Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene traI using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies ϳ100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.Much of the lateral gene transfer between bacteria occurs through the action of conjugative plasmids that encode all of the functions necessary for their hosts to transmit them to recipient cells. Plasmid transfer is achieved through direct cell contact and active transport of DNA by the donor. In gram-negative conjugation systems, typified by the F plasmid of Escherichia coli, only one strand of DNA is translocated, so single-strand cleavage and unwinding of the substrate DNA must occur prior to transfer (9). Strand scission is performed by plasmid-encoded "relaxases" that cleave their cognate plasmid at a specific site called nic within the origin-of-transfer region (oriT). In the case of the F and related plasmids, the accessory proteins TraM, TraY, and integration host factor also bind at oriT as part of the relaxosome complex (15,17,19,32). DNA unwinding is usually performed by a separate helicase, though in some systems, such as the F and R388 plasmids of E. coli, relaxase and helicase activities are both present in the relaxase (11,27,42). After making a single-stranded break at nic, the relaxase is thought to deliver the DNA to a type IV secretory apparatus that can translocate it across the recipient cell membrane. The relaxase remains covalently bound to the nic site and religates the scission once DNA transfer is complete. The replication of single-stranded plasmid DNA in the donor and recipient regenerates the double-stranded DNA plasmid in both cells.The F plasmid relaxase TraI is a well-studied model of structure and function for relaxases. The single-stranded DNA-cleaving activity of TraI is present in the first ϳ310 residues of this 1,756-residue protein, while the helicase motifs are locat...

SummaryBacteria respond to ever-changing environments through several adaptive strategies. This includes mechanisms leading to a high degree of phenotypic variability within a genetically homogeneous population. In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also impedes adherence to endothelial cells and/or matrix proteins. We analysed the regulation of core biosynthesis genes (capA-P) necessary for CP synthesis using single-cell assays (immunofluorescence and promoter-activity). In persistent human carriers, we found a distinct subpopulation of nasal S. aureus to be CP positive. In vitro, cap expression is also heterogeneous and strongly growth-phase dependent. We asked whether this peculiar expression pattern (earlyOff/lateHeterogen) is orchestrated by the quorum system Agr. We show that the Agr-driven effector molecule RNAIII promotes cap expression largely via inactivation of the repressor Rot. High NaCl, deletion of CodY or Sae also resulted in higher cap expression but did not change the earlyOFF/ lateHeterogen expression pattern. Activity of the quorum system itself is largely homogenous and does not account for the observed heterogeneity of cap expression or the strictly growth phase dependent expression. Our findings are in contrast to the prevailing view that quorum sensing is the main driving force for virulence gene expression when bacterial cell densities increase.

No abstract

Type IV secretory systems are a group of bacterial transporters responsible for the transport of proteins and nucleic acids directly into recipient cells. Such systems play key roles in the virulence of some pathogenic organisms and in conjugation-mediated horizontal gene transfer. Many type IV systems require conserved "coupling proteins," transmembrane polypeptides that are critical for transporting secreted substrates across the cytoplasmic membrane of the bacterium. In vitro evidence suggests that the functional form of coupling proteins is a homohexameric, ring-shaped complex. Using a library of tagged mutants, we investigated the structural and functional organization of the F plasmid conjugative coupling protein TraD by coimmunoprecipitation, cross-linking, and genetic means. We present direct evidence that coupling proteins form stable oligomeric complexes in the membranes of bacteria and that the formation of some of these complexes requires other F-encoded functions. Our data also show that different regions of TraD play distinct roles in the oligomerization process. We postulate a model for in vivo oligomerization and discuss the probable participation of individual domains of TraD in each step.