Distribution of Irr, Fur/Mur, MntR, RirA, and IscR Regulators in α-Proteobacteria

<p>Genome abbreviations are given in the second column. The conventional taxonomic tree of α-proteobacteria shown on the left of the table was adapted from the NCBI Taxonomy Homepage (<a href="http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html" target="_blank">http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html</a>) and is in agreement with the tree of α-proteobacteria based on 16S rRNA sequences [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020163#pcbi-0020163-b064" target="_blank">64</a>]. Double plus (“++”) denotes the presence of two genes encoding copies of a given transcription factor.</p

Sequence Logos for the Predicted Irr Recognition Motifs (ICEs) in Various Groups of α-Proteobacteria

<div><p>(A) Rhizobiaceae plus <i>Mesorhizobium, Brucella,</i> and <i>Bartonella</i> species.</p><p>(B) Bradyrhizobiaceae group.</p><p>(C) Rhodobacteraceae group.</p><p>(D) Rhodospirillales group.</p></div

Multiple Alignment of the Upstream Regions of the <i>iscR-suf</i> Operons in the Rhodobacteraceae Species

<p>Multiple Alignment of the Upstream Regions of the <i>iscR-suf</i> Operons in the Rhodobacteraceae Species</p

Validation of the Predicted RirA Recognition Motif in R. leguminosarum by Site-Directed Mutagenesis

<div><p>(A) RirA-box in the common intergenic region of the RirA-regulated <i>vbsC</i> and <i>rpoI</i> genes in R. leguminosarum. The sequence of this region is shown where the transcription start sites are in bold and marked by arrows. The previously identified IRO-boxes for <i>vbsC</i> and <i>rpoI</i> [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020163#pcbi-0020163-b041" target="_blank">41</a>] are under the dashed line brackets. The highly conserved “TGA” and “TCA” in the newly described RirA-box are highlighted.</p><p>(B) Effect of mutating the conserved regions of the RirA-boxes on Fe-responsive expression of <i>rpoI-lacZ</i> and <i>vbsC-lacZ</i> transcriptional fusions. The previously described [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020163#pcbi-0020163-b041" target="_blank">41</a>] plasmids pBIO1328 and pBIO1306 are based on the wide host-range promoter probe plasmid pMP220 [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020163#pcbi-0020163-b080" target="_blank">80</a>] and contain the promoter and regulatory regions of <i>rpoI</i> and <i>vbsC,</i> respectively, fused to its promoter-less <i>lacZ</i> gene. In addition, four new sets of mutant derivatives were made, in which the conserved “TGA” and “TCA” sequences of the RirA-box were substituted, using methods described by Yeoman et al. (2004). Mutant derivatives of pBIO1328 and pBIO1306 with substitutions of the conserved TGA and TCA sequences of the RirA-box were made using the Stratagene ExSite PCR-based Site-directed Mutagenesis kit, with each of these two plasmids being used as template and a suitable oligonucleotide as the mutagenic primer. The mutated forms are shown with dark backgrounds. Each of the six plasmids was individually mobilized into wild type <i>R. leguminosarum.</i> Transconjugants were grown in Fe-replete and Fe-depleted medium and assayed in triplicate for β-galactosidase activity as in Wexler et al. [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020163#pcbi-0020163-b081" target="_blank">81</a>].</p></div

Phylogenetic Tree of α-Proteobacterial Regulators from the Fur Superfamily

<p>(A) Fur/Mur, (B) Irr. Experimentally characterized regulatory proteins are in bold and boxed. Positional clustering (i.e., close linkage) of the <i>mur</i> genes with the target manganese uptake <i>sit</i> operons is shown by background grey. Irr proteins with the heme regulatory motif (see text) at their N-termini are marked with asterisks. Functional annotation of the “mur” and “fur^α” regulators is based on the genomic analysis of their candidate regulatory motifs that occur in 5′ regions of either manganese or iron uptake genes, respectively. Possible role of the B. japonicum regulator Fur in the control of the manganese uptake gene <i>mntH</i> was predicted in this study and is not yet proved.</p

Sequence Logos for the Predicted Regulatory Sites in α-Proteobacteria

<div><p>(A) RirA-box (IRO) in eight species from the Rhizobiales order (four Rhizobiaceae, two <i>Mesorhizobium</i> species, <i>Brucella,</i> and <i>Bartonella</i>).</p><p>(B) Iron-Rhodo-box in the Rhodobacteraceae.</p><p>(C) Mur-box (MRS) in the Rhodobacteraceae/Rhizobiales.</p><p>(D) Fur^α-box in other α-proteobacteria species.</p><p>(E) IscR^α-I motif in the Rhodobacterales, the Rickettsia, <i>Pelagibacter, Oceanicaulis, Caulobacter, Parvularcula, Rhodospirillum,</i> and <i>Magnetospirillum</i> species.</p><p>(F) IscR^α-II motif in the Sphingomonadales and <i>Gluconobacter</i> species.</p></div

Combined Regulatory Network for Iron and Manganese Homeostasis Genes in α-Proteobacteria

<p>The connecting lines denote regulatory interactions, with the thickness reflecting the frequency of the interaction in the analyzed genomes.</p