Computational Reconstruction of Iron- and Manganese-Responsive Transcriptional Networks in α-Proteobacteria

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Abstract
We used comparative genomics to investigate the distribution of conserved DNA-binding motifs in the regulatory regions of genes involved in iron and manganese homeostasis in alpha-proteobacteria. Combined with other computational approaches, this allowed us to reconstruct the metal regulatory network in more than three dozen species with available genome sequences. We identified several classes of cis-acting regulatory DNA motifs (Irr-boxes or ICEs, RirA-boxes, Iron-Rhodo-boxes, Fur-alpha-boxes, Mur-box or MRS, MntR-box, and IscR-boxes) in regulatory regions of various genes involved in iron and manganese uptake, Fe-S and heme biosynthesis, iron storage, and usage. Despite the different nature of the iron regulons in selected lineages of alpha-proteobacteria, the overall regulatory network is consistent with, and confirmed by, many experimental observations. This study expands the range of genes involved in iron homeostasis and demonstrates considerable interconnection between iron-responsive regulatory systems. The detailed comparative and phylogenetic analyses of the regulatory systems allowed us to propose a theory about the possible evolution of Fe and Mn regulons in alpha-proteobacteria. The main evolutionary event likely occurred in the common ancestor of the Rhizobiales and Rhodobacterales, where the Fur protein switched to regulating manganese transporters (and hence Fur had become Mur). In these lineages, the role of global iron homeostasis was taken by RirA and Irr, two transcriptional regulators that act by sensing the physiological consequence of the metal availability rather than its concentration per se, and thus provide for more flexible regulation. The availability of hundreds of complete genomes allows one to use comparative genomics to describe key metabolic processes and regulatory gene networks. Genome context analyses and comparisons of transcription factor binding sites between genomes offer a powerful approach for functional gene annotation. Reconstruction of transcriptional regulatory networks allows for better understanding of cellular processes, which can be substantiated by direct experimentation. Iron homeostasis in bacteria is conferred by the regulation of various iron uptake transporters, iron storage ferritins, and iron-containing enzymes. In high concentrations, iron is poisonous for the cell, so strict control of iron homeostasis is maintained, mostly at the level of transcription by iron-responsive regulators. Despite their general importance, iron regulatory networks in most bacterial species are not well-understood. In this study, Rodionov and colleagues applied comparative genomic approaches to describe the regulatory network formed by genes involved in iron homeostasis in the alpha subclass of proteobacteria, which have extremely versatile lifestyles. These networks are mediated by a set of various DNA motifs (or regulatory signals) that occur in 5′ gene regions and involve at least six different metal-responsive regulators. This study once again shows the power of comparative genomics in the analysis of complex regulatory networks and their evolution.