Elevated Mutation Frequency in Surviving Populations of Carbon-Starved rpoS -Deficient Pseudomonas putida Is Caused by Reduced Expression of Superoxide Dismutase and Catalase

Abstract

ABSTRACT RpoS is a bacterial sigma factor of RNA polymerase which is involved in the expression of a large number of genes to facilitate survival under starvation conditions and other stresses. The results of our study demonstrate that the frequency of emergence of base substitution mutants is significantly increased in long-term-starved populations of rpoS -deficient Pseudomonas putida cells. The increasing effect of the lack of RpoS on the mutation frequency became apparent in both a plasmid-based test system measuring Phe + reversion and a chromosomal rpoB system detecting rifampin-resistant mutants. The elevated mutation frequency coincided with the death of about 95% of the cells in a population of rpoS -deficient P . putida . Artificial overexpression of superoxide dismutase or catalase in the rpoS -deficient strain restored the survival of cells and resulted in a decline in the mutation frequency. This indicated that, compared to wild-type bacteria, rpoS -deficient cells are less protected against damage caused by reactive oxygen species. 7,8-Dihydro-8-oxoguanine (GO) is known to be one of the most stable and frequent base modifications caused by oxygen radical attack on DNA. However, the spectrum of base substitution mutations characterized in rpoS -deficient P . putida was different from that in bacteria lacking the GO repair system: it was broader and more similar to that identified in the wild-type strain. Interestingly, the formation of large deletions was also accompanied by a lack of RpoS. Thus, the accumulation of DNA damage other than GO elevates the frequency of mutation in these bacteria. It is known that oxidative damage of proteins and membrane components, but not that of DNA, is a major reason for the death of cells. Since the increased mutation frequency was associated with a decline in the viability of bacteria, we suppose that the elevation of the mutation frequency in the surviving population of carbon-starved rpoS -deficient P . putida may be caused both by oxidative damage of DNA and enzymes involved in DNA replication and repair fidelity.