Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa
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.
KeywordsSinorhizobium meliloti Sigma factor Symbiosis Nitrogen fixation Heat shock protein
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.
- Better M, Ditta G, Helinski DR (1985) Deletion analysis of Rhizobium meliloti symbiotic promoters. EMBO J 4:2419–2424Google Scholar
- 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 417–429Google Scholar
- 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 1382–1399Google Scholar
- 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.Google Scholar
- 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 147:2399–2408Google Scholar
- 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 14:887–894Google Scholar
- 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 7:99–112Google Scholar
- Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3rd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.Google Scholar
- Udvardi MK, Kahn ML (1992) Evolution of the (Brady) Rhizobium -legume symbiosis: why do bacteroids fix nitrogen? Symbiosis 14:87–101Google Scholar