K. Maneewannakul et al., CHARACTERIZATION OF TRAX, THE F-PLASMID LOCUS REQUIRED FOR ACETYLATION OF F-PILIN SUBUNITS, Journal of bacteriology, 177(11), 1995, pp. 2957-2964
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.