Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti
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O R I GI N A L P A P E R
Y. Ono á H. Mitsui á T. Sato á K. Minamisawa
Two RpoH homologs responsible for the expression of heat shock protein genes in Sinorhizobium meliloti
Received: 30 May 2000 / Accepted: 25 September 2000 / Published online: 9 November 2000 Ó Springer-Verlag 2000
Abstract We identi®ed two rpoH-related genes encoding r32-like proteins from Sinorhizobium meliloti, a nitrogen®xing root-nodule symbiont of alfalfa. The genes, rpoH1 and rpoH2, 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 dierences in synthesis kinetics: synthesis of the ®rst group peaked 5± 10 min, and expression of the other group 30 min, after temperature upshift. In the rpoH1 mutant, inducible synthesis of the former group was markedly reduced, whereas that of the latter group was not aected. Synthesis of both the 66- and 60-kDa proteins was partially reduced. While no appreciable eect was observed in the rpoH2 single mutant, the rpoH2 mutation had a synergistic eect on the 60-kDa protein in the rpoH1± background. The results indicate that two distinct mechanisms are involved in the heat shock response of S. meliloti: one requires the rpoH1 function, while rpoH2 can substitute in part for the rpoH1 function. Moreover, the rpoH1 mutant and rpoH1 rpoH2 double mutant exhibited Nod+ Fix± and Nod± phenotypes, respectively, on alfalfa. Key words Sinorhizobium meliloti á rpoH á Sigma factor á Nitrogen ®xation á Heat shock protein
Communicated by R. Hagemann Y. Ono á H. Mitsui (&) á T. Sato á K. Minamisawa Institute of Genetic Ecology, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan E-mail: [email protected] Tel.: +81-22-2175685; Fax: +81-22-2639845
Introduction In the eubacteria, the core RNA polymerase, with the subunit composition a2bb¢, catalyzes the synthesis of RNA from a DNA template, and the sigma factor mediates promoter-speci®c transcription initiation by RNA polymerase. Most bacterial species has several distinct sigma factors, the primary sigma factor and alternative sigma factors, and the selectivity of RNA polymerase is modulated in the ®rst instance by the species of sigma factor associated with the core enzyme. For example, Escherichia coli has seven species of sigma factors. The primary sigma factor, r70 (the rpoD gene product), is responsible for the transcription of most genes expressed in growing cells and is essential for viability. Each of the other alternative sigma factors directs the transcription of a speci®c set of genes with distinct promoter sequences, resulting in the coordinate regulation of expression of those ge
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