Sequencing and characterization of Salmonella typhi plasmid R27 (incompatibility group HI1) trhC, a transfer gene encoding a potential nucleoside triphosphate-binding domain

Abstract

IncHI1 plasmids are one of the few plasmids known to mediate multiple antibiotic resistance in Salmonella typhi. These plasmids are temperature-sensitive for transfer and R27 is the prototype plasmid. DNA sequencing within the Tra2 region of R27, encoding genes involved in mating pair formation, identified trhC encoding the TrhC protein of 90,000 Da, which was visualized using an in vitro transcription/translation system. Expression of the TrhC protein also identified two smaller protein products of approximately 23 and 25 kDa which are predicted to be protease digestion products. The migration of these smaller products changed when the reactions were run at 28 vs 37 degrees C. The TrhC protein contained a putative nucleotide triphosphate-binding protein and exhibited sequence similarities with several other proteins implicated in bacterial conjugation, including TraC (F), TraB (pKM101), VirB4 (Ti), TrbE (RP4). Phylogenetic analysis showed TrhC was most closely related to TraC. Mini-Tn10 insertions in trhC generated transfer defective mutants, and no pili were specified by the trhC mutants. The trhC gene appeared to be a hot spot for transposon insertion as 37.5% mapped into this open reading frame. One trhC mutant, pDT2990, was able to be complemented by a cloned trhC gene giving a transfer frequency of 1 x 10(-3) transconjugants per recipient in an 18-h mating, whereas the wild-type transfer frequency of R27 was 1 x 10(-2) transconjugants per recipient.