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Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria

Extremophiles volume 15, Article number: 653 (2011) Cite this article

Abstract

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1T. In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146T , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1T, the genes forming the ectABC–ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.

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References

  • Anthony C (1982) The biochemistry of methylotrophs. Academic Press, London, p 251

    Google Scholar 

  • Eshinimaev BT, Tsyrenzhapova IS, Khmelenina VN, Trotsenko YA (2007) Measurement of the content of the osmoprotectant ectoine in methylotrophic bacteria by means of normal-phase high performance liquid chromatography. Appl Biochem Microbiol 43:193–196 (Moscow, English translation)

    Article  CAS  Google Scholar 

  • Buenger J (1999) Ectoine added protection and care for the skin. Eurocosmetics 7:22–24

    Google Scholar 

  • Cánovas D, Vargas C, Csonka LN, Ventosa A, Nieto JJ (1998) Synthesis of glycine betaine from exogenous choline in the moderately halophilic bacterium Halomonas elongata. Appl Environ Microbiol 64:4095–4097

    PubMed  Google Scholar 

  • Doronina NV, Sakharovsky VG, Drachuk SV, Trotsenko YA (1998) Organic osmoprotectors of aerobic halophilic methylobacteria. Mikrobiologiya 67:457–462 (English translation)

    Google Scholar 

  • Doronina NV, Trotsenko YA, Tourova TP (2000) Methylarcula marina gen nov., sp. nov. and Methylarcula terricola sp. nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments. Int J Syst Evol Microbiol 50:1849–1859

    PubMed  CAS  Google Scholar 

  • Doronina NV, TsD Darmaeva, Trotsenko YA (2003a) Methylophaga alcalica sp. nov., a novel alkaliphilic and moderately halophilic, obligately methylotrophic bacterium from the East Mongolian saline soda lake. Int J Syst Evol Microbiol 53:223–229

    PubMed  Article  CAS  Google Scholar 

  • Doronina NV, TsD Darmaeva, Trotsenko YA (2003b) Methylophaga natronica sp. nov., a new alkaliphilic and moderately halophilic, restricted-facultative methylotrophic bacterium from soda lake of the Southern Transbaikal region. Syst Appl Microbiol 26:382–389

    PubMed  Article  CAS  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791

    Article  Google Scholar 

  • Galinski EA (1995) Osmoadaptation in bacteria. Adv Microbial Physiol 37:273–328

    Article  CAS  Google Scholar 

  • Galinski EA, Pfeiffer HP, Trüper HG (1985) 1,4,5,6,-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, a novel cyclic acid from halophilic phototrophic bacteria of genus Ectothiorhodospira. Eur J Biochem 149:135–139

    PubMed  Article  CAS  Google Scholar 

  • Göller K, Galinski EA (1999) Protection of the model enzyme (lactate dehydrogenase) against heat, urea and freeze–thaw treatment by compatible solute addition. J Mol Biol B Enzym 7:37–45

    Article  Google Scholar 

  • Graf R, Anzali S, Buenger J, Pfluecker F, Driller H (2008) The multifunctional role of ectoine as a natural cell protectant. Clinics Dermatol 26:326–333

    Article  Google Scholar 

  • Grant WD (2004) Life at low water activity. Phil Trans R Soc Lond B 359:1249–1267

    Article  CAS  Google Scholar 

  • Hanson RS, Hanson TE (1996) Methanotrophic bacteria. Microbiol Rev 60:439–471

    PubMed  CAS  Google Scholar 

  • Imhoff JF (1993) Osmotic adaptation in halophilic and halotolerant microorganisms. In: Vreeland RH, Hochstein LJ (eds) The biology of halophilic bacteria. The CRC Press, Boca Raton, pp 211–253

    Google Scholar 

  • Jung H (2001) Towards the molecular mechanism of Na/solute symport in prokaryotes. Biochim Biophys Acta 1505:131–143

    PubMed  Article  CAS  Google Scholar 

  • Kalyuzhnaya MG, Khmelenina VN, Kotelnikova S, Holmquist L, Pedersen K, Trotsenko YA (1999) Methylomonas scandinavica sp. nov., a new methanotrophic psychrotrophic bacterium isolated from deep igneous rock ground water of Sweden. Syst Appl Microbiol 22:565–572

    PubMed  CAS  Google Scholar 

  • Kalyuzhnaya MG, Khmelenina VN, Eshinimaev BT, Sorokin D, Fuse H, Lidstrom M, Trotsenko Y (2008) Reclassification of halo(alkali)philic and halo(alkali)tolerant methanotrophs provisionally assigned to the genera Methylomicrobium and Methylobacter and emended description of the genus Methylomicrobium. Int J Syst Evol Microbiol 58:591–596

    PubMed  Article  CAS  Google Scholar 

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

    PubMed  Article  CAS  Google Scholar 

  • Khmelenina VN, Kalyuzhnaya MG, Sakharovsky VG, Suzina NE, Trotsenko YA, Gottschalk G (1999) Osmoadaptation in halophilic and alkaliphilic methanotrophs. Arch Microbiol 172:321–329

    PubMed  Article  CAS  Google Scholar 

  • Khmelenina VN, Shchukin VN, Reshetnikov AS, Mustakhimov II, Suzina NE, Eshinimaev BT, Trotsenko YA (2010) Structural and functional features of methanotrophs from hypersaline and alkaline lakes. Mikrobiologiya 79:472–482 (English translation)

    CAS  Google Scholar 

  • Knap S, Ladenstain R, Galinski EA (1999) Thermal stabilization of bovine ribonuclease A by naturally occurring osmolyte beta-hydroxyectoine and betaine. Extremophiles 3:191–198

    Article  Google Scholar 

  • Ko W-Y, Ryan MD, Akashi H (2003) Molecular phylogeny of the Drosophila melanogaster species subgroup. J Mol Evol 57:562–573

    PubMed  Article  CAS  Google Scholar 

  • Kuhlmann AU, Bremer E (2002) Osmotically regulated synthesis of the compatible solute ectoine in Bacillus pasteurii and related Bacillus spp. Appl Environ Microbiol 68:772–783

    PubMed  Article  CAS  Google Scholar 

  • Lidstrom ME (2006) Aerobic methylotrophic prokaryotes. In: Balows A et al (eds) The Prokaryotes, 3rd edn. Springer Verlag, New York, pp 618–634

    Chapter  Google Scholar 

  • Lippert K, Galinski EA (1992) Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing, and drying. Appl Microbial Biotechnol 37:61–65

    Article  CAS  Google Scholar 

  • Lo C–C, Bonner AC, Xie G, D’Souza M, Jensen AR (2009) Cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways. Microbiol Molec Biol Rev 73:594–651

    Article  CAS  Google Scholar 

  • Louis P, Galinski EA (1997) Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli. Microbiology 143:1141–1149

    PubMed  Article  CAS  Google Scholar 

  • Mustakhimov II, Reshetnikov AS, Khmelenina VN, YuA Trotsenko (2009) EctR—a novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica. Doklady Biochem Biophys 429:305–308 (Moscow, English translation)

    Article  CAS  Google Scholar 

  • Mustakhimov II, Reshetnikov AS, Glukhov AS, Khmelenina VN, Kalyuzhnaya MG, Trotsenko YA (2010) Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. J Bacteriol 192:410–417

    PubMed  Article  CAS  Google Scholar 

  • Ono H, Sawada K, Khunajakr N, Toa T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y (1999) Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J Bacteriol 181:91–99

    PubMed  CAS  Google Scholar 

  • Oren A (2002) Halophilic microorganisms and their environments. Kluwer Scientific Publishers, Dordrecht

    Book  Google Scholar 

  • Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Syst 4:2

    PubMed  Google Scholar 

  • Peters R, Galinski EA, Truper HG (1990) The biosynthesis of ectoine. FEMS Microbiol Lett 71:157–162

    Article  CAS  Google Scholar 

  • Rajan AL, Joseph TC, Thampuran N, James R, Ashok Kumar K, Viswanathan C, Bansal KC (2008) Cloning and heterologous expression of ectoine biosynthesis genes from Bacillus halodurans in Escherichia coli. Biotechnol Lett 30:1403–1407

    Article  CAS  Google Scholar 

  • Reshetnikov AS, Mustakhimov II, Khmelenina VN, Trotsenko YA (2005) Cloning, purification, and characterization of diaminobutyrate acetyltransferase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Biochemistry 70:878–883 (Moscow, English translation)

    PubMed  CAS  Google Scholar 

  • Reshetnikov AS, Khmelenina VN, Trotsenko YA (2006) Characterization of the ectoine biosynthesis genes in obligate haloalkalotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Arch Microbiol 184:286–296

    PubMed  Article  CAS  Google Scholar 

  • Roberts MF (2005) Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Syst 1:5

    PubMed  Article  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork

    Google Scholar 

  • Saum SH, Müller V (2008) Regulation of osmoadaptation in the moderate halophile Halobacillus halophilus: chloride, glutamate and switching osmolyte strategies. Saline Syst 4:4

    PubMed  Article  Google Scholar 

  • Schwibbert K, Marin-Sanguino A, Bagyan I, Heidrich G, Lentzen G, Seitz H, Rampp M, Schuster SC, Klenk HP, Pfeiffer F, Oesterhelt D, Kunte HJ (2010) A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T. Environ Microbiol. doi:10.1111/j.1462-2920.2010.02336.x

  • Severin J, Wohlfarth A, Galinski EA (1992) The predominant role of recently discovered tetrahydropyrimidines for the osmoadaptation of halophilic eubacteria. J Gen Microbiol 138:1629–1638

    CAS  Google Scholar 

  • Sorokin DY, Jones BE, Kuenen JG (2000) A novel obligately methylotrophic, methane-oxidizing Methylomicrobium species from a highly alkaline environment. Extremophiles 4:145–155

    PubMed  Article  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    PubMed  Article  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmaugin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    PubMed  Article  CAS  Google Scholar 

  • Trotsenko YA, Khmelenina VN (2002) Biology and osmoadaptation of haloalkaliphilic methanotrophs. Mikrobiologiya 71:123–132 (English translation)

    CAS  Google Scholar 

  • Vargas C, Argandona M, Reina-Bueno M, Rodriguez-Moya J, Fernandez-Aunion C, Nieto JJ (2008) Unravelling the adaptation responses to osmotic and temperature stress in Chromohalobacter salexigens, a bacterium with broad salinity tolerance. Saline Syst 4:14. doi:10.1186/1746-1448-4-14

    PubMed  Article  Google Scholar 

  • Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544

    PubMed  CAS  Google Scholar 

  • Wohlfarth A, Severin J, Galinski EA (1990) The spectrum of compatible solutes in heterotrophic halophilic eubacteria of the family Halomonadaceae. J Gen Microbiol 136:705–712

    CAS  Google Scholar 

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Acknowledgments

This work was supported by Grants CRDF Rub1-2946-PU-09, RFBR 11-04-00801a, Russian State Contract 16.740.11.0615 and NSF Grant MCB-0604269. The authors thank Nicole Smalley for helpful comments on the earlier draft.

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Affiliations

  1. Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow region, 142290, Russia

    Alexander S. Reshetnikov, Valentina N. Khmelenina, Ildar I. Mustakhimov & Yuri A. Trotsenko

  2. Department of Chemical Engineering, University of Washington, Box 355014, Seattle, WA, USA

    Mary Lidstrom

  3. Department of Microbiology, University of Washington, Box 355014, Seattle, WA, USA

    Marina Kalyuzhnaya & Mary Lidstrom

Authors
  1. Alexander S. Reshetnikov
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  2. Valentina N. Khmelenina
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  3. Ildar I. Mustakhimov
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  4. Marina Kalyuzhnaya
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  5. Mary Lidstrom
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  6. Yuri A. Trotsenko
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Corresponding author

Correspondence to Valentina N. Khmelenina.

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Communicated by M. da Costa.

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792_2011_396_MOESM1_ESM.doc

Fig. S1. Sequencing strategies used for identification of ectoine biosynthetic genes in Methylomicrobium alcaliphilum ML1 (C), Methylophphaga alcalica M8 (A), Methylophaga thalassica ATCC 33146 (B), Methylobacter marinus 7C (D), Methylomicrobium kenyense AMO1 (E) and Methylarcula marina h1 (F). The nucleotide sequences of oligonucleotide primers are given in Table 1. (DOC 94 kb)

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Reshetnikov, A.S., Khmelenina, V.N., Mustakhimov, I.I. et al. Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria. Extremophiles 15, 653 (2011). https://doi.org/10.1007/s00792-011-0396-x

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  • DOI: https://doi.org/10.1007/s00792-011-0396-x

Keywords

  • Methylotrophic bacteria
  • Methanotrophs
  • Compatible solutes
  • Ectoine biosynthesis genes