1887

Abstract

In this study it was demonstrated that a range of transposon mutants of , previously described as having impaired survival in carbon-starved stationary phase, were not markedly affected in O-starved stationary-phase survival. One exception was 329B, a purine auxotroph, which showed a precipitous reduction in viability from 10 to 10 c.f.u. ml during the first 5–10 d in O-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 gene. encodes phosphoribosylpyrophosphate amidotransferase, which catalyses the first committed step in purine biosynthesis. The gene, which encodes a protein with a very high degree of similarity to the PurF homologues of and , was cloned and shown to substantially complement the O-starvation phenotype. The recovery in culturabilty of the mutant in O-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-starved stationary phase for the mutant represents its true phenotype and is not a result of subsequent genetic changes in the culture. It is argued that the 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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-2-473
2001-02-01
2024-11-04
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/2/1470473a.html?itemId=/content/journal/micro/10.1099/00221287-147-2-473&mimeType=html&fmt=ahah

References

  1. Barer M. R. 1997; Viable but non-culturable and dormant bacteria: time to resolve an oxymoron and a misnomer?. J Med Microbiol 46:629–631
    [Google Scholar]
  2. Barer M. R., Harwood C. R. 1999; Bacterial viability and culturability. Adv Microb Physiol 41:93–137
    [Google Scholar]
  3. Bogosian G., Noelle D., Aardema E. V., Bourneuf E. V., Morris P. J. L., O’Neil J. 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]
    [Google Scholar]
  4. Cole S. T., Brosch R., Parkhill J.39 other authors 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
    [Google Scholar]
  5. Cunningham A. F., Spreadbury C. L. 1998; Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton α-crystallin homolog. J Bacteriol 180:801–808
    [Google Scholar]
  6. Dick T., Lee B. H., Murugasu-Oei B. 1998; Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 163:159–164 [CrossRef]
    [Google Scholar]
  7. Duwat P., Ehrlich S. D., Gruss A. 1999; Effects of metabolic flux on stress response pathways in Lactococcus lactis. Mol Microbiol 31:845–858 [CrossRef]
    [Google Scholar]
  8. Garcı́a-Horsman J. A., Barquera B., Rumbley J., Ma J., Gennis R. B. 1994; The superfamily of haem-copper respiratory oxidases. J Bacteriol 176:5587–5600
    [Google Scholar]
  9. Goldman B. S., Gabbert K. K., Kranz R. 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
    [Google Scholar]
  10. Guilhot C., Otal I., Van Rompaey I., Martin C., Gicquel B. 1994; Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176:535–539
    [Google Scholar]
  11. Hartmans S., De Bont J. A. M. 1992; The genus Mycobacterium – nonmedical. In The Prokaryotes, 2nd edn. vol. II pp. 1215–1237Edited by Balows A.others New York: Springer;
    [Google Scholar]
  12. Howard N. S., Gomez J. E., Ko C., Bishai W. R. 1995; Color selection with a hygromycin-resistance-based Escherichia coli–mycobacterial shuttle vector. Gene 166:181–182 [CrossRef]
    [Google Scholar]
  13. Hu Y., Coates A. R. M. 1999a; Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase Mycobacterium tuberculosis. J Bacteriol 181:469–476
    [Google Scholar]
  14. Hu Y., Coates A. 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
    [Google Scholar]
  15. Hu Y., Butcher P. D., Mangan J. A., Rajandream M. A., Coates A. 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
    [Google Scholar]
  16. Hutter B., Dick T. 1998; Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 167:7–11 [CrossRef]
    [Google Scholar]
  17. Keer J., Smeulders M. J., Gray K. M., Williams H. D. 2000; Mutants of Mycobacterium smegmatis impaired in stationary-phase survival. Microbiology 146:2209–2217
    [Google Scholar]
  18. Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. R., Barer M. B. 1988; Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie Leeuwenhoek 73:169–187
    [Google Scholar]
  19. Lee B. H., Murugasu-Oei B., Dick T. 1998; Upregulation of a histone-like protein in dormant Mycobacterium smegmatis. Mol Gen Genet 260:475–479 [CrossRef]
    [Google Scholar]
  20. Lim A., Eleuterio M., Hutter B., Murugasu-Oei B., Dick T. 1999; Oxygen depletion-induced dormancy in Mycobacterium bovis BCG. J Bacteriol 181:2252–2256
    [Google Scholar]
  21. Parrish N. M., Dick J. D., Bishai W. R. 1998; Mechanisms of latency in Mycobacterium tuberculosis. Trends Microbiol 6:107–112 [CrossRef]
    [Google Scholar]
  22. Rallu F., Gruss A., Ehrlich S. D., Maguin E. 2000; Acid- and multistress-resistant mutants of Lactococcus lactis: identification of intracelluar stress signals. Mol Microbiol 35:517–528
    [Google Scholar]
  23. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Siegele D., Kolter R. 1993; Isolation and characterisation of an Escherichia coli mutant defective in resuming growth after starvation. Genes Dev 7:2629–2640 [CrossRef]
    [Google Scholar]
  25. Siegele D., Imlay K. R. C., Imlay J. 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
    [Google Scholar]
  26. Smeulders M. J., Keer J., Speight R. A., Williams H. D. 1999; Adaptation of Mycobacterium smegmatis to stationary phase. J Bacteriol 181:270–283
    [Google Scholar]
  27. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919 [CrossRef]
    [Google Scholar]
  28. Soberon M., Lopez O., Miranda J., Tabche M. L., Morera C. 1997; Genetic evidence for 5 aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase complex cbb 3 production in Rhizobium etli. Mol Gen Genet 254:665–673 [CrossRef]
    [Google Scholar]
  29. Sundar Raj C. V., Ramakrishnan T. 1970; Transduction in Mycobacterium smegmatis. Nature 228:280–281
    [Google Scholar]
  30. Wayne L. G. 1994; Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13:908–914 [CrossRef]
    [Google Scholar]
  31. Wayne L. G., Hayes L. 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
    [Google Scholar]
  32. Whitesides M. D., Oliver J. D. 1997; Resuscitation of Vibrio vulnificus from the viable but nonculturable state. Appl Environ Microbiol 63:1002–1005
    [Google Scholar]
  33. Yuan Y., Crane D. D., Barry C. E. III 1996; Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog. J Bacteriol 178:4484–4492
    [Google Scholar]
/content/journal/micro/10.1099/00221287-147-2-473
Loading
/content/journal/micro/10.1099/00221287-147-2-473
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error