Computational reconstruction of iron- and manganese-responsive transcriptional networks in alpha-proteobacteria

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.

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens

“…Consensus DNA binding sites for Irr (the iron control element [ICE] box) and RirA (the iron response operator [IRO] box) have been determined for the Alphaproteobacteria (53). The presence of ICE and IRO boxes in the genome of A. tumefaciens was analyzed using the RegPredict software package with score cutoffs of 4.9 for ICE box prediction and 3.93 for IRO box prediction (45).…”

“…While nearly all free-living Alphaproteobacteria contain close homologues of fur, evidence suggests that in some branches, this protein is functioning predominantly to regulate the cellular response to manganese levels (3,6,15,48,53). In place of the typical Fur-based iron transcriptional response, many of the Alphaproteobacteria use the iron regulator Irr (53). Irr represents a distinct branch of the Fur family of transcriptional regulators that was first discovered in the soybean-nodulating organism Bradyrhizobium japonicum as a regulator of heme biosynthesis (22,34).…”

“…In addition to Irr, all members of the Rhizobiaceae contain homologues of a different iron-responsive regulator designated RirA, for rhizobial iron regulator A (53). RirA was initially discovered in Rhizobium leguminosarum and is a member of the Rrf2 family of putative transcriptional regulators (31,65).…”

“…The ligand that imparts iron responsiveness to RirA has not been established, although the related Rrf2-type protein IscR binds and responds to Fe-S clusters, and it has been suggested that RirA also binds Fe-S clusters (19,31,58). Agrobacterium tumefaciens is also a well-studied member of the Alphaproteobacteria, related to both B. japonicum and R. leguminosarum (7,53). A. tumefaciens is best known for its ability to cause crown gall neoplasia on plants.…”

“…tumefaciens encodes homologues of fur, rirA, and irr, as well as a large cluster of genes that directs the synthesis of a siderophore (20,54,72). The A. tumefaciens fur homologue is reported to primarily regulate manganese uptake and plays only a minor role in response to iron levels (33,53). The RirA and Irr proteins are in fact the probable iron-responsive regulatory candidates.…”