Sinorhizobium meliloti RpoH1 is required for effective nitrogen-fixing symbiosis with alfalfa

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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.