Molecular genetic analysis of multiple rpoH and groEL genes in Sinorhizobium meliloti

Abstract

The genomes of root-nodulating, nitrogen-fixing rhizobia that have been completely sequenced contain multiple copies of genes that encode the heat-shock transcription factor RpoH and the chaperone GroEL-GroES. Sinorhizobium meliloti maintains two rpoH genes, four groESL operons, and a single groEL gene. Mutations in some of these genes result in symbiotic defects: an rpoH1 mutant cannot fix nitrogen in nodules, an rpoH1 rpoH2 double mutant cannot form nodules, and a groEL1 mutant cannot fix nitrogen in nodules. My work has sought to further characterize the roles of multiple rpoH and groEL genes during growth and symbiosis. In E. coli, groESL is the key target of RpoH. However, I showed that S. meliloti rpoH suppressor mutants do not overproduce GroEL, and overexpression of groESL does not bypass the rpoH mutant defects. In addition, RpoH1 controls expression of only groEL5, which is not required for symbiosis, and RpoH2 does not control expression of any of the groEL genes. Therefore, the requirements for RpoH1 and RpoH2 during symbiosis cannot be explained solely by loss of GroEL-GroES production, and there must be other crucial targets. To determine what genes are controlled by RpoH1 and RpoH2, I performed microarray experiments to compare global gene expression profiles between wild-type and rpoH mutant cells. Although the regulon of RpoH1 is incomplete, the results indicate that the RpoH1 and RpoH2 regulons at least partially overlap with each other and with the E. coli RpoH regulon. To uncover functional redundancies among the groE genes during growth and symbiosis, I constructed strains containing all possible combinations of groEL mutations. Although a groEL1 groEL2 double mutant could not be constructed, the 1^-3^- 4^-5^- and the 2^-3^-4 ^-5^- quadruple mutants are viable, demonstrating that like other bacteria S. meliloti requires one groEL for growth. Analysis of the quadruple mutants during symbiosis indicates that only groEL1 is necessary and sufficient for symbiosis. The groEL1 groESL5 double mutant is temperature sensitive unlike either single mutant, suggesting overlapping roles during stress response. I conclude that groESL1 encodes the housekeeping GroEL-GroES and that groESL5 is specialized for stress response.