F plasmid TraF and TraH are components of an outer membrane complex involved in conjugation.

F plasmid TraF and TraH are required for F pilus assembly and F plasmid transfer. Using flotation sucrose density gradients, we found that TraF and TraH (as well as TraU and TraW) localized to the outer membrane in the presence of the complete F transfer region, especially TraV, the putative anchor. Mutational analysis of TraH revealed two domains that are important for its function and possible interaction with TrbI, which in turn has a role in stabilizing TraH.

Structural basis of specific TraD-TraM recognition during F plasmid-mediated bacterial conjugation.

F plasmid-mediated bacterial conjugation requires interactions between a relaxosome component, TraM, and the coupling protein TraD, a hexameric ring ATPase that forms the cytoplasmic face of the conjugative pore. Here we present the crystal structure of the C-terminal tail of TraD bound to the TraM tetramerization domain, the first structural evidence of relaxosome-coupling protein interactions. The structure reveals the TraD C-terminal peptide bound to each of four symmetry-related grooves on the surface of the TraM tetramer. Extensive protein-protein interactions were observed between the two proteins. Mutational analysis indicates that these interactions are specific and required for efficient F conjugation in vivo. Our results suggest that specific interactions between the C-terminal tail of TraD and the TraM tetramerization domain might lead to more generalized interactions that stabilize the relaxosome-coupling protein complex in preparation for conjugative DNA transfer.

Entry exclusion in F-like plasmids requires intact TraG in the donor that recognizes its cognate TraS in the recipient.

The mating pair stabilization (Mps) protein of the F plasmid, TraG, is unique to F-like type IV secretion systems. TraG is a polytopic inner-membrane protein with a large C-terminal periplasmic domain that is required for piliation and Mps, whereas the N-terminal region is sufficient for pilus synthesis. The C-terminal region of TraG is thought to be cleaved by the host signal peptidase I to give a fragment called TraG* that is responsible for Mps. Using mutational analysis and cell localization studies, it was shown that TraG* is most probably an artifact caused by non-specific degradation. TraS (173 aa in F), which is involved in entry exclusion (Eex), blocks redundant conjugative DNA synthesis and transport between donor cells, suggesting that it interferes with a signalling pathway required to trigger DNA transfer. Using the F and R100 plasmids, TraG in the donor cell was found to recognize TraS in the recipient cell inner membrane, in a plasmid-specific manner. This activity mapped to aa 610-673 in F TraG, the only region that differs significantly from R100 TraG. Expression of traG or traG* in a recipient cell did not affect mating ability or Eex. These results suggest that TraG may be translocated to the recipient cell, where it contacts the inner membrane, initiating transfer, a process that is blocked by TraS.

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM.

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.

Crystallization and preliminary diffraction studies of TraF, a component of the Escherichia coli type IV secretory system.

TraF, a component of the Escherichia coli type IV secretory system, has been crystallized and preliminary X-ray diffraction data have been collected. TraF is a 26 kDa protein encoded by the E. coli F plasmid and is required for conjugative plasmid transfer and the formation of sex pili. The N-terminal domain of TraF has no recognizable sequence features, whereas the C-terminal domain is believed to adopt a thioredoxin fold. However, since the active-site cysteines of thioredoxin-like proteins are not conserved in TraF, its biochemical role remains unclear. TraF crystallizes in space group C2, with unit-cell parameters a = 119.87, b = 34.36, c = 46.21 A, beta = 90.40 degrees , and crystals diffract to 2.3 A resolution.

Analysis and characterization of the IncFV plasmid pED208 transfer region.

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.

F- phenocopies: characterization of expression of the F transfer region in stationary phase.

The phenomenon of 'F- phenocopies' in which F+ cells become transfer-deficient in stationary phase seems contradictory to the proposed role for F transfer in adaptive mutation during stationary phase induced by nutrient limitation. The expression of a range of transfer genes at the transcriptional and translational level in stationary phase has been characterized as well as the degree of nicking at the origin of transfer, oriT. Transfer efficiency rapidly decreased in mid-exponential phase, coincident with a decrease in traM transcripts. Approximately 2 h later, the transcript for traA, encoding F-pilin, also decreased to undetectable levels. The levels of TraA (pilin), TraD, TraJ and TraT remained fairly constant well into stationary phase while the levels of TraM and Tral decreased to undetectable levels in early stationary phase. A null mutation in the gene for the alternative sigma factor, rpoS, did not affect mating efficiency or transcript levels but did increase the stability of TraM and Tral in stationary phase. Nicking at oriT was detected at maximal levels in early stationary phase and at low levels in late stationary phase. The results suggest that the F-pilus transfer apparatus is maintained in the cell envelope after transcription of the transfer region from the main promoter, Py, has ceased with down-regulation of traM transcription being the first step detected in this process. The presence of a low level of nicking at oriT in stationary phase is consistent with a role for F in promoting adaptive mutation.