Characterization of genes and gene products encoded by the F plasmid transfer region : trbI, traW, traU, trbC, traN, and trbE

Abstract

Genes and gene products encoded by the central part of the F transfer region (between traC and traF) were investigated. The nucleotide sequence of the entire region was determined and all gene products were identified and characterized. Three of the tra cistrons in which amber mutations were available and three previously unidentified (trb) genes were found to be included. These mapped in the order: traC trbI traW traU trbC traN trbE traF. Complementation analyses showed traW to be distal to traC rather than proximal as had been suggested in earlier work. The traW gene was shown to express a 210 amino acid, 23,630 Da protein with a 17-19 amino acid signal sequence. The sequence of traW546 amber mutant DNA contained a C [arrow T change in the Gln codon for amino acid 141. Product analysis in maxicells showed that signal processing was inhibited by ethanol; in its absence, mature TraW (21,618-21,760 Da) was found in periplasmic fractions. Similar experiments showed that the product encoded by traU (330 amino acids; 36,786 Da) and trbC (212 amino acids; 23,433 Da) were also processed to 34,275 Da and 21,226 Da mature proteins that were also located in the periplasm. Mutations affecting these three periplasmic proteins are known to affect both F transfer and piliation properties. The traN product, which encoded a 602 amino acid, 65,714 Da precursor, was also subject to signal processing to a 63,823 Da mature protein. However, TraN, which is involved in stable aggregate formation, was found to be an outer membrane protein. Additional experiments showed that TraN spans the outer membrane and is sensitive to hydrolysis when cells are exposed to protease K. The traN548 amber mutation was found to result from a C [arrow] T transition in the 131st codon (Gln) in the traN sequence. In addition, products encoded by trbI (128 amino acids; 14,132 Da) and trbE (86 amino acids; 9,910 Da) were identified...