Edinburgh Napier University

  Although the starvation or non-growth state is probably the most common physiological state of bacteria, it has been studied in relatively few organisms. In spite of its importance in pathogenesis, bioremediation and several industrial processes, limited research has been performed on Rhodococcus under starvation/stationary phase conditions. The objectives of this study were to analyse the physiological adaptation of Rhodococcus erythropolis SQ1 to starvation/stationary phase, and to generate and screen a bank of mutants to identify genetic elements involved in this adaptation. It was found that R. erythropolis SQ1 can survive for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1 % w/v) or low (0.1 % w/v) glucose concentrations. Early stationary phase R. erythropolisSQ1 cells grown in 0.1 % glucose also exhibited enhanced resistance to heat and oxidative stress compared with exponential phase cells. A mutant bank of 898 R. erythropolis SQ1 mutants was generated and screened; four mutants were of particular interest. The culturability of mutants 4G6 and 10D3 dropped to <0.1 % of the maximum CFU/ml at 27 days incubation, and to <3 % of the maximum CFU/ml for mutants 1B2 and 1H1, when grown in 1 % glucose medium. No drop in culturability was observed when mutants were grown in 0.1 % glucose. Mutant 4G6 had a transposon insertion in uvrB (UvrB, part of the DNA excision repair mechanism), while the insertion for mutant 10D3 was immediately downstream of a putative guaB gene, which, based on bioinformatic analyses, is followed by another putative IMP dehydrogenase (guaB-like) and/or a cholesterol oxidase gene. In mutant 1H1 the transposon inserted 272 nucleotides downstream of a gene encoding a putative phosphoglycerate mutase and upstream of putative thioredoxin and cytochrome c biogenesis genes. In conclusion, R. erythropolis SQ1 was shown to present a classic starvation/stationary phase survival response, with the associated increase in resistance to various external stresses. A mutant bank has been generated which can be used in the future to analyse other phenotypes of interest. Several genes linked to starvation/stationary phase survival were identified. These findings show that a wide variety of genes are involved in starvation/stationary phase survival. Indeed, over 100 such genes have been identified in Escherichia coli and Mycobacterium tuberculosis.