Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-β-1,3-1,4-glucanases and ExoH resembles membrane proteins

  • 76 Accesses

  • 57 Citations

Abstract

Nucleotide sequencing of a 4.15 kb DNA fragment from megaplasmid 2 of Rhizobium meliloti 2011 revealed the location of the genes exoH, exoK and exoL. The putative proteins encoded by these genes have molecular weights of 41, 30, and 44 kDa, respectively. The hydrophobicity profile of the ExoH amino acid sequence resembles that of transmembrane proteins. The predicted exoL gene product does not contain hydrophobic regions, indicating a cytoplasmic localization. The exoK gene product is characterized by a putative signal peptide and exhibits significant homology to endo-β-1,3 1,4-glucanases of bacilli and Clostridium thermocellum. R. meliloti exoK mutants induced pink nodules and synthesized a reduced amount of exopolysaccharide (EPS). Colonies of this mutant showed a delay in the appearance of the Calcofluor white fluorescence. In addition, the formation of the characteristic halo was strongly delayed. R. meliloti exoL and exoH mutants induced pseudonodules. The exoH, but not the exoL mutant, synthesized an EPS that could be precipitated by cetyl pyridinium chloride (CPC) and also by ethanol. Plasmid integration mutagenesis revealed promoter regions preceding exoH, exoK and exoL.

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

References

  1. Aman P, McNeil M, Franzen L, Darvill AG, Albersheim P (1981) Structural elucidation using HPLC-MS and GLC-MS of the acidic polysaccharide secreted by Rhizobium meliloti strain 1021. Carbohydr Res 95:263–282

  2. Arnold W, Pühler A (1988) A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene 70:171–179

  3. Bauer WD (1981) Infection of legumes by Rhizobia. Annu Rev Plant Physiol 32:407–449

  4. Beringer JE (1974) R-factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198

  5. Borriss R, Buettner K, Maentsaelae P (1990) Structure of the beta1,3-1,4-glucanase gene of Bacillus macerans: Homologies to other beta-glucanases. Mol Gen Genet 222:278–283

  6. Buendia AM, Enenkel B, Köplin R, Niehaus K, Arnold W, Pühler A (1991) The Rhizobium meliloti exoZ/exoB fragment of megaplasmid 2: ExoB functions as a UDP-glucose-4-epimerase and ExoZ shows homology to NodX of Rhizobium leguminosarum biovar viciae strain TOM. Mol Microbiol 5:1519–1530

  7. Casse F, Boucher C, Julliot JS, Michel M, Dénarié J (1979) Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J Bacteriol 113:229–242

  8. Chaplin MF, Kennedy SF (1986) Carbohydrate analysis. A practical approach. IRL Press, Oxford, Washington DC

  9. Chou PY, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 47:45–148

  10. Eisenberg D, Schwarz E, Komaromy M, Wall R (1984) Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol 179:125–142

  11. Engelman DM, Henderson R, McLochlen AD, Wallace BA (1980) Path of the polypeptide in bacteriorhodopsin. Proc Natl Acad Sci USA 77:2023–2027

  12. Finan TM, Hirsch AM, Leigh JA, Johansen E, Kuldau GA, Deegan S, Walker GC, Signer ER (1985) Symbiotic mutants of Rhizobium meliloti that uncouple plant from bacterial differentiation. Cell 40:869–877

  13. Forshauer S, Green GN, Boyd D, McGovern K, Beckwith J (1988) Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of E. coli. J Mol Biol 200:267–271

  14. Hawley DK, McClure WR (1983) Compilation and analysis of Escherichia coli promoter sequences. Nucleic Acids Res 11:2237–2255

  15. Heery DM, Gannon F, Powell R (1990) A simple method for subcloning DNA fragments from gel slices. Trends Genet 6:173

  16. Henikoff S (1984) Unidirectional digestions with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28:351–359

  17. Hofemeister J, Kurtz A, Borriss R, Knowles J (1986) The β-gluca-nase gene from Bacillus amyloliquefaciens shows extensive homology with that of Bacillus subtilis. Gene 49:177–187

  18. Hohn B (1979) In vitro packaging of lambda and cosmid DNA. Methods Enzymol 68:299–309

  19. Hynes MF, Simon R, Müller P, Niehaus K, Labes M, Pühler A (1986) The two megaplasmids of Rhizobium meliloti are involved in the effective modulation of alfalfa. Mol Gen Genet 202:356–362

  20. Keller M, Arnold W, Kapp D, Müller P, Niehaus K, Schmidt M, Quandt J, Weng WM, Pühler A (1990) Rhizobium meliloti genes involved in exopolysaccharide production and infection of alfalfa nodules. In: Silver S, Chakrabarty AM, Iglewski B, Kaplan S (eds) Pseudomonas: biotransformations, pathogenesis, and evolving biotechnology. ASM, Washington, pp 91–97

  21. Leigh JA, Lee CC (1988) Characterization of polysaccharides of Rhizobium meliloti exo mutants that form ineffective nodules. J Bacteriol 170:3327–3332

  22. Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharide deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82:6231–6235

  23. Leigh JA, Reed JW, Hanks JF, Hirsch AM, Walker GC (1987) Rhizobium meliloti mutants that fail to succinylate their Calcofluor-binding exopolysaccharide are deficient in nodule invasion. Cell 51:579–587

  24. Lipman DJ, Pearson WR (1985) Rapid and sensitive protein similarity searches. Science 277:1435–1441

  25. Lloberas J, Perez-Pons JA, Querol E (1991) Molecular cloning, expression and nucleotide sequence of the endo-β-1,3-1,4-Rhizobium meliloti 2011 revealed the location of the genes d-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide. Eur J Biochem 197:337–343

  26. Long SR (1989) Rhizobium-legume nodulation: life together in the underground. Cell 56:203–214

  27. Long S, Reed JW, Himawan J, Walker GC (1988) Genetic analysis of a cluster of genes required for synthesis of the Calcofluorbinding exopolysaccharide of Rhizobium meliloti. J Bacteriol 170:4239–4248

  28. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

  29. Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122

  30. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

  31. Morrison DA (1977) Transformation in Escherichia coli: cryogenic preservation of competent cells. J Bacteriol 132:349–351

  32. Müller P, Hynes M, Kapp D, Niehaus K, Pühler A (1988) Two classes of Rhizobium meliloti infection mutants differ in exopolysaccharide production and in coinoculation properties with modulation mutants. Mol Gen Genet 211:17–26

  33. Murphy N, McConnell DJ, Cantwell BA (1984) The DNA sequence of the gene and genetic control sites for the excreted B. subtilis enzyme β-glucanase. Nucleic Acids Res 12:5355–5367

  34. Pridmore RD (1987) New and versatile cloning vectors with a kanamycin resistance marker. Gene 56:309–312

  35. Priefer U (1984) Isolation of plasmid DNA. In: Pühler A, Timmis KN (eds) Advanced molecular genetics. Springer-Verlag, Berlin, pp 14–25

  36. Priefer U, Simon R, Pühler A (1984) Cloning with cosmids. In: Puhler A, Timmis KN (eds) Advanced molecular genetics. Springer-Verlag, Berlin, pp 190–201

  37. Reuber TL, Long S, Walker GC (1991a) Regulation of Rhizobium meliloti exo genes in free-living cells and in planta examined by using TnphoA fusions. J Bacteriol 173:426–434

  38. Reuber TL, Reed JW, Glazebrook J, Urzainqui A, Walker GC (1991b) Analysis of the roles of R. meliloti exopolysaccharides in nodulation. In: Hennecke H, Verma DPS (eds) Advances in molecular genetics of plant-microbe interactions vol. 1. Kluwer Publ., Dordrecht, pp 182–188

  39. Rolfe BG, Gresshoff PM, Shine J (1980) Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci Lett 19:277–284

  40. Rostas K, Kondorosi E, Horvath B, Simoncsits A, Kondorosi A (1986) Conservation of extended promoter regions of modulation genes in Rhizobium. Proc Natl Acad Sci USA 83:1747–1761

  41. Schimming S, Schwarz WH, Staudenbauer WL (1992) Structure of the Clostridium thermocellum gene licB and the encoded beta1,3-1,4-glucanase: A catalytic region homologous to Bacillus lichenases joined to the reiterated domain of clostridial cellulases. Eur J Biochem 204:13–19

  42. Simon R (1984) High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet 196:413–420

  43. Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784–791

  44. Somasegaran P, Hoben J (1985) Methods in Legume-Rhizobium Technology, NIFTAL. USA Lib Congress 87–106109

  45. Staden R (1986) The current status and portability of our sequence handling software. Nucleic Acids Res 14:217–232

  46. Staden R, McLachlan AD (1982) Codon preference and its use in identifying protein coding regions in large DNA sequences. Nucleic Acids Res 10:141–156

  47. Thöny B, Hennecke H (1989) The -24/- 12 promoter comes of age. FEMS Microbiol Rev 63:341–358

  48. Tolmasky ME, Staneloni RJ, Leloir LF (1982) Lipid-bound saccharides in Rhizobium meliloti. J Biol Chem 257:6751–6757

  49. Urzainqui A, Walker GC (1992) Exogeneous suppression of the symbiotic deficiencies of Rhizobium meliloti exo mutants. J Bacteriol 174:3403–3406

  50. Vieira J, Messing J (1982) The pUC plasmids, and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

  51. Vincent JM (1970) A manual for the practical study of root nodule bacteria. (BP handbook no 15 Oxford)

  52. von Heijne G (1986) A new method for predicting sequence cleavage sites. Nucleic Acids Res 14:4683–4690

  53. Zimmermann J, Voss H, Schwager C, Stegemann J, Erfle H, Stucky K, Kristensen T, Ansorge W (1990) A simplified method protocol for fast plasmid DNA sequencing. Nucleic Acids Res 18:1067

Download references

Author information

Correspondence to Alfred Pühler.

Additional information

Communicated by A. Kondorosi

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Becker, A., Kleickmann, A., Arnold, W. et al. Analysis of the Rhizobium meliloti exoH/exoK/exoL fragment: ExoK shows homology to excreted endo-β-1,3-1,4-glucanases and ExoH resembles membrane proteins. Molec. Gen. Genet. 238, 145–154 (1993). https://doi.org/10.1007/BF00279541

Download citation

Key words

  • Rhizobium meliloti
  • Acidic exopolysaccharide (EPS)
  • Alfalfa nodule infection
  • Nucleotide sequence
  • Endo-β-1,3-1,4-glucanase