Jan Manchak

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.

Mutational analysis of F‐pilin reveals domains for pilus assembly, phage infection and DNA transfer

The F‐pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F‐pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F‐pilus tip, and the RNA phages, R17 and Qβ, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F‐pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F‐pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45–60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.

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.

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 Å 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.

Construction of derivatives of the F plasmid pOX–tra715: characterization of traY and traD mutants that can be complemented in trans

A derivative of the F plasmid, pOX38–tra715, expresses the entire F tra operon from a foreign promoter (PT7) derived from phage T7. A series of plasmids related to pOX38–tra715 were constructed which carry either deletion mutations or point mutations in traY. When the PT7 promoter was induced, these plasmids expressed the F pilus but were transfer deficient unless TraY was supplied in trans from compatible plasmids. Insertion of a kanamycin‐resistance cassette in the traY gene of the pOX38 plasmid, which contains the wild‐type PY promoter, resulted in loss of F piliation and transfer ability. Introduction of TraY in trans partially restored piliation and transfer suggesting that TraY has a role in positively regulating the PY promoter. pOX38–tra719–traD411, which contains a chloramphenicol‐resistance cassette in place of the kanamycin‐resistance cassette in pOX38–tra715 and a mutation in traD, was constructed to demonstrate the utility of this series of plasmids in studying the long (30 kb) F tra operon.

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.

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.