Genetic analysis of a temperature-sensitive Salmonella typhimurium rho mutant with an altered rho-associated polycytidylate-dependent adenosine triphosphatase activity

Abstract

A conditional-lethal rho mutant of Salmonella typhimurium LT2 has been isolated. The mutation was selected as a suppressor of the polarity of an insertion sequence (IS)2-induced mutation (gal3) carried on an F' plasmid. In addition to suppression of IS2-induced polarity, the rho-ill mutation suppressed nonsense and frameshift polarity. The rho-associated polycytidylic acid-dependent adenosine triphosphatase activity in the mutant strain was elevated 15-fold above that in the parental strain, and the mutant rho protein was thermally unstable. A temperature-resistant revertant of the mutant strain did not suppress polarity and contained normal levels of polycytidylic acid-dependent adenosine triphosphatase, suggesting that the phenotype of the rho-ill-bearing strain is the consequence of a single mutation. The rho-ill mutation was located on the S. typhimurium linkage map midway between the ilv and cya loci by phage P22 cotransduction studies. F' plasmid maintenance was not impaired in the mutant strain, and the mutation was recessive to the wild-type allele. The rho-ill mutation did not alter in vivo expression of either the tryptophan or histidine operons. This study; see text B. Ames Wild-type; B. Ames R. 0. Burns (5) J. Roth (15) J. Roth e Genetic symbols are those of Sanderson and Hartman (33). The his operator, formerly hisO, is written hi8Go. b (P1) or (P22) indicates source of transducing lysate. ' Nomenclature conventions for insertion mutations follow the suggestions of Chumley et al. (8).

Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium

“…Strains carrying mutations in the rho gene are conditionally lethal and exhibit a temperature-sensitive phenotype (9,24). E. coli AD1600 and S. typhimurium SC684 (Table 1) were grown in LB broth at 32°C to optical densities at 550 nm of 0.4 and 0.7, respectively.…”

“…Evidence for the presence of a Rho-like activity in gramnegative species relies upon enzymatic assays and Western blot (immunoblot) experiments using an antibody directed against Rho of E. coli (4). Only in Salmonella typhimurium has Rho factor been partially characterized at the genetic and biochemical levels (15,24,25). The existence of a Rho-like activity in gram-positive bacteria has not been convincingly demonstrated (4, 26).…”

Internal promoters of the his operon in Salmonella typhimurium

“…We sought these cryptic message starts by looking for internal his promoters in strains carrying a rho mutation. By making use of transductional linkage between rho and ilv, strains were constructed that contained a rho mutation (rho-Hll [12]) and a his deletion that removed Pl or both Pl and P2. The ability of these strains to express his genes was tested by complementation to determine whether Rho-strains initiated transcripts at sites other than the previously described internal promoters.…”

Regulation of Salmonella typhimurium ilvYC genes

“…If antitermination occurs, readthrough transcription of the adjacent galK gene should be detected as increased galK expression. The pDU71-determined galK activity was increased to 15 U when measured in a S. typhimurium rho mutant, AA111 (12). This level of activity was much lower than that seen with ilvY-galK fusion plasmids such as pDU71.1, suggesting that most transcription from py was still properly terminated.…”