Research for novel symbiotic genes using RNA polymerase sigma factor rpoH mutants of rhizobia.

Sinorhizobium meliloti是一种根瘤固氮细菌。一株S.meliloti是rpoH_1基因的突变株，该基因编码一种σ^<32>样蛋白，在寄主植物紫花苜蓿上形成无效的根瘤。我们对rpoH_1突变体进行了与共生相关的表型表征。与野生型相比，rpoH_1突变体形成的紫花苜蓿根瘤的乙炔还原活性大大降低。虽然根瘤菌在根尖分生组织下方的区域内定植，但我们在近端相邻区域的许多宿主细胞中发现了超微结构异常和类细菌退化的迹象。在结节的近端，只有少数结节细胞含有类细菌。相比之下，rpoH_1突变体在微氧培养中诱导固氮基因表达正常。这些结果表明，rpoH_1突变引起了内共生过程中类杆菌的早期衰老，但不影响入侵过程或氮酶机制的合成。rpoH_1突变体对各种药物和酸性pH值的敏感性增加，表明rpoH_1是保护细菌细胞免受宿主环境胁迫所必需的。由于RpoH_1在一些热休克蛋白（Hsps）的合成中是必需的，因此我们检测了几个热休克蛋白同源基因的转录。我们发现，热休克后，groESL_5、lon和clpB的转录依赖于rrpoh_1，并且在这些基因转录起始位点上游的-35和-10区域发现了保守的核苷酸序列。虽然groESL_5的表达几乎完全依赖于RpoH_1，但我们发现一个groESL_5突变株仍然能够在苜蓿上正常的共生固氮。

Sinorhizobium meliloti is a root-nodulating nitrogen-fixing bacterium. An S.meliloti strain that is mutant for the rpoH_1 gene, which encodes a σ^<32>-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 abnormality 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 RpoH_1 is required to protect the bacterial cell against environmental stresses encountered within the host. Since RpoH_1 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 RrpoH_1-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 RpoH_1, we found that a groESL_5 mutant strain is still capable of normal symbiotic nitrogen fixation on alfalfa.