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 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.
Maneewannakul
K
K
Department of Medical Microbiology and Immunology, Texas A&M University Health Science Center, College Station 77843, USA.
Maneewannakul
S
S
Ippen-Ihler
K
K
D000107:Acetylation; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005144:F Factor; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D012333:RNA, Messenger / Q000235:genetics; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C444040:F pilin, E coli; D012333:RNA, Messenger; C093803:TraX protein, bacteria; D036781:Fimbriae Proteins