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Characterization of a cold-sensitive hisW mutant of Salmonella typhimurium
Abstract:
Previous studies of hisW mutants of Salmonella typhimurium have led to the suggestion that such strains are defective in tRNA maturation. (J. E. Brenchley and J. Ingraham, J. Bacteriol. 114:528-536, 1973). In this study, we report that one hisW strain is defective in the accumulation of all stable RNA species. Polyacrylamide gel electrophoresis of radiolabeled RNA indicated tha at the nonpermissive temperature (20 degrees C) all stable RNa species in the cold-sensitive hisW3333 mutant were synthesized and rapidly degraded. We propose that the cold sensitivity of this strain is caused by such a restriction in stable RNA accumulation at low temperature. In vitro and in vivo studies demonstrated that the RNA degraded in this strain was synthesized de novo and was not preexisting RNA. Furthermore, physiological and genetic recovery from the cold-sensitive hisW phenotype resulted in relatively normal RNA synthesis and accumulation. Thus, the RNA alterations observed in this strain were not explained by defects in a tRNA modification enzyme. Rather, these findings suggest the existence of defective RNA processing and that a control mechanism for the overall synthesis or accumulation of stable RNA species is altered in the hisW3333 mutant.
Insights
Salmonella typhimurium hisW mutants show cold sensitivity due to rapid degradation of all stable RNA species at low temperatures. This suggests a defect in RNA processing rather than tRNA modification.
Area of Science:
- Microbiology
- Molecular Biology
- Bacterial Genetics
Background:
- Previous research suggested hisW mutants of Salmonella typhimurium are deficient in transfer RNA (tRNA) maturation.
- The hisW gene's specific role in RNA metabolism remained unclear.
Purpose of the Study:
- To investigate the molecular basis of cold sensitivity in a specific hisW mutant (hisW3333) of Salmonella typhimurium.
- To determine if the defect lies in tRNA maturation or a broader RNA regulation pathway.
Main Methods:
- Utilized polyacrylamide gel electrophoresis to analyze radiolabeled RNA synthesis and degradation in the hisW3333 mutant at permissive and non-permissive temperatures.
- Conducted in vitro and in vivo studies to differentiate between de novo synthesized and pre-existing RNA.
- Examined RNA synthesis and accumulation during physiological and genetic recovery from the cold-sensitive phenotype.
Main Results:
- The hisW3333 mutant exhibited cold sensitivity at 20°C, characterized by the synthesis and rapid degradation of all stable RNA species.
- Degraded RNA was confirmed to be newly synthesized (de novo), not pre-existing.
- Recovery from the cold-sensitive phenotype restored normal RNA synthesis and accumulation.
Conclusions:
- The cold sensitivity of the hisW3333 mutant is attributed to a defect in stable RNA accumulation at low temperatures.
- Findings indicate a problem with RNA processing or a regulatory mechanism controlling stable RNA synthesis/accumulation, not a tRNA modification enzyme defect.