Promoter Selectivity of the Bradyrhizobium japonicum RpoH Transcription Factors In Vivo and In Vitro

Expression of the dnaKJ and groESL(1) heat shock operons of Bradyrhizobium japonicum depends on a ς(32)-like transcription factor. Three such factors (RpoH(1), RpoH(2), and RpoH(3)) have previously been identified in this organism. We report here that they direct transcription from some but not all ς(32)-type promoters when the respective rpoH genes are expressed in Escherichia coli. All three RpoH factors were purified as soluble C-terminally histidine-tagged proteins, although the bulk of overproduced RpoH(3) was insoluble. The purified proteins were recognized by an anti-E. coli ς(32) serum. While RpoH(1) and RpoH(2) productively interacted with E. coli core RNA polymerase and produced E. coli groE transcript in vitro, RpoH(3) was unable to do so. B. japonicum core RNA polymerase was prepared and reconstituted with the RpoH proteins. Again, RpoH(1) and RpoH(2) were active, and they initiated transcription at the B. japonicum groESL(1) and dnaKJ promoters. In all cases, the in vitro start site was shown to be identical to the start site determined in vivo. Promoter competition experiments revealed that the B. japonicum dnaKJ and groESL(1) promoters were suboptimal for transcription by RpoH(1)- or RpoH(2)-containing RNA polymerase from B. japonicum. In a mixture of different templates, the E. coli groESL promoter was preferred over any other promoter. Differences were observed in the specificities of both sigma factors toward B. japonicum rpoH-dependent promoters. We conclude that the primary function of RpoH(2) is to supply the cell with DnaKJ under normal growth conditions whereas RpoH(1) is responsible mainly for increasing the level of GroESL(1) after a heat shock.