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A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase

Department of Biology, Imperial College of Science, Technology and Medicine, Imperial College Road, London SW7 2AZ, UK¹

Author for correspondence: Huw D. Williams. Tel: +44 20 75945383. Fax: +44 20 75842056. e-mail: h.d.williams{at}ic.ac.uk

In this study it was demonstrated that a range of transposon mutants of Mycobacterium smegmatis, previously described as having impaired survival in carbon-starved stationary phase, were not markedly affected in O₂-starved stationary-phase survival. One exception was 329B, a purine auxotroph, which showed a precipitous reduction in viability from 10⁸ to 10³ c.f.u. ml^-1 during the first 5–10 d in O₂-starved stationary phase. This was followed by an equally rapid recovery in culturability to a level within 10–100-fold of wild-type levels by 10–20 d into stationary phase. Transduction of the mutation into a clean genetic background demonstrated that the phenotype was due to the transposon insertion, which was shown to be in the purF gene. purF encodes phosphoribosylpyrophosphate amidotransferase, which catalyses the first committed step in purine biosynthesis. The M. smegmatis purF gene, which encodes a protein with a very high degree of similarity to the PurF homologues of Mycobacterium tuberculosis and Mycobacterium leprae, was cloned and shown to substantially complement the O₂-starvation phenotype. The recovery in culturabilty of the purF mutant in O₂-starved stationary phase did not involve movement of the transposon. In addition, when cells that had recovered culturability were retested, their survival kinetics in stationary phase were identical to the original culture, indicating that their recovery was not explained by the accumulation of suppressor mutations. It is concluded that the survival curve in O₂-starved stationary phase for the purF mutant represents its true phenotype and is not a result of subsequent genetic changes in the culture. It is argued that the purF cells lose culturability for a finite period of time in stationary phase. Whether this is due to a fraction of the population dying and then regrowing using a previously undiscovered fermentation pathway, or becoming transiently dormant, or entering an active nonculturable state and subsequently undergoing resuscitation cannot be distinguished at this stage.

Keywords: dormancy, Mycobacterium tuberculosis, purine, VBNC, oxygen

The GenBank accession number for the sequence reported in this paper is AJ278609.

^a Present address: LGC, Queens Road, Middlesex TW11 0LY, UK.

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