Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti

Abstract.

We identified two rpoH-related genes encoding σ³²-like proteins from Sinorhizobium meliloti, a nitrogen-fixing root-nodule symbiont of alfalfa. The genes, rpoH ₁ and rpoH ₂, are functionally similar to rpoH of Escherichia coli because they partially complemented an E. coli rpoH null mutant. We obtained evidence indicating that these genes are involved in the heat shock response in S. meliloti. Following an increase in temperature, synthesis of several putative heat shock proteins (Hsps) was induced in cultures of wild-type cells: the most prominent were 66- and 60-kDa proteins, both of which are suggested to represent GroEL species. The other Hsps could divided into two groups based on differences in synthesis kinetics: synthesis of the first group peaked 5–10 min, and expression of the other group 30 min, after temperature upshift. In the rpoH ₁ mutant, inducible synthesis of the former group was markedly reduced, whereas that of the latter group was not affected. Synthesis of both the 66- and 60-kDa proteins was partially reduced. While no appreciable effect was observed in the rpoH ₂ single mutant, the rpoH ₂ mutation had a synergistic effect on the 60-kDa protein in the rpoH ₁ ^– background. The results indicate that two distinct mechanisms are involved in the heat shock response of S. meliloti: one requires the rpoH ₁ function, while rpoH ₂ can substitute in part for the rpoH ₁ function. Moreover, the rpoH ₁ mutant and rpoH ₁ rpoH ₂ double mutant exhibited Nod⁺ Fix^– and Nod^– phenotypes, respectively, on alfalfa.