Microbial Ecology

, Volume 59, Issue 2, pp 296–310 | Cite as

Structural Changes and Cellular Localization of Resuscitation-Promoting Factor in Environmental Isolates of Micrococcus luteus

  • Viktoria Koltunov
  • Charles L. GreenblattEmail author
  • Anna V. Goncharenko
  • Galya R. Demina
  • Benjamin Y. Klein
  • Michael Young
  • Arseny S. Kaprelyants
Environmental Microbiology


Dormancy among nonsporulating actinobacteria is now a widely accepted phenomenon. In Micrococcus luteus, the resuscitation of dormant cells is caused by a small secreted protein (resuscitation-promoting factor, or Rpf) that is found in “spent culture medium.” Rpf is encoded by a single essential gene in M. luteus. Homologs of Rpf are widespread among the high G + C Gram-positive bacteria, including mycobacteria and streptomycetes, and most organisms make several functionally redundant proteins. M. luteus Rpf comprises a lysozyme-like domain that is necessary and sufficient for activity connected through a short linker region to a LysM motif, which is present in a number of cell-wall-associated enzymes. Muralytic activity is responsible for resuscitation. In this report, we characterized a number of environmental isolates of M. luteus, including several recovered from amber. There was substantial variation in the predicted rpf gene product. While the lysozyme-like and LysM domains showed little variation, the linker region was elongated from ten amino acid residues in the laboratory strains to as many as 120 residues in one isolate. The genes encoding these Rpf proteins have been characterized, and a possible role for the Rpf linker in environmental adaptation is proposed. The environmental isolates show enhanced resistance to lysozyme as compared with the laboratory strains and this correlates with increased peptidoglycan acetylation. In strains that make a protein with an elongated linker, Rpf was bound to the cell wall, rather than being released to the growth medium, as occurs in reference strains. This rpf gene was introduced into a lysozyme-sensitive reference strain. Both rpf genes were expressed in transformants which showed a slight but statistically significant increase in lysozyme resistance.


Lysozyme Reference Strain Peptidoglycan Linker Region Environmental Isolate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors are grateful to the Center for the Study of Emerging Diseases and the “Program for Molecular and Cellular Biology” of the Russian Academy of Science for their generous financial support. We thank Maria Ines Zylber for helping with the figures.


  1. 1.
    Artzatbanov VYu, Ostrovsky DN (1990) The distribution of electron flow in the branched respiratory chain of Micrococcus luteus. Biochem J 266:481–486PubMedGoogle Scholar
  2. 2.
    Barsukov LI, Kulikov VI, Bergelson LD (1976) Lipid transfer proteins as a tool in the study of membrane structure inside–outside distribution of the phospholipids in the protoplasmic membrane of Micrococcus lysodeikticus. Biochem Biophys Res Commun 71:704–711CrossRefPubMedGoogle Scholar
  3. 3.
    Bateman A, Bycroft M (2000) The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD). J Mol Biol 299:1113–1119CrossRefPubMedGoogle Scholar
  4. 4.
    Bera A, Biswas R, Herbert S, Gotz F (2006) The presence of peptidoglycan O-acetyltransferase in various staphylococcal species correlates with lysozyme resistance and pathogenicity. Infect Immun 74:4598–46045CrossRefPubMedGoogle Scholar
  5. 5.
    Blokpoel MCJ, Murphy HN, O'Toole R, Wiles S, Runn ESC, Stewart GR, Young DB, Robertson BD (2005) Tetracycline-inducible gene regulation in mycobacteria. Nucl Acids Res 33:e22CrossRefPubMedGoogle Scholar
  6. 6.
    Brumfitt W, Wardlaw AC Park JT (1958) Development of lysozyme-resistance in Micrococcus lysodeikticus and its association with an increased O-acetyl content of the cell wall. Nature 181:1783–1784CrossRefPubMedGoogle Scholar
  7. 7.
    Cohen-Gonsaud M, Keep NH, Davies AP, Ward J, Henderson B, Labesse G (2004) Resuscitation-promoting factors possess a lysozyme-like domain. Trends Biochem Sci 29:7–10CrossRefPubMedGoogle Scholar
  8. 8.
    Colobert L (1957) Alleged extraction of protoplasts from Micrococcus lysodeikticus and Sarcina lutea by controlled action of lysozyme. C R Séances Soc Biol Fil 151:114–116PubMedGoogle Scholar
  9. 9.
    Dupont C, Clarke AJ (1991) Dependence of lysozyme-catalysed solubilization of Proteus mirabilis peptidoglycan on the extent of O-acetylation. Eur J Biochem 195:763–769CrossRefPubMedGoogle Scholar
  10. 10.
    Greenblatt CL, Baum J, Klein BY, Nachshon S, Koltunov V, Cano RJ (2004) Micrococcus luteus—survival in amber. Microb Ecol 48:120–127CrossRefPubMedGoogle Scholar
  11. 11.
    Greenblatt CL, Davis A, Clement BG, Kitts CL, Cox T, Cano RJ (1999) Diversity of microorganisms isolated from amber. Microb Ecol 38:58–68CrossRefPubMedGoogle Scholar
  12. 12.
    Grossowicz N, Ariel M (1963) Mechanism of protection of cells by spermine against lysozyme-induced lysis. J Bacteriol 85:293–300PubMedGoogle Scholar
  13. 13.
    Hartmann M, Barsch A, Niehaus K, Puhler A, Tauch A, Kalinowski J (2004) The glycosylated cell surface protein Rpf2, containing a resuscitation-promoting factor motif, is involved in intercellular communication of Corynebacterium glutamicum. Arch Microbiol 182:299–312CrossRefPubMedGoogle Scholar
  14. 14.
    Kaprelyants AS, Kell DB (1993) Dormancy in stationary-phase cultures of Micrococcus luteus: flow cytometric analysis of starvation and resuscitation. Appl Environ Microbiol 59:3187–3196PubMedGoogle Scholar
  15. 15.
    Keep NH, Ward JM, Cohen-Gonsaud M, Henderson B (2006) Wake up! Peptidoglycan lysis and bacterial non-growth states. Trends Microbiol 14:271–276CrossRefPubMedGoogle Scholar
  16. 16.
    Koltunova V (2008) Doctoral thesis, Hebrew UniversityGoogle Scholar
  17. 17.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  18. 18.
    Litwack G (1958) Development of Micrococcus lysodeikticus resistant to lysozyme. Nature 181:1348–1350CrossRefPubMedGoogle Scholar
  19. 19.
    Matsuda M, Togo M, Kagawa S, Moore JE (2001) PCR cloning of the resuscitation-promoting factor (Rpf) gene from Micrococcus luteus, sequencing and expression in Escherichia coli. Microbios 104:55–61PubMedGoogle Scholar
  20. 20.
    Mukamolova GV, Kaprelyants AS, Young DI, Young M, Kell DB (1998) A bacterial cytokine. Proc Natl Acad Sci U S A 95:8916–8921CrossRefPubMedGoogle Scholar
  21. 21.
    Mukamolova GV, Murzin AG, Salina EG, Demina GR, Kell DB, Kaprelyants AS, Young M (2006) Muralytic activity of Micrococcus luteus Rpf and its relationship to physiological activity in promoting bacterial growth and resuscitation. Mol Microbiol 59:84–98CrossRefPubMedGoogle Scholar
  22. 22.
    Mukamolova GV, Turapov OA, Kazarian K, Telkov M, Kaprelyants AS, Kell DB, Young M (2002) The rpf gene of Micrococcus luteus encodes an essential secreted growth factor. Mol Microbiol 46:611–621CrossRefPubMedGoogle Scholar
  23. 23.
    Mukamolova GV, Turapov OA, Young DI, Kaprelyants AS, Kell DB, Young M (2002) A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol 46:623–635CrossRefPubMedGoogle Scholar
  24. 24.
    Peleg A, Shifrin Y, Ophir I, Nadler-Yona C, Nov S, Koby S, Baruch K, Altuvia S, Elgrably-Weiss M, Abe CM, Knutton S, Saper MA, Rosenshine I (2005) Identification of an Escherichia coli operon for formation of the O-antigen capsule. J Bacteriol 187:5259–5266CrossRefPubMedGoogle Scholar
  25. 25.
    Perraudin JP, Prieels JP (1982) Lactoferrin binding to lysozyme-treated Micrococcus luteus. Biochim Biophys Acta 718:42–48PubMedGoogle Scholar
  26. 26.
    Rosenthal RS, Blundell JK, Perkins HR (1982) Strain-related differences in lysozyme sensitivity and extent of O-acetylation of gonococcal peptidoglycan. Infect Immun 37:826–829PubMedGoogle Scholar
  27. 27.
    Salina EG, Vostroknutova GN, Shleeva MO, Kaprelyants AS (2006) Cell–cell interactions during the formation and reactivation of “nonculturable” mycobacteria. Microbiology 75:432–437CrossRefGoogle Scholar
  28. 28.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  29. 29.
    Votyakova TV, Artzatbanov VYu, Mukamolova GV, Kaprelyants AS (1994) On the relationship between bacterial cell integrity and respiratory chain activity: a fluorescence anisotropy study. Arch Biochem Biophys 314:280–283CrossRefPubMedGoogle Scholar
  30. 30.
    Weis WI, Drickamer K (1996) Structural basis of lectin-carbohydrate recognition. Ann Rev Biochem 65:441–473CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Viktoria Koltunov
    • 1
  • Charles L. Greenblatt
    • 1
    Email author
  • Anna V. Goncharenko
    • 2
  • Galya R. Demina
    • 2
  • Benjamin Y. Klein
    • 3
  • Michael Young
    • 4
  • Arseny S. Kaprelyants
    • 2
  1. 1.Kuvin Centre and Department of Microbiology and Molecular GeneticsHebrew UniversityJerusalemIsrael
  2. 2.Bakh Institute of BiochemistryRussian Academy of SciencesMoscowRussia
  3. 3.Department of NeuroscienceColumbia UniversityNew YorkUSA
  4. 4.Institute of Biological Environmental and Rural SciencesUniversity of WalesAberystwythUK

Personalised recommendations