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

Abstract

The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…Before secretion to the cell surface, the pilin precursor proteins (100-150 aa in size) undergo several processing steps starting with the cleavage of the N-terminal signal peptide. In case of the F plasmid pilin (TraA) signal peptide cleavage is followed by N-acetylation of the N-terminus (Moore et al, 1993). The pilins of A. tumefaciens (VirB2) and RP4 (TrbC) undergo an unusual kind of modiWcation: N-and C-termini of the precursors form a peptide bond resulting in cyclic proteins, composed of 74 and 78 aa, respectively (Eisenbrandt et al, 1999).…”

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…Pilin subunits in other systems undergo a variety of further post-translational modi®cations. TraA of the F-pilus is acetylated at the C-terminus (Moore et al, 1993), and the subunit of type IV adhesive pili in Neisseria meningitidis is glycosylated (Virji et al, 1993). VirB2, as well as its IncP homolog TrbC, forms an intramolecular bond between its N-and C-termini producing a cyclic polypeptide, a rare reaction in prokaryotes (Eisenbrandt et al, 1999).…”

Functional and Mutational Analysis of Conjugative Transfer Region 2 (Tra2) from the IncHI1 Plasmid R27

“…The Mpf apparatus is a membrane-associated protein complex that functions in producing conjugative pili and subsequently transferring plasmid DNA (15). The Mpf apparatus consists of 12 or 13 proteins (15,18), including the pilin subunit and usually a pilin-processing protein (i.e., acetylase [35] or peptidase [20]). The processed pilin subunits insert into the inner membrane before being assembled onto the donor surface by all, or some, of the Mpf apparatus components (32,37).…”