CHARACTERIZATION OF TRAX, THE F-PLASMID LOCUS REQUIRED FOR ACETYLATION OF F-PILIN SUBUNITS

Acetylation of F-pilin subunits has previously been shown to depend up on expression of the F plasmid transfer operon gene traX. To assess th e requirement for pilin acetylation in conjugative transfer of F, we c onstructed traY::kan insertion mutations and crossed them onto the tra nsmissible F derivative pOX38. Under standard conditions, the function of traX seemed to be dispensable. Although pilin synthesized by mutan t plasmids pOX38-fraX482 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 pro ducts showed that TraX(+) plasmids expressed two approximately 24- (Tr aX1) and 22-kDa (TraX2) polypeptides that localized in the cytoplasmic membranes of cells. No product that was similar in size to the produc t predicted from the traX open reading frame (27.5 kDa) was detected. Therefore, we used site-directed mutagenesis, stop codon linker insert ions, and phoA fusion analysis to investigate traX expression. Both Tr aX1 and TraX2 appeared to be encoded by the traX open reading frame. I nsertion of a stop codon linker into the traX C-terminal coding region led to synthesis of two correspondingly truncated products, and fusio ns to phoA indicated that only the traX reading frame was translated. Expression was also very dependent on the traX M1 start codon; when th is was altered, no protein products were observed. However, pilin acet ylation activity was still detectable, indicating that some other in-f rame start codon(s) can also be used. All sequences that are essential for activity are contained between traX codons 29 and 225. Sequence a nalysis indicated that traX mRNA is capable of forming a variety of ba se-paired structures. We suggest that traX expression is translational ly controlled and that F-pilin acetylation activity may be regulated b y physiological conditions in cells.