Figure 1.
Distribution of Irr, Fur/Mur, MntR, RirA, and IscR Regulators in α-Proteobacteria
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 (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html) and is in agreement with the tree of α-proteobacteria based on 16S rRNA sequences [64]. Double plus (“++”) denotes the presence of two genes encoding copies of a given transcription factor.
Figure 2.
Phylogenetic Tree of α-Proteobacterial Regulators from the Fur Superfamily
(A) Fur/Mur, (B) Irr. Experimentally characterized regulatory proteins are in bold and boxed. Positional clustering (i.e., close linkage) of the mur genes with the target manganese uptake sit 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 “furα” 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 mntH was predicted in this study and is not yet proved.
Figure 3.
Phylogenetic Tree of the α-proteobacterial Regulators from the Rrf2 Superfamily
(A) RirA/NsrR, (B) IscR. Genome abbreviations are listed in Figure 1. nsrR/rirA homologs positionally linked with the nitrosative stress response genes are underlined.
Figure 4.
Sequence Logos for the Predicted Regulatory Sites in α-Proteobacteria
(A) RirA-box (IRO) in eight species from the Rhizobiales order (four Rhizobiaceae, two Mesorhizobium species, Brucella, and Bartonella).
(B) Iron-Rhodo-box in the Rhodobacteraceae.
(C) Mur-box (MRS) in the Rhodobacteraceae/Rhizobiales.
(D) Furα-box in other α-proteobacteria species.
(E) IscRα-I motif in the Rhodobacterales, the Rickettsia, Pelagibacter, Oceanicaulis, Caulobacter, Parvularcula, Rhodospirillum, and Magnetospirillum species.
(F) IscRα-II motif in the Sphingomonadales and Gluconobacter species.
Figure 5.
Validation of the Predicted RirA Recognition Motif in R. leguminosarum by Site-Directed Mutagenesis
(A) RirA-box in the common intergenic region of the RirA-regulated vbsC and rpoI 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 vbsC and rpoI [41] are under the dashed line brackets. The highly conserved “TGA” and “TCA” in the newly described RirA-box are highlighted.
(B) Effect of mutating the conserved regions of the RirA-boxes on Fe-responsive expression of rpoI-lacZ and vbsC-lacZ transcriptional fusions. The previously described [41] plasmids pBIO1328 and pBIO1306 are based on the wide host-range promoter probe plasmid pMP220 [80] and contain the promoter and regulatory regions of rpoI and vbsC, respectively, fused to its promoter-less lacZ 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 R. leguminosarum. Transconjugants were grown in Fe-replete and Fe-depleted medium and assayed in triplicate for β-galactosidase activity as in Wexler et al. [81].
Figure 6.
Sequence Logos for the Predicted Irr Recognition Motifs (ICEs) in Various Groups of α-Proteobacteria
(A) Rhizobiaceae plus Mesorhizobium, Brucella, and Bartonella species.
(B) Bradyrhizobiaceae group.
(C) Rhodobacteraceae group.
(D) Rhodospirillales group.
Figure 7.
Occurrence of Candidate Regulatory Elements and Genes Involved in the Iron and Manganese Homeostasis in α-Proteobacteria
Only conserved members of the predicted Fe/Mn regulons are shown. Genes are arranged by their functional role. Genomes are arranged by taxonomic lineages. When the gene is present in the genome, the background colors denote the presence of the specific recognition motif in its upstream region. If the gene is preceded by two different regulatory motifs, it is shown by a diagonally separated bicolor square. Differential regulation of two mntH paralogs in Bradyrhizobium sp. BTAi1 by Fur/Mur and MntR regulons is shown by vertically separated bicolor square. Genes without any candidate iron or manganese regulatory motifs described in this study are indicated by grey background color. Empty crossings denote the absence of an orthologous gene in the genome.
Figure 8.
Sequence Logos for the Predicted MntR Recognition Motifs in α-Proteobacteria
Table 1.
Comparison of the Predicted Irr Regulon and ICE Motifs with the Published Expression Microarray Data for Iron- and Irr-Affected Genes in Bradyrhizobium japonicum
Figure 9.
Multiple Alignment of the Upstream Regions of the iscR-suf Operons in the Rhodobacteraceae Species
Figure 10.
Combined Regulatory Network for Iron and Manganese Homeostasis Genes in α-Proteobacteria
The connecting lines denote regulatory interactions, with the thickness reflecting the frequency of the interaction in the analyzed genomes.