Construction and analysis of F plasmid traR, trbJ, and trbH mutants

Abstract

F plasmid derivatives carrying kan insertion mutations in the transfer region genes traR, trbJ, and trbH were constructed. Standard tests indicated that these loci are not essential for F pilus production or F transfer among Escherichia coli K-12 hosts. Among the traR and trbH mutants tested, the orientation of the kan cassette had no effect on the mutant phenotype. In each case, there was no significant effect on the appearance of F pili, the transfer frequency, or the plating efficiency of F-pilus-specific phages. The trbJ insertion carrying a kan gene oriented in the direction opposite to tra transcription had very little effect on phage sensitivity but markedly reduced the plasmid transfer efficiency. However, the kan insertion mutation at the same site, in the tra orientation, did not seem to affect either property. Analysis of clones carrying trbJ sequences regulated by a phage T7 promoter showed that trbJ expresses an approximately 11-kDa protein product. The TrbJ protein was not expressed from clones carrying a kan insertion or stop codon linker insertion in the trbJ sequence. However, it was expressed from clones that did not include sequences at the beginning of the 113-codon open reading frame in this region. Our data indicated that translation of trbJ must be initiated at the more distal GUG codon in this frame. This would result in expression of a 93-amino-acid polypeptide.