Skip to main content
Log in

Salt Tolerance in Astragalus cicer Microsymbionts: The Role of Glycine Betaine in Osmoprotection

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

In this study, we have investigated intrinsic salt tolerance of Astragalus cicer microsymbionts (USDA3350, ACMP18) and the role of exogenous glycine betaine in osmoprotection in these bacteria. Salt stress was imposed by NaCl concentrations ranging from 0.5 to 2 %. A. cicer mesorhizobia were capable of tolerating up to 2 % sodium chloride with a population count that was inversely proportional to the salt content. When the extracellular concentration of NaCl was raised to 2 %, the generation time of the UDSA3350 strain in the mid-exponential phase of growth was 3.9-times greater than that in the no-salt control medium, whereas the ACMP18 strain survived under the same conditions but did not multiply. Application of 1 mM glycine betaine into the salt-stressed rhizobium cultures increased the number of culturable bacteria, pointing out that this molecule was involved in restoration of osmotic balance. The decline in A. cicer symbiont viability in the medium with sodium chloride and the osmoprotective role of glycine betaine for these bacteria was confirmed in the experiment using the live/dead Bac Light Bacterial Vibility Kit. Data presented in this study showed the presence of proU-like genes in the genomes of A. cicer rhizobia with high sequence similarity to the genes of the ProU-like system in Sinorhizobium meliloti and the proU operon of Escherichia coli.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2

Similar content being viewed by others

References

  1. Alloing G, Travers I, Sagot B, Le Rudulier D, Dupont L (2006) Proline betaine uptake in Sinorhizobium meliloti: characterization of Prb, an opp-like ABC transporter regulated by both proline betaine and salinity stress. J Bacteriol 188(17):6308–6317

    Article  PubMed  CAS  Google Scholar 

  2. Alexander E, Pham D, Steck TR (1999) The viable-but-nonculturable condition is induced by cooper in Agrobacterium tumefaciens and Rhizobium leguminosarum. Appl Environ Microbiol 65:3754–3756

    PubMed  CAS  Google Scholar 

  3. Bernard T, Pocard JA, Perroud B, Le Rudulier D (1986) Variations in the response of salt-stressed Rhizobium strains to betaines. Arch Microbiol 143:359–364

    Article  CAS  Google Scholar 

  4. Boncompagni E, Østreås M, Poggi MC, Le Rudulier D (1999) Occurrence of choline and glycine betaine uptake and metabolism in the family Rhizobiaceae and their roles in osmoprotection. Appl Environ Microbiol 65:2072–2077

    PubMed  CAS  Google Scholar 

  5. Boncompagni E, Dupont L, Mignot T, Østeräs M, Lambert A, Poggi MC, Le Rudulier D (2000) Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake. J Bacteriol 182(13):3717–3725

    Article  PubMed  CAS  Google Scholar 

  6. Boscari A, Mandon K, Dupont L, Poggi MC, Le Rudulier D (2002) BetS is a major glycine betaine/proline betaine transporter required for early osmotic adjustment in Sinorhizobium meliloti. J Bacteriol 184:2654–2663

    Article  PubMed  CAS  Google Scholar 

  7. Botsford JL, Lewis TA (1990) Osmoregulation in Rhizobium meliloti: production of glutamic acid in response to osmotic stress. Appl Environ Microbiol 56:488–494

    PubMed  CAS  Google Scholar 

  8. Brígido C, Alexandre A, Oliveira S (2012) Transcriptional analysis of major chaperone genes in salt-tolerant and salt-sensitive mesorhizobia. Microbiol Res 167(10):623–629

    Article  PubMed  Google Scholar 

  9. Cleland D, Krader P, McCree C, Tang J, Emerson D (2004) Glycine betaine as a cryoprotectant for prokaryotes. J Microbiol Methods 58:31–38

    Article  PubMed  CAS  Google Scholar 

  10. Csonka LN, Epstein W (1996) Osmoregulation. In: Neidhardt FC (ed) Escherichia coli and Salmonella: Cellular and Molecular Biology. ASM Press, Washington DC, pp 1210–1223

    Google Scholar 

  11. Dardanelli MS, Gonzáles PS, Bueno MA, Ghittoni NE (2000) Synthesis, accumulation and hydrolysis of trehalose during growth of peanut rhizobia in hyperosmotic media. J Basic Microbiol 40:149–156

    Article  PubMed  CAS  Google Scholar 

  12. Dattananda CS, Gowrishankar J (1989) Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus. J Bacteriol 171:1915–1922

    PubMed  CAS  Google Scholar 

  13. Dupont L, Garcia I, Poggi MC, Alloing G, Mandon K, Le Rudulier D (2004) The Sinorhizobium meliloti ABC transporter Chois highly specific for choline and expressed in bacteroids from Medicago sativa nodules. J Bacteriol 186(18):5988–5996

    Article  PubMed  CAS  Google Scholar 

  14. Elkan GH (1992) Taxonomy of the rhizobia. Can J Microbiol 38:446–450

    Article  Google Scholar 

  15. Elsheikh EAE (1998) Effects of salt rhizobia and bradyrhizobia: a review. Ann Appl Biol 132:507–524

    Article  Google Scholar 

  16. Elsheikh EAE, Wood M (1990) Rhizobia and bradyrhizobia under salt stress: possible role of trehalose in osmoregulation. Lett Appl Microbiol 10:127–129

    Article  CAS  Google Scholar 

  17. Fox MA, Karunakaran R, Leonard ME, Mouhsine B, Williams A, East AK, Downie JA, Poole PS (2008) Rhizobia and bradyrhizobia under salt stress: possible role of trechalose in osmoregulation. FEMS Microbiol Lett 287(2):212–220

    Article  PubMed  CAS  Google Scholar 

  18. Fujihara S, Yoneyama T (1994) Response of Rhizobium fredii P220 to osmotic shock: interrelationships between K+, Mg2+, glutamate and homospermidine. Microbiol 140:1909–1916

    Article  CAS  Google Scholar 

  19. Gloux K, Le Rudulier D (1989) Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti. Arch Microbiol 151:143–148

    Article  CAS  Google Scholar 

  20. Goldman A, Lecoeur L, Massage B, Delarue M, Schoonejans E, Tapfer D (1994) Symbiotic plasmid genes essential to the catabolism of proline betaine, or stachydine, are also required for efficient nodulation by Rhizobium meliloti. FEMS Microbiol Lett 115:305–312

    Article  Google Scholar 

  21. Gowrishankar J (1989) Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli. J Bacteriol 171:1923–1931

    PubMed  CAS  Google Scholar 

  22. Kempf B, Bremer E (1998) Uptake and synthesis of compatible solutes as microbial stress responses to high osmolality environments. Arch Microbiol 170:319–330

    Article  PubMed  CAS  Google Scholar 

  23. Laranjo M, Oliveira S (2011) Tolerance of Mesorhizobium type strains to different environmental stresses. Antonie van Leeuvenhoek 99(3):651–662

    Article  CAS  Google Scholar 

  24. Le Rudulier D, Gloux K, Riou N (1991) Identification of an osmotically induced periplasmic glycine betaine-binding protein from Rhizobium meliloti. Biochim Biophys Acta 1061:197–205

    Article  PubMed  Google Scholar 

  25. Lippert K, Galinski EA (1992) Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol 37:61–64

    Article  CAS  Google Scholar 

  26. Lucht JM, Bremer E (1994) Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system ProU. FEMS Microbiol Rev 14:3–20

    Article  PubMed  CAS  Google Scholar 

  27. Mandon K, Østerås M, Boncompagni E, Trinchant JC, Spennato G, Poggi MC, Le Rudulier D (2003) The Sinorhizobium meliloti glycine betaine biosynthetic genes (betCBA) are induced by choline and highly expressed in bacteroids. Mol Plant-Microbe Interact 16:709–719

    Article  PubMed  CAS  Google Scholar 

  28. McDougald D, Rice SA, Weichart D, Kjelleberg S (1998) Nonculturability: adaption or debilitation. FEMS Microbiol Ecol 25:1–9

    Article  CAS  Google Scholar 

  29. Miller KJ, Wood JM (1996) Osmoadaptation by rhizosphere bacteria. Ann Rev Microbiol 50:101–106

    Article  CAS  Google Scholar 

  30. Pichereau V, Pocard JA, Hamelin J, Blanco C, Bernard T (1998) Differential effects of dimethylsulfoniopropionate, dimethylsulfonioacetate, and other S-Methylated compounds on the growth of Sinorhizobium meliloti at low and high osmolarities. Appl Environ Microbiol 64:1420–1429

    PubMed  CAS  Google Scholar 

  31. Pitcher DG, Saunders NA, Owen RJ (1989) Rapid extraction of bacterial genomic DNA with guanifium thiocyanate. Lett Appl Microbiol 8:151–156

    Article  CAS  Google Scholar 

  32. Racher KI, Voegele RT, Marshall EV (1999) Purification and reconstitution of an osmosensor: transporter ProP of Escherichia coli senses and responds to osmotic shifts. Biochemistry 38:1676–1684

    Article  PubMed  CAS  Google Scholar 

  33. Singleton PW, Elswaify SA, Bohlool BB (1982) Effect of salinity on Rhizobium growth and survival. Appl Environ Microbiol 44:884–890

    PubMed  CAS  Google Scholar 

  34. Sleator RD, Hill C (2001) Bacterial osmoadaptation: the role of osmolytes in bacteria stress and virulence. FEMS Microbiol 26:49–71

    Google Scholar 

  35. Smith LT, Pocard JA, Bernard T, Le Rudulier D (1988) Osmotic control of glycine betaine biosynthesis and degradation in Rhizobium meliloti. J Bacteriol 170:3142–3149

    PubMed  CAS  Google Scholar 

  36. Soussi M, Ocaña A, Lluch C (1998) Effects of salt stress on growth, photosynthesis and nitrogen fixation in chick-pea (Cicer arietinum L.). J Exper Bot 49:1329–1337

    CAS  Google Scholar 

  37. Talibart R, Jebbar M, Gouffi K, Pichereau V, Gouesbet G, Blanco C, Bernard T, Pocard JA (1997) Transient accumulation of glycine betaine and dynamics of endogenous osmolytes in salt-stressed cultures of Sinorhizobium meliloti. Appl Environ Microbiol 63:4657–4663

    PubMed  CAS  Google Scholar 

  38. Tempest DW, Meers JL, Brown CM (1970) Influence of environment on the content and composition of microbial free amino acid pools. J Gen Microbiol 64:171–185

    PubMed  CAS  Google Scholar 

  39. Toffanin A, Basaglia M, Ciardi C, Vian P, Povolo S, Casella S (2000) Energy content decrease and viable-not-culturable status induced by xygen limitation coupled to the presence of nitrogen oxides in Rhizobium hedysari. Biol Fertil Soils 31:484–488

    Article  CAS  Google Scholar 

  40. Trotman AP, Weaver RW (1995) Tolerace of clover rhizobia to heat and desiccation stresses in soil. Soil Sci Soc Am J 59:466–470

    Article  CAS  Google Scholar 

  41. Vincent J (1970) A manual for the practical study of root nodule bacteria. UK, Blackwell Scientific Publication Ltd, Oxford

    Google Scholar 

  42. Vriezen JAC, deBruijn FJ, Nüsslein K (2012) Desiccation induces viable but non-culturable cells in Sinorhizobium meliloti 1021. AMB Express 2:6

    Article  PubMed  Google Scholar 

  43. Vriezen JAC, deBruijn FJ, Nüsslein K (2007) Responses of rhizobia to desiccation in relation to osmotic stress, oxygen and temperature. Appl Environ Microbiol 73:3451–3459

    Article  PubMed  CAS  Google Scholar 

  44. Wdowiak S, Małek W (2000) Numerial analysis of Astragalus cicer microsymbionts. Curr Microbiol 41:142–148

    Article  PubMed  CAS  Google Scholar 

  45. Wood JM, Bremer E, Csonka LN, Kramer R, Poolman B, van der Heide T, Smith LT (2001) Osmosensing and osmoregulatory compatible solute accumulation by bacteria. Comp Biochem Physiol 130:437–460

    Article  CAS  Google Scholar 

  46. Yap SF, Lim ST (1983) Response of Rhizobium sp. UMKL 20 to sodium chloride stress. Arch Microbiol 135:224–228

    Article  CAS  Google Scholar 

  47. Yelton MM, Yang SS, Edie SA, Lim ST (1983) Characterization of an effective salt tolerant fast growing strain of Rhizobium japonicum. J Gen Microbiol 129:1537–1547

    Google Scholar 

  48. Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate. Microbiol Mol Biol Rev 63:968–989

    PubMed  CAS  Google Scholar 

  49. Zou N, Dart PJ, Marcar NE, Bushby HVA (1995) Interaction of salinity and rhizobial strain on growth and nitrogen fixation by Acacia ampliceps. Soil Biol Biochem 27:409–413

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sylwia Wdowiak-Wróbel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wdowiak-Wróbel, S., Leszcz, A. & Małek, W. Salt Tolerance in Astragalus cicer Microsymbionts: The Role of Glycine Betaine in Osmoprotection. Curr Microbiol 66, 428–436 (2013). https://doi.org/10.1007/s00284-012-0293-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-012-0293-2

Keywords

Navigation