Abstract
Sinorhizobium meliloti is a root-nodulating, nitrogen-fixing bacterium. An S. meliloti strain that is mutant for the rpoH 1 gene, which encodes a σ32-like protein, elicits the formation of ineffective nodules on the host plant alfalfa. We characterized the rpoH 1 mutant for phenotypes related to symbiosis. Alfalfa nodules formed by the rpoH 1 mutant exhibited greatly reduced levels of acetylene reduction activity compared to the wild-type nodules. Whereas intracellular colonization by rhizobia was observed in a zone just below the apical meristem, we found ultrastructural abnormalities and signs of degeneration of bacteroids within many host cells in the proximally adjacent zone. In the proximal part of the nodule, only a few nodule cells contained bacteroids. In contrast, the rpoH 1 mutant showed normal induction of nitrogen fixation gene expression in microaerobic culture. These results suggest that the rpoH 1 mutation causes early senescence of bacteroids during the endosymbiotic process, but does not affect the invasion process or the synthesis of the nitrogenase machinery. The rpoH 1 mutant exhibited increased sensitivity to various agents and to acid pH, suggesting that RpoH1 is required to protect the bacterial cell against environmental stresses encountered within the host. Since RpoH1 was previously reported to be required for the synthesis of some heat shock proteins (Hsps), we examined the transcription of several genes for Hsp homologs. We found that transcription of groESL 5, lon , and clpB after heat shock was RpoH1-dependent, and conserved nucleotide sequences were found in the –35 and –10 regions upstream of the transcription start sites of these genes. Although groESL 5 expression is almost completely dependent on RpoH1, we found that a groESL 5 mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.
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References
Ampe F, Kiss E, Sabourdy F, Batut J (2003) Transcriptome analysis of Sinorhizobium meliloti during symbiosis. Genome Biol 4:R15
Becker A, Schmidt M, Jager 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
Better M, Ditta G, Helinski DR (1985) Deletion analysis of Rhizobium meliloti symbiotic promoters. EMBO J 4:2419–2424
Brewin NJ (1998) Tissue and cell invasion by Rhizobium: the structure and development of infection threads and symbiosomes. In: Spaink HP, Kondorosi A, Hooykaas PJJ (eds) The Rhizobiaceae: molecular biology of model plant-associated bacteria. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 417–429
Campbell GRO, Reuhs BL, Walker GC (2002) Chronic intracellular infection of alfalfa nodules by Sinorhizobium meliloti requires correct lipopolysaccharide core. Proc Natl Acad Sci USA 99:3938–3943
David M, Daveran M-L, Batut J, Dedieu A, Domergue O, Ghai J, Hertig C, Boistard P, Kahn D (1988) Cascade regulation of nif gene expression in Rhizobium meliloti. Cell 54:671–683
Ditta G, Schmidhauser T, Yakobson E, Lu P, Liang X-W, Finlay DR, Guiney D, Helinski DR (1985) Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid 13:149–153
Ferguson GP, Roop II RM, Walker GC (2002) Deficiency of a Sinorhizobium meliloti bacA mutant in alfalfa symbiosis correlates with alteration of the cell envelope. J Bacteriol 184:5625–5632
Fischer H-M (1994) Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev 58:352–386
Galibert F, et al (2001) The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293:668–672
Glazebrook J, Walker GC (1991) Genetic techniques in Rhizobium meliloti. Methods Enzymol 204:398–418
Gross CA (1996) Function and regulation of the heat shock proteins. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low KB, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger E (eds) Escherichia coli and Salmonella typhimurium: cellular and molecular biology (2nd edn). ASM Press, Washington DC, pp 1382–1399
Gustafsson P, Nordström K, Normark S (1973) Outer penetration barrier of Escherichia coli K-12: kinetics of the uptake of gentian violet by wild type and envelope mutants. J Bacteriol 116:893–900
Hirsch AM, Smith CA (1987) Effects of Rhizobium meliloti nif and fix mutants on alfalfa root nodule development. J Bacteriol 169:1137–1146
Hirsch AM, Bang M, Ausubel FM (1983) Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti. J Bacteriol 155:367–380
Kaneko T, et al (2000) Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7:331–338
Kusukawa N, Yura T (1988) Heat shock protein GroE of Escherichia coli: key protective roles against thermal stress. Genes Dev 2:874–882
Lagares A, Caetano-Anollés G, Niehaus K, Lorenzen J, Ljunggren HD, Pühler A, Favelukes G (1992) A Rhizobium meliloti lipopolysaccharide mutant altered in competitiveness for nodulation of alfalfa. J Bacteriol 174:5941–5952
Leigh JA, Signer ER, Walker GC (1985) Exopolysaccharide-deficient mutants of R. meliloti that form ineffective nodules. Proc Natl Acad Sci USA 82:6231–6235
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
Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Moulin L, Munive A, Dreyfus B, Boivin-Masson C (2001) Nodulation of legumes by members of the β-subclass of Proteobacteria. Nature 411:948–950
Nakahigasi K, Ron EZ, Yanagi H, Yura T (1999) Differential and independent roles of a σ32 homolog (RpoH) and an HrcA repressor in the heat shock response of Agrobacterium tumefaciens. J Bacteriol 181:7509–7515
Narberhaus F, Weiglhofer W, Fischer HM, Hennecke H (1996) The Bradyrhizobium japonicum rpoH 1 gene encoding a σ32-like protein is part of a unique heat shock gene cluster together with groESL 1 and three small heat shock genes. J Bacteriol 178:5337–5346
Narberhaus F, Krummenacher P, Fischer HM, Hennecke H (1997) Three disparately regulated genes for σ32-like transcription factors in Bradyrhizobium japonicum. Mol Microbiol 24:93–104
Oke V, Rushing BG, Fisher EJ, Moghadam-Tabrizi M, Long SR (2001) Identification of the heat-shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti. Microbiol 147:2399–2408
Oláh B, Kiss E, Györgypál Z, Borzi J, Cinege G, Csanádi G, Batut J, Kondorosi A, Dusha I (2001) Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti. Mol Plant-Microbe Interact 14:887–894
Ono Y, Mitsui H, Sato T, Minamisawa K (2001) Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti. Mol Gen Genet 264:902–912
Perez-Galdona R, Kahn ML (1994) Effects of organic acids and low pH on Rhizobium meliloti 104A14. Microbiology 140:1231–1235
Perotto S, Brewin NJ, Kannenberg EL (1994) Cytological evidence for a host defence response that reduces cell and tissue invasion in pea nodules by lipopolysaccharide-defective mutants of Rhizobium leguminosarum strain 3841. Mol Plant-Microbe Interact 7:99–112
Ronson CW, Nixon BT, Albright LM, Ausubel FM (1987) Rhizobium meliloti ntrA (rpoN) gene is required for diverse metabolic functions. J Bacteriol 169:2424–2431
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3rd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
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
Segal G, Ron EZ (1996) Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat. J Bacteriol 178:3634–3640
Summers ML, Botero LM, Busse SC, McDermott TR (2000) The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa. J Bacteriol 182:2551–2558
Udvardi MK, Kahn ML (1992) Evolution of the (Brady) Rhizobium -legume symbiosis: why do bacteroids fix nitrogen? Symbiosis 14:87–101
Vasse J, deBilly F, Camut S, Truchet G (1990) Correlation between ultrastructural differentiation of bacteroids and nitrogen fixation in alfalfa nodules. J Bacteriol 172:4295–4306
Zimmerman JL, Szeto WW, Ausubel FM (1983) Molecular characterization of Tn5-induced symbiotic (Fix–) mutants of Rhizobium meliloti. J Bacteriol 156:1025–1034
Acknowledgements
We thank Bin Ye for kind instruction in microscopic analysis. We thank Donald R. Helinski for providing pMB210 and pGD926, Frederick M. Ausubel for Rm1491 and Rm1681, Daniel Kahn for pGMI931, and Alfred Pühler for pMS266. We thank Gordana Bothe for providing sequence data for the clpA region before publication. This work was supported in part by Special Coordination Funds for Promoting Science and Technology to H.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Grant-in-Aid for Scientific Research to H.M. (No. 15580056) from the Japan Society for the Promotion of Science.
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Mitsui, H., Sato, T., Sato, Y. et al. Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa. Mol Genet Genomics 271, 416–425 (2004). https://doi.org/10.1007/s00438-004-0992-x
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DOI: https://doi.org/10.1007/s00438-004-0992-x