Different roles of two groEL homologues in methylotrophic utiliser of dichloromethane Methylorubrum extorquens DM4

  • Yulia E. Firsova
  • Maria L. TorgonskayaEmail author
Original Paper


The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound—dichloromethane (DCM). The evaluation of activity of putative PgroE1 and PgroE2 promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from PgroE1 was heat-inducible, while the activity of PgroE2 was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.


Aerobic methylotrophic bacteria Chaperonins Dichloromethane 60-kDa heat shock protein Stress response 



This work was supported by Russian Foundation for Basic Research (Grants 15-01-04458-a and 18-04-01148-a).

Author’s contribution

ML Torgonskaya designed the experiments, coordinated the study, analysed the DNA sequences of groESL operons and their upstream regions. YE Firsova carried out the generation of the mutant and reporter strains, characterised the phenotype of ∆groEL2 mutant and registered the GFP expression from promoters. Both authors contributed to data analysis and manuscript preparation. The final manuscript was reviewed and approved by both authors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10482_2019_1320_MOESM1_ESM.doc (172 kb)
Supplementary material 1 (DOC 172 kb)


  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. CrossRefPubMedGoogle Scholar
  2. Barnett MJ, Bittner AM, Toman CJ, Oke V, Long SR (2012) Dual RpoH sigma factors and transcriptional plasticity in a symbiotic bacterium. J Bacteriol 194:4983–4994. CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bittner AN, Foltz A, Oke V (2007) Only one of five groEL genes is required for viability and successful symbiosis in Sinorhizobium meliloti. J Bacteriol 189:1884–1889. CrossRefPubMedGoogle Scholar
  4. Bosch G, Skovran E, Xia Q, Wang T, Taub F, Miller JA, Lidstrom ME, Hackett M (2008) Comprehensive proteomics of Methylobacterium extorquens AM1 metabolism under single carbon and non-methylotrophic conditions. Proteomics 8:3494–3505. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Cha HJ, Srivastava R, Vakharia VN, Rao G, Bentley WE (1999) Green fluorescent protein as a noninvasive stress probe in resting Escherichia coli cells. Appl Environ Microbiol 65:409–414PubMedPubMedCentralGoogle Scholar
  7. Chen IA, Chu K, Palaniappan K, Pillay M, Ratner A, Huang J, Huntemann M, Varghese N, White JR, Seshadri R, Smirnova T, Kirton E, Jungbluth SP, Woyke T, Eloe-Fadrosh EA, Ivanova NN, Kyrpides NC (2019) IMG/M vol 5.0: an integrated data management and comparative analysis system for microbial genomes and microbiomes. Nucleic Acids Res 47:D666–D677. CrossRefPubMedGoogle Scholar
  8. Chongcharoen R, Smith FJ, Flint KP, Dalton H (2005) Adaptation and acclimatization to formaldehyde in methylotrophs capable of high-concentration formaldehyde detoxification. Microbiology 151:2615–2622. CrossRefPubMedGoogle Scholar
  9. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR, da Costa MS, Rooney AP, Yi H, Xu XW, De Meyer S, Trujillo ME (2018) Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 68:461–466. CrossRefPubMedGoogle Scholar
  10. Csáki R, Bodrossy L, Klem J, Murrell JC, Kovács KL (2003) Genes involved in the copper-dependent regulation of soluble methane monooxygenase of Methylococcus capsulatus (Bath): cloning, sequencing and mutational analysis. Microbiology 149:1785–1795. CrossRefPubMedGoogle Scholar
  11. De Marco P, Pacheco CC, Figueiredo AR, Moradas-Ferreira P (2004) Novel pollutant-resistant methylotrophic bacteria for use in bioremediation. FEMS Microbiol Lett 234:75–80. CrossRefPubMedGoogle Scholar
  12. Dennis JJ, Zylstra GJ (1998) Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of Gram-negative bacterial genomes. Appl Environ Microbiol 64:2710–2715PubMedPubMedCentralGoogle Scholar
  13. Doronina NV, Trotsenko YA, Tourova TP, Kuznetzov BB, Leisinger T (2000) Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.—novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane. Syst Appl Microbiol 23:210–218. CrossRefPubMedGoogle Scholar
  14. Eom CY, Kim E, Ro YT, Kim SW, Kim YM (2005) Cloning and molecular characterization of groESL heat-shock operon in methylotrophic bacterium Methylovorus sp. strain SS1 DSM 11726. J Biochem Mol Biol 38:695–702. CrossRefPubMedGoogle Scholar
  15. Fayet O, Ziegelhoffer T, Georgopulos C (1989) The groES and groEL heat shock gene products of Escherichia coli are essential for bacterial growth at all temperatures. J Bacteriol 171:1379–1385. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783–791. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Firsova YE, Torgonskaya ML, Doronina NV, Trotsenko YA (2005) Effects of DNA-damaging agents on aerobic methylobacteria capable and incapable of utilizing dichloromethane. Appl Biochem Microbiol 41:480–485. CrossRefGoogle Scholar
  18. Firsova YE, Fedorov DN, Trotsenko YA (2011) Analysis of the 3′- region of the dcmA gene of dichloromethane dehalogenase of Methylobacterium dichloromethanicum DM4. Microbiology 80:805–811. CrossRefGoogle Scholar
  19. Firsova YE, Torgonskaya ML, Trotsenko YA (2015) Functionality of the xoxF Gene in Methylobacterium dichloromethanicum DM4. Microbiology (Moscow) 84:796–803. CrossRefGoogle Scholar
  20. Firsova YE, Torgonskaya ML, Trotsenko YA (2017) Functionality of METDI5511 gene in Methylobacterium dichloromethanicum DM4. Appl Biochem Microbiol 53:194–200. CrossRefGoogle Scholar
  21. Goloubinoff P, Christeller JT, Gatenby AA, Lorimer GH (1989) Reconstitution of active dimeric ribulose biphosphate carboxylase from an unfolded state depends on two chaperonin proteins and Mg-ATP. Nature 342:884–889. CrossRefPubMedGoogle Scholar
  22. Gourion B, Francez-Charlot A, Vorholt JA (2008) PhyR is involved in the general stress response of Methylobacterium extorquens AM1. J Bacteriol 190:1027–1035. CrossRefPubMedGoogle Scholar
  23. Goyal K, Qamra R, Mande SC (2006) Multiple gene duplication and rapid evolution in the groEL gene: functional implications. J Mol Evol 63:781–787. CrossRefPubMedGoogle Scholar
  24. Green PN, Ardley JK (2018) Review of the genus Methylobacterium and closely related organisms: a proposal that some Methylobacterium species be reclassified into a new genus, Methylorubrum gen. nov. Int J Syst Evol Microbiol 68:2727–2748. CrossRefPubMedGoogle Scholar
  25. Gruber TM, Gross CA (2003) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57:441–466. CrossRefPubMedGoogle Scholar
  26. Hayer-Hartl M, Bracher A, Hartl FU (2016) The GroEL-GroES chaperonin machine: a nano cage for protein folding. Trends Biochem Sci 41:62–76. CrossRefPubMedGoogle Scholar
  27. Hecker M, Schumann W, Völker U (1996) Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol 19:417–428. CrossRefPubMedGoogle Scholar
  28. Hemmingsen SM, Woolford C, van der Vies SM, Tilly K, Dennis DT, Georgopoulos CP, Hendrix RW, Ellis RJ (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333:330–334. CrossRefPubMedGoogle Scholar
  29. Hendrickson EL, Beck DAC, Wang T, Lidstrom ME, Hackett M, Chistoserdova L (2010) Expressed genome of Methylobacillus flagellatus as defined through comprehensive proteomics and new insights into methylotrophy. J Bacteriol 192:4859–4867. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8:275–282. CrossRefPubMedGoogle Scholar
  31. Jörg G, Bertau M (2004) Thiol-tolerant assay for quantitative colorimetric determination of chloride released from whole-cell biodehalogenations. Anal Biochem 328:22–28. CrossRefPubMedGoogle Scholar
  32. Kato Y, Asahara M, Arai D, Goto K, Yokota A (2005) Reclassification of Methylobacterium chloromethanicum and Methylobacterium dichloromethanicum as later subjective synonyms of Methylobacterium extorquens and of Methylobacterium lusitanum as a later subjective synonym of Methylobacterium rhodesianum. J Gen Appl Microbiol 51:287–299. CrossRefPubMedGoogle Scholar
  33. Kayser MF, Vuilleumier S (2001) Dehalogenation of dichloromethane by dichloromethane dehalogenase/glutathione S-transferase leads to formation of DNA adducts. J Bacteriol 183:5209–5212. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Kolb S (2009) Aerobic methanol-oxidizing bacteria in soil. FEMS Microbiol Lett 300:1–10. CrossRefPubMedGoogle Scholar
  35. Kumar CM (2017) Prokaryotic multiple chaperonins: the mediators of functional and evolutionary diversity. In: Kumar CM, Mande CS (eds) Heat shock proteins. Prokaryotic chaperonins. Multiple copies and multitude functions, vol 11. Springer, Singapore, pp 39–51. CrossRefGoogle Scholar
  36. Kumar CMS, Mande SC, Mahajan G (2015) Multiple chaperonins in bacteria—novel functions and non-canonical behaviors. Cell Stress Chaperones 20:555–574. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35:1547–1549. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Lee WT, Terlesky KC, Tabita FR (1997) Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon. J Bacteriol 179:487–495. CrossRefPubMedPubMedCentralGoogle Scholar
  39. Li M, Wong SL (1992) Cloning and characterization of the groESL operon from Bacillus subtilis. J Bacteriol 174:3981–3992. CrossRefPubMedPubMedCentralGoogle Scholar
  40. López-Leal G, Tabche ML, Castillo-Ramírez S, Mendoza-Vargas A, Ramírez-Romero MA, Dávila G (2014) RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock. BMC Genom 15:770. CrossRefGoogle Scholar
  41. Lund PA (2009) Multiple chaperonins in bacteria—why so many? FEMS Microbiol Rev 33:785–800. CrossRefPubMedGoogle Scholar
  42. Lund P, Tramonti A, De Biase D (2014) Coping with low pH: molecular strategies in neutralophilic bacteria. FEMS Microbiol Rev 38:1091–1125. CrossRefPubMedGoogle Scholar
  43. Martínez-Salazar JM, Sandoval-Calderón M, Guo X, Castillo-Ramírez S, Reyes A, Loza MG, Rivera J, Alvarado-Affantranger X, Sánchez F, González V, Dávila G, Ramírez-Romero MA (2009) The Rhizobium etli RpoH1 and RpoH2 sigma factors are involved in different stress responses. Microbiology 155:386–397. CrossRefPubMedGoogle Scholar
  44. Marx CJ, Lidstrom ME (2001) Development of improved versatile broad-host-range vectors for use in methylotrophs and other Gram-negative bacteria. Microbiology 147:2065–2075. CrossRefPubMedGoogle Scholar
  45. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M (2013) Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform 14:60. CrossRefGoogle Scholar
  46. Miller WG, Lindow SE (1997) An improved GFP cloning cassette designed for prokaryotic transcriptional fusions. Gene 191:149–153. CrossRefPubMedGoogle Scholar
  47. Mizobata T, Kawata Y (2018) The versatile mutational « repertoire » of Escherichia coli GroEL, a multidomain chaperonin nanomachine. Biophys Rev 10:631–640. CrossRefPubMedGoogle Scholar
  48. Motojima F (2015) How do chaperonins fold protein? Biophysics 11:93–102. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Muller EEL, Hourcade E, Louhichi-Jelail Y, Hammann P, Vuilleumier S, Bringel F (2011) Functional genomics of dichloromethane utilization in Methylobacterium extorquens DM4. Environ Microbiol 13:2518–2535. CrossRefPubMedGoogle Scholar
  50. Narberhaus F, Kaeser R, Nocker A, Hennecke H (1998a) A novel DNA element that controls bacterial heat shock gene expression. Mol Microbiol 28:315–323. CrossRefPubMedGoogle Scholar
  51. Narberhaus F, Kowarik M, Beck C, Hennecke H (1998b) Promoter selectivity of the Bradyrhizobium japonicum RpoH transcription factors in vivo and in vitro. J Bacteriol 180:2395–2401PubMedPubMedCentralGoogle Scholar
  52. Nocker A, Krstulovic NP, Perret X, Narberhaus F (2001) ROSE elements occur in disparate rhizobia and are functionally interchangeable between species. Arch Microbiol 176:44–51. CrossRefPubMedGoogle Scholar
  53. Rodríguez-Quiñones F, Maguire M, Wallington EJ, Gould PS, Yerko V, Downie JA, Lund PA (2005) Two of the three groEL homologues in Rhizobium leguminosarum are dispensable for normal growth. Arch Microbiol 183:253–265. CrossRefPubMedGoogle Scholar
  54. Roncarati D, Scarlato V (2017) Regulation of heat-shock genes in bacteria: from signal sensing to gene expression output. FEMS Microbiol Rev 41:549–574. CrossRefPubMedGoogle Scholar
  55. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  56. Schäfer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Seo JH, Kang DG, Cha HJ (2003) Comparison of cellular stress levels and green-fluorescent-protein expression in several Escherichia coli strains. Biotechnol Appl Biochem 37:103–107. CrossRefPubMedGoogle Scholar
  58. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Nat Biotechnol 1:784–791. CrossRefGoogle Scholar
  59. Tittabutr P, Payakapong W, Teaumroong N, Boonkerd N, Singleton PW, Borthakur D (2006) The alternative sigma factor RpoH2 is required for salt tolerance in Sinorhizobium sp. strain BL3. Res Microbiol 157:811–818. CrossRefPubMedGoogle Scholar
  60. Torgonskaya ML, Doronina NV, Hourcade E, Trotsenko YA, Vuilleumier S (2011) Chloride-associated adaptive response in aerobic methylotrophic dichloromethane-utilising bacteria. J Basic Microbiol 51:296–303. CrossRefPubMedGoogle Scholar
  61. Trotsenko YA, Khmelenina VN (2002) Biology and osmoadaptation of halophilic methanotrophs. Microbiol 71:123–132. CrossRefGoogle Scholar
  62. Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10:828–840. CrossRefPubMedGoogle Scholar
  63. Vuilleumier S (2002) Coping with a halogenated one-carbon diet: aerobic dichloromethane-mineralising bacteria. In: Agathos SN, Reineke W (eds) Biotechnology for the environment: strategy and fundamentals focus on biotechnology, vol 3A. Springer, DordrechtGoogle Scholar
  64. Walter S (2002) Structure and function of the GroEL chaperone. Cell Mol Life Sci 59:1589–1597CrossRefPubMedGoogle Scholar
  65. Ward N, Larsen Ø, Sakwa J, Bruseth L, Khouri H, Durkin AS, Dimitrov G, Jiang L, Scanlan D, Kang KH, Lewis M, Nelson KE, Methé B, Wu M, Heidelberg JF, Paulsen IT, Fouts D, Ravel J, Tettelin H, Ren Q, Read T, DeBoy RT, Seshadri R, Salzberg SL, Jensen HB, Birkeland NK, Nelson WC, Dodson RJ, Grindhaug SH, Holt I, Eidhammer I, Jonasen I, Vanaken S, Utterback T, Feldblyum TV, Fraser CM, Lillehaug JR, Eisen JA (2004) Genomic insights into methanotrophy: the complete genome sequence of Methylococcus capsulatus (Bath). PLoS Biol 2:e303. CrossRefPubMedPubMedCentralGoogle Scholar
  66. Xu Z, Horwich AL, Sigler PB (1997) The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature 388:741–750. CrossRefPubMedGoogle Scholar
  67. Zhou YN, Kusukawa N, Erickson JW, Gross CA, Yura T (1988) Isolation and characterization of Escherichia coli mutants that lack the heat shock sigma factor sigma 32. J Bacteriol 170:3640–3649. CrossRefPubMedPubMedCentralGoogle Scholar
  68. Zuber U, Schumann W (1994) CIRCE, a novel heat shock element involved in regulation of heat shock operon DnaK of Bacillus subtilis. J Bacteriol 176:1359–1363. CrossRefPubMedPubMedCentralGoogle Scholar
  69. Zügel U, Kaufmann SHE (1999) Role of heat shock proteins in protection from and pathogenesis of infectious diseases. Clin Microbiol Rev 12:19–39. CrossRefPubMedPubMedCentralGoogle Scholar

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Authors and Affiliations

  1. 1.Laboratory of Radioactive Isotopes, G.K. Skryabin Institute of Biochemistry and Physiology of MicroorganismsFRC Pushchino Center for Biological Research of Russian Academy of SciencesPushchinoRussia

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