1887

Abstract

Random transposon mutagenesis led to the isolation of a novel mutant that is defective in nitrogen fixation during symbiosis with . The mutated locus, designated , encodes a putative cell-envelope protein displaying no significant sequence similarity to proteins with known functions. This mutant elicits the formation of nodule-like bumps and root-hair curling, but not the elongation of infection threads, on roots. This is reminiscent of the phenotypes of rhizobial mutants impaired in cyclic -glucan biosynthesis. The mutant exhibits partially reduced content of cell-associated glucans and intermediate deficiency of motility under hypo-osmotic conditions as compared to a glucan-deficient mutant. Second-site pseudorevertants of the mutant were isolated by selecting for restoration of symbiotic nitrogen fixation. A subset of pseudorevertants restored both symbiotic capability and glucan content to levels comparable to that of the wild-type. These results suggest that the Cep product acts on a successful symbiosis by affecting cell-associated glucan content.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.2007/008631-0
2007-12-01
2019-11-13
Loading full text...

Full text loading...

/deliver/fulltext/micro/153/12/3983.html?itemId=/content/journal/micro/10.1099/mic.0.2007/008631-0&mimeType=html&fmt=ahah

References

  1. Banba, M., Siddique, A.-B. M., Kouchi, H., Izui, K. & Hata, S. ( 2001; ). Lotus japonicus forms early senescent root nodules with Rhizobium etli. Mol Plant Microbe Interact 14, 173–180.[CrossRef]
    [Google Scholar]
  2. Becker, A., Schmidt, M., Jäger, W. & Pühler, A. ( 1995; ). New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162, 37–39.[CrossRef]
    [Google Scholar]
  3. Bhagwat, A. A., Tully, R. E. & Keister, D. L. ( 1992; ). Isolation and characterization of an ndvB locus from Rhizobium fredii. Mol Microbiol 6, 2159–2165.[CrossRef]
    [Google Scholar]
  4. Bhagwat, A. A., Tully, R. E. & Keister, D. L. ( 1993; ). Identification and cloning of a cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus from Bradyrhizobium japonicum. FEMS Microbiol Lett 114, 139–144.
    [Google Scholar]
  5. Bhagwat, A. A., Gross, K. C., Tully, R. E. & Keister, D. L. ( 1996; ). β-Glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing β-(1→6) linkages. J Bacteriol 178, 4635–4642.
    [Google Scholar]
  6. Bhagwat, A. A., Mithöfer, A., Pfeffer, P. E., Kraus, C., Spickers, N., Hotchkiss, A., Ebel, J. & Keister, D. L. ( 1999; ). Further studies of the role of cyclic β-glucans in symbiosis. An ndvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1→3)-β-glucosyl. Plant Physiol 119, 1057–1064.[CrossRef]
    [Google Scholar]
  7. Bittner, A. N., 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.[CrossRef]
    [Google Scholar]
  8. Bohin, J.-P. ( 2000; ). Osmoregulated periplasmic glucans in Proteobacteria. FEMS Microbiol Lett 186, 11–19.[CrossRef]
    [Google Scholar]
  9. Breedveld, M. W. & Miller, K. J. ( 1994; ). Cyclic β-glucans of members of the family Rhizobiaceae. Microbiol Rev 58, 145–161.
    [Google Scholar]
  10. Breedveld, M. W. & Miller, K. J. ( 1995; ). Synthesis of glycerophosphorylated cyclic (1,2)-β-glucans in Rhizobium meliloti strain 1021 after osmotic shock. Microbiology 141, 583–588.[CrossRef]
    [Google Scholar]
  11. Breedveld, M. W., Zevenhuizen, L. P. T. M. & Zehnder, A. J. B. ( 1990; ). Osmotically induced oligo- and polysaccharide synthesis by Rhizobium meliloti SU-47. J Gen Microbiol 136, 2511–2519.[CrossRef]
    [Google Scholar]
  12. Breedveld, M. W., Hadley, J. A. & Miller, K. J. ( 1995; ). A novel cyclic β-1,2-glucan mutant of Rhizobium meliloti. J Bacteriol 177, 6346–6351.
    [Google Scholar]
  13. Broughton, W. J. & Dilworth, M. J. ( 1971; ). Control of leghaemoglobin synthesis in snake beans. Biochem J 125, 1075–1080.
    [Google Scholar]
  14. Cangelosi, G. A., Martinetti, G. & Nester, E. W. ( 1990; ). Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic β-1,2-glucan. J Bacteriol 172, 2172–2174.
    [Google Scholar]
  15. Charles, T. C., Newcomb, W. & Finan, T. M. ( 1991; ). ndvF, a novel locus located on megaplasmid pRmeSU47b (pEXO) of Rhizobium meliloti, is required for normal nodule development. J Bacteriol 173, 3981–3992.
    [Google Scholar]
  16. Chen, R., Bhagwat, A. A. & Keister, D. ( 2003; ). A motility revertant of the ndvB mutant of Bradyrhizobium japonicum. Curr Microbiol 47, 431–433.
    [Google Scholar]
  17. D'Antuono, A. L., Casabuono, A., Couto, A., Ugalde, R. A. & Lepek, V. C. ( 2005; ). Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis. Mol Plant Microbe Interact 18, 446–457.[CrossRef]
    [Google Scholar]
  18. de Iannino, N. I., Briones, G., Iannino, F. & Ugalde, R. A. ( 2000; ). Osmotic regulation of cyclic 1,2-β-glucan synthesis. Microbiology 146, 1735–1742.
    [Google Scholar]
  19. Delgado, M. J., Bedmar, E. J. & Downie, J. A. ( 1998; ). Genes involved in the formation and assembly of rhizobial cytochromes and their role in symbiotic nitrogen fixation. Adv Microb Physiol 40, 191–231.
    [Google Scholar]
  20. Dombrecht, B., Vanderleyden, J. & Michiels, J. ( 2001; ). Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in Gram-negative bacteria. Mol Plant Microbe Interact 14, 426–430.[CrossRef]
    [Google Scholar]
  21. Dunlap, J., Minami, E., Bhagwat, A. A., Keister, D. L. & Stacey, G. ( 1996; ). Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic β-glucan synthesis. Mol Plant Microbe Interact 9, 546–555.[CrossRef]
    [Google Scholar]
  22. Dylan, T., Ielpi, L., Stanfield, S., Kashyap, L., Douglas, C., Yanofsky, M., Nester, E., Helinski, D. R. & Ditta, G. ( 1986; ). Rhizobium meliloti genes required for nodule development are related to chromosomal virulence genes in Agrobacterium tumefaciens. Proc Natl Acad Sci U S A 83, 4403–4407.[CrossRef]
    [Google Scholar]
  23. Dylan, T., Helinski, D. R. & Ditta, G. S. ( 1990a; ). Hypoosmotic adaptation in Rhizobium meliloti requires β-(1→2)-glucan. J Bacteriol 172, 1400–1408.
    [Google Scholar]
  24. Dylan, T., Nagpal, P., Helinski, D. R. & Ditta, G. S. ( 1990b; ). Symbiotic pseudorevertants of Rhizobium meliloti ndv mutants. J Bacteriol 172, 1409–1417.
    [Google Scholar]
  25. Finan, T. M., Kunkel, B., De Vos, G. F. & Signer, E. R. ( 1986; ). Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167, 66–72.
    [Google Scholar]
  26. Fraysse, N., Couderc, F. & Poinsot, V. ( 2003; ). Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. Eur J Biochem 270, 1365–1380.[CrossRef]
    [Google Scholar]
  27. Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A. & other authors ( 2001; ). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293, 668–672.[CrossRef]
    [Google Scholar]
  28. Geiger, O., Weissborn, A. C. & Kennedy, E. P. ( 1991; ). Biosynthesis and excretion of cyclic glucans by Rhizobium meliloti 1021. J Bacteriol 173, 3021–3024.
    [Google Scholar]
  29. Glazebrook, J. & Walker, G. C. ( 1991; ). Genetic techniques in Rhizobium meliloti. Methods Enzymol 204, 398–418.
    [Google Scholar]
  30. González, V., Santamaría, R. I., Bustos, P., Hernández-González, I., Medrano-Soto, A., Moreno-Hagelsieb, G., Janga, S. C., Ramírez, M. A., Jiménez-Jacinto, V. & other authors ( 2006; ). The partitioned Rhizobium etli genome: genetic and metabolic redundancy in seven interacting replicons. Proc Natl Acad Sci U S A 103, 3834–3839.[CrossRef]
    [Google Scholar]
  31. Hadri, A.-E. & Bisseling, T. ( 1998; ). Responses of the plant to Nod factors. In The Rhizobiaceae: Molecular Biology of Model Plant-Associated Bacteria, pp. 403–416. Edited by H. P. Spaink, A. Dondorosi & P. J. J. Hooykaas. Dordrecht, The Netherlands: Kluwer Academic Publishers.
  32. Halling, S. M., Peterson-Burch, B. D., Bricker, B. J., Zuerner, R. L., Qing, Z., Li, L.-L., Kapur, V., Alt, D. P. & Olsen, S. C. ( 2005; ). Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J Bacteriol 187, 2715–2726.[CrossRef]
    [Google Scholar]
  33. Handberg, K. & Stougaard, J. ( 1992; ). Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2, 487–496.[CrossRef]
    [Google Scholar]
  34. Hattori, Y., Omori, H., Hanyu, M., Kaseda, N., Mishima, E., Kaneko, T., Tabata, S. & Saeki, K. ( 2002; ). Ordered cosmid library of the Mesorhizobium loti MAFF303099 genome for systematic gene disruption and complementation analysis. Plant Cell Physiol 43, 1542–1557.[CrossRef]
    [Google Scholar]
  35. Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, A. & other authors ( 2000; ). Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7, 331–338.[CrossRef]
    [Google Scholar]
  36. Koehler, L. H. ( 1952; ). Differentiation of carbohydrates by anthrone reaction rate and color intensity. Anal Chem 24, 1576–1579.[CrossRef]
    [Google Scholar]
  37. Lepek, V., Navarro de Navarro, Y. & Ugalde, R. A. ( 1990; ). Synthesis of β(1-2)glucan in Rhizobium loti. Arch Microbiol 155, 35–41.[CrossRef]
    [Google Scholar]
  38. Lodwig, E. & Poole, P. ( 2003; ). Metabolism of Rhizobium bacteroids. Crit Rev Plant Sci 22, 37–78.[CrossRef]
    [Google Scholar]
  39. Long, S., McCune, S. & Walker, G. C. ( 1988; ). Symbiotic loci of Rhizobium meliloti identified by random TnphoA mutagenesis. J Bacteriol 170, 4257–4265.
    [Google Scholar]
  40. Manoil, C. & Beckwith, J. ( 1985; ). TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A 82, 8129–8133.[CrossRef]
    [Google Scholar]
  41. Miller, J. H. ( 1992; ). A Short Course in Bacterial Genetics: a Laboratory Manual and Handbook for Escherichia coli and Related Bacteria. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  42. Miller, K. J., Kennedy, E. P. & Reinhold, V. N. ( 1986; ). Osmotic adaptation by Gram-negative bacteria: possible role for periplasmic oligosaccharides. Science 231, 48–51.[CrossRef]
    [Google Scholar]
  43. Miller, K. J., Reinhold, V. N., Weissborn, A. C. & Kennedy, E. P. ( 1987; ). Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues. Biochim Biophys Acta 901, 112–118.[CrossRef]
    [Google Scholar]
  44. Miller, K. J., Gore, R. S. & Benesi, A. J. ( 1988; ). Phosphoglycerol substituents present on the cyclic β-1,2-glucans of Rhizobium meliloti 1021 are derived from phosphatidylglycerol. J Bacteriol 170, 4569–4575.
    [Google Scholar]
  45. Miller, K. J., Gore, R. S., Johnson, R., Benesi, A. J. & Reinhold, V. N. ( 1990; ). Cell-associated oligosaccharides of Bradyrhizobium spp. J Bacteriol 172, 136–142.
    [Google Scholar]
  46. Mithöfer, A., Bhagwat, A. A., Feger, M. & Ebel, J. ( 1996; ). Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3–1,6-β-glucans from the symbiont Bradyrhizobium japonicum. Planta 199, 270–275.
    [Google Scholar]
  47. Mitsui, H., Sato, T., Sato, Y., Ito, N. & Minamisawa, K. ( 2004; ). Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa. Mol Genet Genomics 271, 416–425.[CrossRef]
    [Google Scholar]
  48. Müller, P., Klaucke, A. & Wegel, E. ( 1995; ). TnphoA-induced symbiotic mutants of Bradyrhizobium japonicum that impair cell and tissue differentiation in Glycine max nodules. Planta 197, 163–175.
    [Google Scholar]
  49. Niwa, S., Kawaguchi, M., Imaizumi-Anraku, H., Chechetka, S. A., Ishizaka, M., Ikuta, A. & Kouchi, H. ( 2001; ). Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501. Mol Plant Microbe Interact 14, 848–856.[CrossRef]
    [Google Scholar]
  50. Park, J. T. & Johnson, M. J. ( 1949; ). A submicrodetermination of glucose. J Biol Chem 181, 149–151.
    [Google Scholar]
  51. Prentki, P. & Krisch, H. M. ( 1984; ). In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29, 303–313.[CrossRef]
    [Google Scholar]
  52. Pugsley, A. P. ( 1993; ). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50–108.
    [Google Scholar]
  53. Quandt, J., Hillemann, A., Niehaus, K., Arnold, W. & Pühler, A. ( 1992; ). An osmorevertant of a Rhizobium meliloti ndvB deletion mutant forms infection threads but is defective in bacteroid development. Mol Plant Microbe Interact 5, 420–427.[CrossRef]
    [Google Scholar]
  54. Roset, M. S., Ciocchini, A. E., Ugalde, R. A. & Iñón de Iannino, N. ( 2004; ). Molecular cloning and characterization of cgt, the Brucella abortus cyclic β-1,2-glucan transporter gene, and its role in virulence. Infect Immun 72, 2263–2271.[CrossRef]
    [Google Scholar]
  55. Rumley, M. K., Therisod, H., Weissborn, A. C. & Kennedy, E. P. ( 1992; ). Mechanisms of regulation of the biosynthesis of membrane-derived oligosaccharides in Escherichia coli. J Biol Chem 267, 11806–11810.
    [Google Scholar]
  56. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  57. Schäfer, A., Tauch, A., Jäger, 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.[CrossRef]
    [Google Scholar]
  58. Stock, J. B., Rauch, B. & Roseman, S. ( 1977; ). Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem 252, 7850–7861.
    [Google Scholar]
  59. Tully, R. E., Keister, D. L. & Gross, K. C. ( 1990; ). Fractionation of the β-linked glucans of Bradyrhizobium japonicum and their response to osmotic potential. Appl Environ Microbiol 56, 1518–1522.
    [Google Scholar]
  60. Young, J. P. W., Crossman, L. C., Johnston, A. W. B., Thomson, N. R., Ghazoui, Z. F., Hull, K. H., Wexler, M., Curson, A. R., Todd, J. D. & other authors ( 2006; ). The genome of Rhizobium leguminosarum has recognizable core and accessory components. Genome Biol 7, R34 [CrossRef]
    [Google Scholar]
  61. Zevenhuizen, L. P. T. M. & Van Neerven, A. R. W. ( 1983; ). (1→2)-β-d-Glucan and acidic oligosaccharides produced by Rhizobium meliloti. Carbohydr Res 118, 127–134.[CrossRef]
    [Google Scholar]
  62. Zorreguieta, A., Cavaignac, S., Geremia, R. A. & Ugalde, R. A. ( 1990; ). Osmotic regulation of β(1-2)-glucan synthesis in members of the family Rhizobiaceae. J Bacteriol 172, 4701–4704.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.2007/008631-0
Loading
/content/journal/micro/10.1099/mic.0.2007/008631-0
Loading

Data & Media loading...

Supplements

Determination of sugar composition of carbohydrates in the major peak from the gel filtration of ML001 (wild-type) cell extract (Fig. 2a of main paper). The sample was hydrolysed in 2 M trifluoroacetic acid at 96 °C for 6 h. The liberated sugars were trimethylsilylated with silylation reagent (TMSH-C; GL Sciences), and separated on a capillary dimethylpolysiloxane column (OV-1, 25 m×0.25 mm×0.3 μm; GL Sciences) at a flow rate of 1.22 ml min of carrier gas (He), which was mounted on a G-3500 gas chromatograph (Hitachi High-Technologies). The column temperature was programmed to 1 min at 140 °C followed by a 2 °C min increase to 220 °C, and the injector and detector temperatures were both 220 °C. A chromatogram of the sample is shown together with that of mixture of glucose and galactose used as standards. Triplets derived from glucose and galactose are marked by asterisks and plus signs, respectively, in the chromatograms. The relative amount of glucose was calculated to be 76% from peak areas as the ratio of glucose to all the materials detected. The other peaks were not characterized to determine whether they are derived from sugar components or different organic compounds. [ PDF] (320 kb) Thin-layer chromatography profiles of cell-associated glucans from strains. A sample containing a fixed amount (15 μg) in glucose equivalents, which was derived from the major peak on the gel-filtration chromatography of ethanol extract of cells, was spotted onto Silica gel 60 TLC plates (Merck). The plates were developed with 1-butanol/ethanol/water (5:5:4), sprayed with 5% (v/v) sulfuric acid in ethanol, and heated at 120 °C for 30 min as described by Breedveld (1995). Lane 1, ML001 (wild-type); lane 2, MYa179 ( ::Tn mutant); lane 3, MYa179 complemented with pYK33 ( ); lane 4, YML1011 ( :: mutant). Positions of anionic glucans and neutral glucans are indicated. [ PDF] (420 kb) A novel cyclic β-1,2-glucan mutant of . , 6346-6351.

PDF

Determination of sugar composition of carbohydrates in the major peak from the gel filtration of ML001 (wild-type) cell extract (Fig. 2a of main paper). The sample was hydrolysed in 2 M trifluoroacetic acid at 96 °C for 6 h. The liberated sugars were trimethylsilylated with silylation reagent (TMSH-C; GL Sciences), and separated on a capillary dimethylpolysiloxane column (OV-1, 25 m×0.25 mm×0.3 μm; GL Sciences) at a flow rate of 1.22 ml min of carrier gas (He), which was mounted on a G-3500 gas chromatograph (Hitachi High-Technologies). The column temperature was programmed to 1 min at 140 °C followed by a 2 °C min increase to 220 °C, and the injector and detector temperatures were both 220 °C. A chromatogram of the sample is shown together with that of mixture of glucose and galactose used as standards. Triplets derived from glucose and galactose are marked by asterisks and plus signs, respectively, in the chromatograms. The relative amount of glucose was calculated to be 76% from peak areas as the ratio of glucose to all the materials detected. The other peaks were not characterized to determine whether they are derived from sugar components or different organic compounds. [ PDF] (320 kb) Thin-layer chromatography profiles of cell-associated glucans from strains. A sample containing a fixed amount (15 μg) in glucose equivalents, which was derived from the major peak on the gel-filtration chromatography of ethanol extract of cells, was spotted onto Silica gel 60 TLC plates (Merck). The plates were developed with 1-butanol/ethanol/water (5:5:4), sprayed with 5% (v/v) sulfuric acid in ethanol, and heated at 120 °C for 30 min as described by Breedveld (1995). Lane 1, ML001 (wild-type); lane 2, MYa179 ( ::Tn mutant); lane 3, MYa179 complemented with pYK33 ( ); lane 4, YML1011 ( :: mutant). Positions of anionic glucans and neutral glucans are indicated. [ PDF] (420 kb) A novel cyclic β-1,2-glucan mutant of . , 6346-6351.

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error