Reconstruction of the Core and Extended Regulons of Global Transcription Factors

The processes underlying the evolution of regulatory networks are unclear. To address this question, we used a comparative genomics approach that takes advantage of the large number of sequenced bacterial genomes to predict conserved and variable members of transcriptional regulatory networks across phylogenetically related organisms. Specifically, we developed a computational method to predict the conserved regulons of transcription factors across α-proteobacteria. We focused on the CRP/FNR super-family of transcription factors because it contains several well-characterized members, such as FNR, FixK, and DNR. While FNR, FixK, and DNR are each proposed to regulate different aspects of anaerobic metabolism, they are predicted to recognize very similar DNA target sequences, and they occur in various combinations among individual α-proteobacterial species. In this study, the composition of the respective FNR, FixK, or DNR conserved regulons across 87 α-proteobacterial species was predicted by comparing the phylogenetic profiles of the regulators with the profiles of putative target genes. The utility of our predictions was evaluated by experimentally characterizing the FnrL regulon (a FNR-type regulator) in the α-proteobacterium Rhodobacter sphaeroides. Our results show that this approach correctly predicted many regulon members, provided new insights into the biological functions of the respective regulons for these regulators, and suggested models for the evolution of the corresponding transcriptional networks. Our findings also predict that, at least for the FNR-type regulators, there is a core set of target genes conserved across many species. In addition, the members of the so-called extended regulons for the FNR-type regulators vary even among closely related species, possibly reflecting species-specific adaptation to environmental and other factors. The comparative genomics approach we developed is readily applicable to other regulatory networks

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

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