylococci represent a medical concern, especially when related to biofilms located in implanted medical devices, such as prostheses and catheters. Unfortunately, their frequent resistance to high doses of antibiotics makes the treatment of these infections a difficult task. Moreover, biofilms represent a hot spot for horizontal gene transfer (HGT) by bacterial conjugation. In this work, 25 biofilm-forming clinical staphylococcal isolates were studied. We found that Staphylococcus epidermidis isolates showed a higher biofilm-forming capacity than Staphylococcus aureus isolates. Additionally, horizontal transfer and relaxase genes of two common staphylococcal plasmids, pSK41 and pT181, were detected in all isolates. In terms of antibiotic resistance genes, aac6-aph2a, ermC, and tetK genes, which confer resistance to gentamicin, erythromycin, and tetracycline, respectively, were the most prevalent. The horizontal transfer and antibiotic resistance genes harbored on these staphylococcal clinical strains isolated from biofilms located in implanted medical devices points to the potential risk of the development and dissemination of multiresistant bacteria.

Subcellular location of the coupling protein TrwB and the role of its transmembrane domain.

Conjugation is the most important mechanism for horizontal gene transfer and it is the main responsible for the successful adaptation of bacteria to the environment. Conjugative plasmids are the DNA molecules transferred and a multiprotein system encoded by the conjugative plasmid itself is necessary. The high number of proteins involved in the process suggests that they should have a defined location in the cell and therefore, they should be recruited to that specific point. One of these proteins is the coupling protein that plays an essential role in bacterial conjugation. TrwB is the coupling protein of R388 plasmid that is divided in two domains: i) The N-terminal domain referred as transmembrane domain and ii) a large cytosolic domain that contains a nucleotide-binding motif similar to other ATPases. To investigate the role of these domains in the subcellular location of TrwB, we constructed two mutant proteins that comprised the transmembrane (TrwBTM) or the cytoplasmic (TrwBΔN70) domain of TrwB. By immunofluorescence and GFP-fusion proteins we demonstrate that TrwB and TrwBTM mutant protein were localized to the cell pole independently of the remaining R388 proteins. On the contrary, a soluble mutant protein (TrwBΔN70) was localized to the cytoplasm in the absence of R388 proteins. However, in the presence of other R388-encoded proteins, TrwBΔN70 localizes uniformly to the cell membrane, suggesting that interactions between the cytosolic domain of TrwB and other membrane proteins of R388 plasmid may happen. Our results suggest that the transmembrane domain of TrwB leads the protein to the cell pole.

The transmembrane domain of the T4SS coupling protein TrwB and its role in protein-protein interactions.

Bacteria use type IV secretion systems to transfer genetic material and proteins from donor to recipient cells, using proteins encoded by conjugative plasmids. Among those proteins the so-called Type IV Coupling Protein plays a central role in the process. One of the best studied members of this family is TrwB, the conjugative coupling protein of R388 plasmid. Previous studies indicated that the transmembrane domain of TrwB plays a role beyond the mere anchoring of the protein to the membrane. TrwB has also been shown to interact with other conjugative proteins, such as the VirB10-like protein of R388 TrwE. The goal of this study is to elucidate the role of the different domains of TrwB and TrwE in their biological function, and in both self- and TrwB-TrwE interactions. To this aim, a series of TrwB and TrwE deletion mutant proteins were constructed. Conjugation and interaction studies revealed that the transmembrane domain of TrwB, and particularly its second transmembrane helix, is needed for TrwB self-interaction and for R388 conjugative transfer and that there are contacts between TrwB and TrwE in the membrane. On the contrary, the lack of the TMD of TrwE does not completely abolish R388 conjugation although the interaction between TrwE-TrwB is lost. These results identify protein-protein interactions inside the membrane needed for T4SS function.