SummaryF 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.
Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing 13-hydroxybutyrate and 13-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. IV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of IV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-'), while the maximum PEIA production rate was 1.1 g liter-' h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PEIA (Yp/s) remained at maximum theoretical efficiency (.0.33 g of PEIA per g of glucose consumed). Up to 45 mol% 1V in the polymer was obtained by growing strain UWD in medium containing c50 mM valerate as the sole C source, but the PHA produced amounted to <1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%. The results strongly support a route involving 13-oxidation in the production of 1V in A. vinelandii PEIA. The results show that strain UWD can form PHA copolymers of potential use as bioplastics and that the substrate cost per kilogram of PHA formed in beet molasses medium should be less than half of that per kilogram of PHIA formed in glucose medium.
Summary 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.
The phenomenon of 'F' phenocopies' in which F+ cells become transferdeficient 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 a t 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 t o undetectable levels. The levels of TraA (pilin), TraD, Tral and TraT remained fairly constant well into stationary phase while the levels of TraM and Tral decreased t o undetectable levels in early stationary phase. A null mutation in the gene for the alternative G 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 a t 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 a t oriT in stationary phase is consistent with a role for F in promoting adaptive mutation.
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