A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase The GenBank accession number for the sequence reported in this paper is AJ278609.

Abstract

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 O 2 -starved stationary-phase survival. One exception was 329B, a purine auxotroph, which showed a precipitous reduction in viability from 10 8 to 10 3 c.f.u. ml N1 during the first 5-10 d in O 2 -starved stationary phase. This was followed by an equally rapid recovery in culturability to a level within 10-100-fold of wildtype 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 O 2 -starvation phenotype. The recovery in culturabilty of the purF mutant in O 2 -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 O 2 -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.

Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation

“…4). It has been shown previously that these mutants lose culturability in stationary phase after growth under oxygen-limited conditions (Keer et al, 2001;O'Toole et al, 2003). This behaviour was reproduced in the present study (Fig.…”

“…Two possible explanations have been proposed to explain the recovery in viability of purF and devR mutants of Myc. smegmatis: (a) resuscitation of NC cells and (b) regrowth of surviving cells (Keer et al, 2001;O'Toole et al, 2003). The present study, in which MPN conditions were employed, confirms the first possibility.…”

“…For plasmid-containing strains, growth media were supplemented with hygromycin (50 mg ml 21 ). Sauton's medium (0?5 g KH 2 PO 4 l 21 , 0?5 g MgSO 4 l 21 , 4 g L-asparagine l 21 , 60 ml glycerol, 0?05 g ferric ammonium citrate l 21 , 2 g citric acid l 21 , 0?1 ml of 1 % ZnSO 4 l 21 ; Connell, 1994) )] containing kanamycin (10 mg ml 21 ) or hygromycin (50 mg ml 21 ) in 30 ml culture tubes for 48 h as described by Keer et al (2001). Kanamycin (10 mg ml 21 ) was also added during growth of the mutants in mHdeB medium.…”

“…We therefore examined whether mutant strains, in which metabolic adjustments are necessitated by genetic change, are more susceptible to loss of culturability than the wild-type. Williams and his colleagues (Keer et al, 2001;O'Toole et al, 2003) have reported what appeared to be a transient decrease in culturability of Myc. smegmatis mutants defective either in purine metabolism (purF) or in a two-component regulation system (devR) when they were grown under oxygenlimited conditions.…”

Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase

“…Recently, Keer et al (2000) have found that several mutants of Mycobacterium smegmatis, defective in stationary phase survival under aerobic or anaerobic conditions, revealed a transient decrease in viability followed by an increase in culturability. The authors suggest at least two possible explanations for this effect : (a) re-growth (cryptic growth) of viable cells, and (b) formation of ' non-culturable ' (and possibly dormant) bacteria in stationary phase and their subsequent resuscitation (Keer et al, 2000(Keer et al, , 2001. Since the authors did not measure culturability by MPN counts, direct evidence was neither sought nor obtained for resuscitation of non-culturable cells.…”