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.
BogosianG., NoelleD., AardemaE. V., BourneufE. V., MorrisP. J. L., O’NeilJ. P.2000; Recovery of hydrogen peroxide-sensitive culturable cells of Vibrio vulnificus gives the appearance of resuscitation from a viable but nonculturable state. J Bacteriol 182:5070–5075[CrossRef]
ColeS. T., BroschR., ParkhillJ.39 other authors1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544[CrossRef]
GoldmanB. S., GabbertK. K., KranzR. G.1996; The temperature-sensitive growth and survival phenotypes of Escherichia coli cydDC and cydAB strains are due to deficiencies in cytochrome bd and are corrected by catalase and reducing agents. J Bacteriol 178:6348–6351
GuilhotC., OtalI., Van RompaeyI., MartinC., GicquelB.1994; Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176:535–539
HartmansS., De BontJ. A. M.1992; The genus Mycobacterium – nonmedical. In The Prokaryotes, 2nd edn. vol. II pp. 1215–1237Edited byBalowsA.others New York: Springer;
HowardN. S., GomezJ. E., KoC., BishaiW. R.1995; Color selection with a hygromycin-resistance-based Escherichia coli–mycobacterial shuttle vector. Gene 166:181–182[CrossRef]
HuY., CoatesA. R. M.1999a; Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase Mycobacterium tuberculosis. J Bacteriol 181:469–476
HuY., CoatesA. R. M.1999b; Transcription of the stationary-phase-associated hspX gene of Mycobacterium tuberculosis is inversely related to synthesis of the 16-kilodalton protein. J Bacteriol 181:1380–1387
HuY., ButcherP. D., ManganJ. A., RajandreamM. A., CoatesA. R. M.1999; Regulation of hmp gene transcription in Mycobacterium tuberculosis: effects of oxygen limitation and nitrosative and oxidative stress. J Bacteriol 181:3486–3493
KellD. B., KaprelyantsA. S., WeichartD. H., HarwoodC. R., BarerM. B.1988; Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Leeuwenhoek 73:169–187
RalluF., GrussA., EhrlichS. D., MaguinE.2000; Acid- and multistress-resistant mutants of Lactococcus lactis: identification of intracelluar stress signals. Mol Microbiol 35:517–528
SiegeleD., KolterR.1993; Isolation and characterisation of an Escherichia coli mutant defective in resuming growth after starvation. Genes Dev 7:2629–2640[CrossRef]
SiegeleD., ImlayK. R. C., ImlayJ. A.1996; The stationary-phase-exit defect of cydC (surB) mutants is due to the lack of a functional terminal cytochrome oxidase. J Bacteriol 178:6091–6096
SoberonM., LopezO., MirandaJ., TabcheM. L., MoreraC.1997; Genetic evidence for 5 aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase complex cbb3 production in Rhizobium etli. Mol Gen Genet 254:665–673[CrossRef]
WayneL. G., HayesL. G.1996; An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64:2062–2069
YuanY., CraneD. D., BarryC. E.III1996; Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog. J Bacteriol 178:4484–4492
A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phaseThe GenBank accession number for the sequence reported in this paper is AJ278609.