Characterization of a fixLJ-regulated Bradyrhizobium japonicum gene sharing similarity with the Escherichia coli fnr and Rhizobium meliloti fixK genes

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Abstract
We describe the cloning, sequencing, regulation, and mutational analysis of a Bradyrhizobium japonicum fixK-like gene whose product belongs to the family of Fnr-Crp-related regulatory proteins. The predicted 237-amino-acid FixK protein was found to share between 28 and 38% sequence 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 activate transcription from an fnr-dependent promoter in the E. coli background; this activation was sixfold higher in anaerobic cultures than in aerobically grown cells, a finding that suggested oxygen sensitivity of the FixK protein and was consistent with the presence of a cysteine-rich, putatively oxygen-responsive domain at its N-terminal end. Similar to the situation in Rhizobium meliloti, expression of the fixK gene 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 have the phenotypic characteristics of B. japonicum fixL and fixJ mutants: the fixK mutant was neither Fix- in symbiosis with soybean plants nor defective in anaerobic respiration with nitrate as the terminal electron acceptor. Also, the fixK mutant was unaffected in the expression of one of the two B. japonicum sigma 54 genes, rpoN1, which was previously shown to be controlled by the fixLJ genes. When fixK was introduced into the B. japonicum fixJ mutant and expressed therein from a constitutive promoter (i.e., uncoupling it from regulation by FixJ), the FixK protein thus synthesized fully restored anaerobic nitrate respiration in that strain. We interpret this to mean that the B. japonicum wild type has two homologs of fixLJ-regulated fixK genes which can functionally substitute for each other.