CHARACTERIZATION OF A FIXLJ-REGULATED BRADYRHIZOBIUM-JAPONICUM GENE SHARING SIMILARITY WITH THE ESCHERICHIA-COLI FNR AND RHIZOBIUM-MELILOTIFIXK GENES

We describe the cloning, sequencing, regulation, and mutational analys is of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 23 7-amino-acid FixK protein was found to share between 28 and 38% sequen ce identity with the Escherichia coli Fnr protein, other bacterial Fnr -like proteins (FnrN, Anr, and HlyX), and two rhizobial FixK proteins. The B. japonicum fixK-like gene, when expressed from a lac promoter, could functionally complement an fnr mutant strain of E. coli and acti vate transcription from an fnr-dependent promoter in the E. coli backg round; this activation was sixfold higher in anaerobic cultures than i n aerobically grown cells, a finding that suggested oxygen sensitivity of the FixK protein and was consistent with the presence of a cystein e-rich, putatively oxygen-responsive domain at its N-terminal end. Sim ilar to the situation in Rhizobium meliloti, expression of the fixK ge ne in B. japonicum was shown to be induced at low O2 tension and this induction was dependent on the two-component regulatory system FixLJ. Despite this dependency, however, a B. japonicum fixK mutant did not h ave the phenotypic characteristics of B. japonicum fixL and fixJ mutan ts: the fixK mutant was neither Fix-in symbiosis with soybean plants n or defective in anaerobic respiration with nitrate as the terminal ele ctron acceptor. Also, the fixK mutant was unaffected in the expression of one of the two B. japonicum sigma(54) genes, rpoN1, which was prev iously shown to be controlled by the fixLJ genes. When fixK was introd uced into the B. japonicum fixJ mutant and expressed therein from a co nstitutive promoter (i.e., uncoupling it from regulation by FixJ), the FixK protein thus synthesized fully restored anaerobic nitrate respir ation in that strain. We interpret this to mean that the B. japonicum wild type has two homologs of fixLJ-regulated fixK genes which can fun ctionally substitute for each other.