Dual RpoH sigma factors and transcriptional plasticity in a symbiotic bacterium.

Sinorhizobium meliloti can live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (σ) factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative σ factors, including two putative RpoH ("heat shock") σ factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2, and rpoH1 rpoH2 mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2. We mapped transcript start sites of 69 rpoH-dependent genes using 5' RACE (5' rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferred S. meliloti RpoH promoter consensus sequences share features of Escherichia coli RpoH promoters but lack extended -10 motifs.

Only one of five groEL genes is required for viability and successful symbiosis in Sinorhizobium meliloti.

Many bacterial species contain multiple copies of the genes that encode the chaperone GroEL and its cochaperone, GroES, including all of the fully sequenced root-nodulating bacteria that interact symbiotically with legumes to generate fixed nitrogen. In particular, in Sinorhizobium meliloti there are four groESL operons and one groEL gene. To uncover functional redundancies of these genes during growth and symbiosis, we attempted to construct strains containing all combinations of groEL mutations. Although a double groEL1 groEL2 mutant cannot be constructed, we demonstrate that the quadruple groEL1 groESL3 groEL4 groESL5 and groEL2 groESL3 groEL4 groESL5 mutants are viable. Therefore, like E. coli and other species, S. meliloti requires only one groEL gene for viability, and either groEL1 or groEL2 will suffice. The groEL1 groESL5 double mutant is more severely affected for growth at both 30 degrees C and 40 degrees C than the single mutants, suggesting overlapping functions in stress response. During symbiosis the quadruple groEL2 groESL3 groEL4 groESL5 mutant acts like the wild type, but the quadruple groEL1 groESL3 groEL4 groESL5 mutant acts like the groEL1 single mutant, which cannot fully induce nod gene expression and forms ineffective nodules. Therefore, the only groEL gene required for symbiosis is groEL1. However, we show that the other groE genes are expressed in the nodule at lower levels, suggesting minor roles during symbiosis. Combining our data with other data, we conclude that groESL1 encodes the housekeeping GroEL/GroES chaperone and that groESL5 is specialized for stress response.

Multiple groESL operons are not key targets of RpoH1 and RpoH2 in Sinorhizobium meliloti.

Among the rhizobia that establish nitrogen-fixing nodules on the roots of host plants, many contain multiple copies of genes encoding the sigma factor RpoH and the chaperone GroEL/GroES. In Sinorhizobium meliloti there are two rpoH genes, four groESL operons, and one groEL gene. rpoH1 mutants are defective for growth at high temperature and form ineffective nodules, rpoH1 rpoH2 double mutants are unable to form nodules, and groESL1 mutants form ineffective nodules. To explore the roles of RpoH1 and RpoH2, we identified mutants that suppress both the growth and nodulation defects. These mutants do not suppress the nitrogen fixation defect. This implies that the functions of RpoH1 during growth and RpoH1/RpoH2 during the initiation of symbiosis are similar but that there is a different function of RpoH1 needed later during symbiosis. We showed that, unlike in Escherichia coli, overexpression of groESL is not sufficient to bypass any of the RpoH defects. Under free-living conditions, we determined that RpoH2 does not control expression of the groE genes, and RpoH1 only controls expression of groESL5. Finally, we completed the series of groE mutants by constructing groESL3 and groEL4 mutants and demonstrated that they do not display symbiotic defects. Therefore, the only groESL operon required by itself for symbiosis is groESL1. Taken together, these results suggest that GroEL/GroES production alone cannot explain the requirements for RpoH1 and RpoH2 in S. meliloti and that there must be other crucial targets.

Identification of the heat-shock sigma factor RpoH and a second RpoH-like protein in Sinorhizobium meliloti.

Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors. The first gene, rpoH1, complements an Escherichia coli rpoH mutation. Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth. The second gene, designated rpoH2, is 42% identical to the S. meliloti rpoH1 gene. Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa. An rpoH2-gusA fusion increases in expression during the stationary phase of growth. The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes.