Synthesis of F pilin

Abstract

Transfer of the Escherichia coli fertility plasmid, F, is dependent on expression of F pili. Synthesis of F-pilin subunits is known to involve three F plasmid transfer (tra) region products: traA encodes the 13-kDa precursor protein, TraQ permits this to be processed to the 7-kDa pilin polypeptide, and TraX catalyzes acetylation of the pilin amino terminus. Using cloned tra sequences, we performed a series of pulse-chase experiments to investigate the effect of TraQ and TraX on the fate of the traA product. In TraQ- cells, the traA gene product was found to be very unstable. While traA polypeptides of various sizes were detected early in the chase period, almost all were degraded within 5 min. Rapid traA product degradation was also observed in TraX+ cells, although an increased percentage of these products persisted during the chase. In TraQ+ cells, most of the traA product was processed to the 7-kDa pilin polypeptide within the 1-min pulse period; this product [7(Q)] was not degraded but was increasingly converted to an 8-kDa form [8(Q)] as the chase continued, suggesting that host enzymes can modify the pilin polypeptide. Similar results were observed in TraQ+ TraX+ cells, but the primary 7-kDa product appeared to be N-acetylated pilin (Ac-7). An 8-kDa product (Ac-8) was also detected, but this band did not increase in intensity during the chase. We suggest a pathway in which TraQ prevents the traA product from folding to a readily degradable conformation and assists its entry into the membrane, Leader peptidase I cleaves the traA product signal sequence, and a subset of the pilin polypeptides becomes modified by host enzymes; TraX then acetylates the N terminal of both the modified and unmodified pilin polypeptides.

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

“…Thus, 8(Q) seems unlikely to be a processing intermediate that retains part of the TraA signal sequence. Further studies suggest that the 8-kDa form of pilin is derived by modifying the 7-kDa pilin polypeptide (24). As both the Ac-8 and Ac-7 forms of F pilin are present in preparations of purified F pili, the subunits in these filaments may be more heterogeneous than previously thought.…”

“…Since the products of both traX and traQ appear to be inner membrane proteins (4,48,49), these proteins may normally be associated with each other. TraQ prevents degradation of the traA product and may assist membrane entry of the pilin precursor in a conformation appropriate for signal-peptide cleavage (23,24). A closely associated traX product would then be positioned for catalyzing acetylation of the pilin polypeptide.…”

Comparative Genome Analysis of Vibrio vulnificus, a Marine Pathogen

“…Notably, several genes related to plasmid conjugation (traB, -C, -D, -E, -F, -G, -H, -I, -K, -L, -N, -U, -W, -Y, trbB, -C) are present in the pYJ016 sequence. The orientation and arrangement of these tra genes are strikingly similar to those of the F plasmid (Kennedy et al 1977), except that the pYJ016 plasmid lacks traM, -J, -A, -P, -V, -R, -E, -Q, -S, and -T that are involved in pilus formation (Moore et al 1981;Maneewannakul et al 1993) and associated functions. Despite this apparent deficiency, the pYJ016 plasmid was transferred between bacteria by conjugation in the laboratory system (L.-I Hor, unpubl.…”

Towards a structural biology of bacterial conjugation

“…An exception is traQ ; of all the tra genes so far tested, only traQ was required for accumulation of membrane F-pilin. TraQ, itself an inner membrane protein, has been suggested as interacting with the traA gene product in the inner membrane, perhaps to position TraA for processing to mature F-pilin (Maneewannakul et al, 1993). Additional interactions between F-pilin precursors and F-pilin, on the one hand, and both Tra and host proteins on the other, have been postulated (Firth et al, 1996;Paiva and Silverman, 1996), but none of these interactions has been demonstrated.…”