1887

Abstract

Conditions were investigated that promote the formation of ‘non-culturable’ (NC) cells of () in stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's–de Bont medium), bacteria adopted a stable NC state after 3–4 days incubation in stationary phase. Such conditions are not specific as and mutants of also showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's–de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-type resume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture with () cells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains of harbouring plasmids containing the gene also adopt a similar NC state after growth to stationary phase in modified Hartman's–de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis of Rpf. Resuscitation was not observed in the control strain harbouring a plasmid lacking . In contrast to wild-type, the NC cells of and mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.26893-0
2004-06-01
2024-03-28
Loading full text...

Full text loading...

/deliver/fulltext/micro/150/6/mic1501687.html?itemId=/content/journal/micro/10.1099/mic.0.26893-0&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. Barer M. R., Gribbon L. T., Harwood C. R., Nwoguh C. E. 1993; The viable but not culturable hypothesis and medical bacteriology. Rev Med Microbiol 4:183–191 [CrossRef]
    [Google Scholar]
  4. Barer M. R., Kaprelyants A. S., Weichart D. H., Harwood C. R., Kell D. B. 1998; Microbial stress and culturability: conceptual and operational domains. Microbiology 144:2009–2010 [CrossRef]
    [Google Scholar]
  5. Connell N. D. 1994; Mycobacterium: isolation, maintenance, transformation, and mutant selection. Methods Cell Biol 45:107–125
    [Google Scholar]
  6. de Man J. C. 1975; The probability of most probable numbers. Eur J Appl Microbiol 1:67–78 [CrossRef]
    [Google Scholar]
  7. Dye C., Scheele S., Dolin P., Pathania V., Raviglione R. C. 1999; Consensus statement. Global burden of tuberculosis – estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA (J Am Med Assoc) 282:677–686 [CrossRef]
    [Google Scholar]
  8. Gangadharam P. R. J. 1995; Mycobacterial dormancy. Tuber Lung Dis 76:477–479 [CrossRef]
    [Google Scholar]
  9. Grange J. M. 1992; The mystery of the mycobacterial persistor. Tuber Lung Dis 73:249–251 [CrossRef]
    [Google Scholar]
  10. Hu Y. M., Butcher P. D., Sole K., Mitchison D. A., Coates A. R. M. 1998; Protein synthesis is shut down in dormant Mycobacterium tuberculosis and is reversed by oxygen or heat shock. FEMS Microbiol Lett 158:139–145 [CrossRef]
    [Google Scholar]
  11. Kaprelyants A. S., Kell D. B. 1992; Rapid assessment of bacterial viability and vitality using rhodamine 123 and flow cytometry. J Appl Bacteriol 72:410–422 [CrossRef]
    [Google Scholar]
  12. Kaprelyants A. S., Kell D. B. 1993; Dormancy in stationary-phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59:3187–3196
    [Google Scholar]
  13. Kaprelyants A. S., Kell D. B. 1996; Do bacteria need to communicate with each other for growth?. Trends Microbiol 4:237–242 [CrossRef]
    [Google Scholar]
  14. Kaprelyants A. S., Gottschal J. C., Kell D. B. 1993; Dormancy in non-sporulating bacteria. FEMS Microbiol Rev 104:271–286 [CrossRef]
    [Google Scholar]
  15. Kaprelyants A. S., Mukamolova G. V., Kell D. B. 1994; Estimation of dormant Micrococcus luteus cells by penicillin lysis and by resuscitation in cell-free spent medium at high dilution. FEMS Microbiol Lett 115:347–352 [CrossRef]
    [Google Scholar]
  16. Kaprelyants A. S., Mukamolova G. V., Kormer S. S., Weichart D. H., Young M., Kell D. B. 1999; Intercellular signalling and the multiplication of prokaryotes: bacterial cytokines. In Microbial Signalling and Communication (Society for General Microbiology Symposium no. 57) pp . 33–69 Edited by England R., Hobbs G., Bainton N. D., Roberts McL. Cambridge: Cambridge University Press;
    [Google Scholar]
  17. Keer J., Smeulders M. J., Williams H. D. 2001; A purF mutant of Mycobacterium smegmatis has impaired survival during oxygen-starved stationary phase. Microbiology 147:473–481
    [Google Scholar]
  18. Kell D. B., Young M. 2000; Bacterial dormancy and culturability: the role of autocrine growth factors. Curr Opin Microbiol 3:238–243 [CrossRef]
    [Google Scholar]
  19. Kell D. B., Kaprelyants A. S., Weichart D. H., Harwood C. R., Barer M. R. 1998; Viability and activity in readily culturable bacteria: a review and discussion of the practical issues. Antonie van Leeuwenhoek 73:169–187 [CrossRef]
    [Google Scholar]
  20. Kell D. B., Mukamolova G. V., Finan C. L., Zhao H., Goodacre R., Kaprelyants A. S., Young M. 2003; Resuscitation of ‘uncultured’ microorganisms. In Microbial Diversity and Bioprospecting Edited by Bull A. T. Washington, DC: American Society for Microbiology;
    [Google Scholar]
  21. 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]
  22. 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]
  23. McCune R. M., Feldmann F. M., Lambert H. P., McDermott W. 1966a; Microbial persistence. I. The capacity of tubercle bacilli to survive sterilization in mouse tissues. J Exp Med 123:445–468 [CrossRef]
    [Google Scholar]
  24. McCune R. M., Feldmann F. M., McDermott W. 1966b; Microbial persistence. II. Characteristics of the sterile state of tubercle bacilli. J Exp Med 123:469–486 [CrossRef]
    [Google Scholar]
  25. Mukamolova G. V., Kormer S. S., Yanopolskaya N. D., Kaprelyants A. S. 1995; Properties of dormant cells in stationary-phase cultures of Micrococcus luteus during prolonged incubation. Microbiology (English translation of Mikrobiologiya) 64:284–288
    [Google Scholar]
  26. Mukamolova G. V., Yanopolskaya N. D., Kell D. B., Kaprelyants A. S. 1998a; On resuscitation from the dormant state of Micrococcus luteus. Antonie van Leeuwenhoek 73:237–243 [CrossRef]
    [Google Scholar]
  27. Mukamolova G. V., Kaprelyants A. S., Young D. I., Young M., Kell D. B. 1998b; A bacterial cytokine. Proc Natl Acad Sci U S A 95:8916–8921 [CrossRef]
    [Google Scholar]
  28. Mukamolova G. V., Turapov O. A., Young D. I., Kaprelyants A. S., Kell D. B., Young M. 2002a; A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol 46:623–635 [CrossRef]
    [Google Scholar]
  29. Mukamolova G. V., Turapov O. A., Kazaryan K., Telkov M., Kaprelyants A. S., Kell D. B., Young M. 2002b; The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Mol Microbiol 46:611–621 [CrossRef]
    [Google Scholar]
  30. Mukamolova G. V., Kaprelyants A. S., Kell D. B., Young M. 2003; Adoption of the transiently non-culturable state – a bacterial survival strategy?. Adv Microb Physiol 47:65–129
    [Google Scholar]
  31. Nystrom T. 1995; The trials and tribulations of growth arrest. Trends Microbiol 3:131–136 [CrossRef]
    [Google Scholar]
  32. Nystrom T. 2001; Not quite dead enough: on bacterial life, culturability, senescence, and death. Arch Microbiol 176:159–164 [CrossRef]
    [Google Scholar]
  33. Nystrom T. 2003; Conditional senescence in bacteria: death of the immortals. Mol Microbiol 48:17–23 [CrossRef]
    [Google Scholar]
  34. O'Toole R., Smeulders M. J., Blokpoel M. C., Kay E. J., Lougheed K., Williams H. D. 2003; A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis. J Bacteriol 185:1543–1554 [CrossRef]
    [Google Scholar]
  35. Parish T., Mahenthiralingam E., Draper P., Davis E. O., Colston M. J. 1997; Regulation of the inducible acetamidase gene of Mycobacterium smegmatis. Microbiology 143:2267–2276 [CrossRef]
    [Google Scholar]
  36. Parrish N. M., Dick J. D., Bishai W. R. 1998; Mechanisms of latency in Mycobacterium tuberculosis. Trends Microbiol 6:107–112 [CrossRef]
    [Google Scholar]
  37. Shleeva M. O., Bagramyan K., Telkov M. V., Mukamolova G. V., Young M., Kell D. B., Kaprelyants A. S. 2002; Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase. Microbiology 148:1581–1591
    [Google Scholar]
  38. 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]
  39. Snapper S., Melton R., Kieser T., Jacobs W. R. 1990; Isolation and characterisation of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919 [CrossRef]
    [Google Scholar]
  40. Votyakova T. V., Kaprelyants A. S., Kell D. B. 1994; Influence of viable cells on the resuscitation of dormant cells in Micrococcus luteus cultures held in an extended stationary phase: the population effect. Appl Environ Microbiol 60:3284–3291
    [Google Scholar]
  41. Wayne L. G. 1994; Dormancy of Mycobacterium tuberculosis and latency of disease. Eur J Clin Microbiol Infect Dis 13:908–914 [CrossRef]
    [Google Scholar]
  42. Wayne L. G., Hayes L. G. 1996; An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through 2 stages of nonreplicating persistence. Infect Immun 64:2062–2069
    [Google Scholar]
  43. Wayne L. G., Sohaskey C. D. 2001; Nonreplicating persistence of Mycobacterium tuberculosis. Annu Rev Microbiol 55:139–163 [CrossRef]
    [Google Scholar]
  44. Zhu W., Plikaytis B. B., Shinnick T. M. 2003; Resuscitation factors from mycobacteria: homologs of Micrococcus luteus proteins. Tuberculosis (Edinb) 83:261–269 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.26893-0
Loading
/content/journal/micro/10.1099/mic.0.26893-0
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