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

苜蓿中华根瘤菌（Sinorhizobium meliloti）是一种根际固氮菌。苜蓿根瘤菌（S.meliloti）的rpoH_1基因突变株<32>能在寄主苜蓿上形成无效根瘤。rpoH_1基因编码类σ^ -蛋白。我们对rpoH_1突变体进行了与共生相关的表型表征。与野生型根瘤相比，由rpoH_1突变体形成的根瘤表现出大大降低的乙炔还原活性水平。而根瘤菌的细胞内定植观察到在一个区下面的顶端分生组织，我们发现超微结构异常和退化的迹象，在许多宿主细胞在近端相邻区。在结节的近端部分，只有少数结节细胞含有类杆菌。与此相反，rpoH_1突变体在微氧培养中表现出正常的固氮基因表达诱导。这些结果表明，rpoH_1突变引起类杆菌在内共生过程中的早衰，但不影响入侵过程或固氮酶的合成。rpoH_1突变体表现出对各种试剂和酸性pH的敏感性增加，表明RpoH_1是保护细菌细胞免受宿主内遇到的环境胁迫所必需的。由于RpoH_1是合成热休克蛋白（Hsps）所必需的，我们研究了Hsps同源基因的转录。我们发现热激后groESL_5、lon和clpB基因的转录依赖于RrpoH_1，并且在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groESL_5突变株仍然能够在苜蓿上正常共生固氮。

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.