Characterization of traX, the F plasmid locus required for acetylation of F-pilin subunits

Abstract

Acetylation of F-pilin subunits has previously been shown to depend upon expression of the F plasmid transfer operon gene traX. To assess the requirement for pilin acetylation in conjugative transfer of F, we constructed traX::kan insertion mutations and crossed them onto the transmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable. Although pilin synthesized by mutant plasmids pOX38-traX482 and pOX38-traX483 was not acetylated, F-pilus production and F-pilus-specific phage infection appeared to be normal and transfer occurred at wild-type frequency. Analysis of labeled products showed that TraX ؉ plasmids expressed two approximately 24-(TraX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells. No product that was similar in size to the product predicted from the traX open reading frame (27.5 kDa) was detected. Therefore, we used site-directed mutagenesis, stop codon linker insertions, and phoA fusion analysis to investigate traX expression. Both TraX1 and TraX2 appeared to be encoded by the traX open reading frame. Insertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusions to phoA indicated that only the traX reading frame was translated. Expression was also very dependent on the traX M1 start codon; when this was altered, no protein products were observed. However, pilin acetylation activity was still detectable, indicating that some other in-frame start codon(s) can also be used. All sequences that are essential for activity are contained between traX codons 29 and 225. Sequence analysis indicated that traX mRNA is capable of forming a variety of base-paired structures. We suggest that traX expression is translationally controlled and that F-pilin acetylation activity may be regulated by physiological conditions in cells.

Comparative Genomics of the Conjugation Region of F-like Plasmids: Five Shades of F

“…An exception to this rule was traX , a gene that encodes an acetylase of pilin subunits. This gene was deemed non-essential for F plasmid conjugation (Maneewannakul et al, 1995), yet it was detected in 88% of the plasmids analyzed.…”

Towards a structural biology of bacterial conjugation

“…However, at least in some conditions, domain I is available to antibodies only when the ␣-amino group of the amino-terminal alanine residue is not acetylated. Normally, when acetylation occurs in a reaction requiring the traX gene product (Maneewannakul et al, 1995), domain I appears to be masked, in so far as antibodies to domain I epitopes fail to bind, as judged by immunogold electron microscopy (Frost et al, 1986;Grossman et al, 1990). Additionally, filaments observed in bacterial cultures, without purification, and composed of acetylated subunits tend to remain isolated, with little tendency to aggregate, whereas those composed of unacetylated subunits form massive aggregates (Grossman and Silverman, 1989).…”

Processing and Maturation of the Pilin of the Type IV Secretion System Encoded within the Gonococcal Genetic Island

“…Pilin subunits can undergo different modifications like methylation (47), acetylation (48), and glycosylation (49). Moreover a subset of pili from T4SSs matures into a circular form (19).…”