Genomic Organization of Genes Regulated by NsrR, NorR, and DNR in β-Proteobacteria

<p>Magenta, green, and blue circles denote candidate NsrR, NorR, and DNR sites, respectively. Candidate σ⁵⁴ promoters associated with NorR sites are shown by angle arrows. Experimentally known sites of NorR and DNR are marked by “s.” Additional sites of the NarP and FNR factors are indicated by purple squares and black triangles, respectively.</p

Maximum Likelihood Phylogenetic Tree of the Hybrid Cluster (Prismane) Proteins

<p>Genes predicted to be regulated by the nitrogen oxides–related factors are highlighted by respective colors. Additionally, predicted FNR-regulated genes are denoted by black circles. Genes positionally linked to the NADH oxidoreductase <i>hcr</i> and the hypothetical ferredoxin <i>frdX</i> genes are shown by red and blue lines, respectively. Archaeal genes are shown by pointed lines.</p

Sequence Logos for the Identified NorR-Binding Sites in Various Species of Proteobacteria

<p>Sequence Logos for the Identified NorR-Binding Sites in Various Species of Proteobacteria</p

Regulatory Interactions between Genes for Dissimilatory Nitrogen Oxides Metabolism in Bacteria

<div><p>(A) Distribution of regulators and regulated genes. The number of cases when a gene is regulated by a specific transcription factor is indicated by the length of a colored bar in the histogram. The white bar in the histogram shows the cases when the gene is present in a genome possessing at least one of the studied regulons, but is not regulated by any of them.</p><p>(B) Combined regulatory network. Arrows denote regulatory interactions, with the thickness reflecting the frequency of the interaction in the analyzed genomes. Experimentally established (for DNR, NnrR, NsrR, and NorR) and predicted based on the regulon content (for HcpR) signal molecules are shown in filled ovals, and the protein family for each transcription factor is shown below.</p></div

Genomic Organization of Genes Regulated by NnrR and NsrR in α-Proteobacteria

<p>Orange and magenta circles denote candidate NnrR and NsrR sites, respectively. Experimentally known NnrR sites are marked by “s.”</p

Maximum Likelihood Phylogenetic Tree of the FNR/CRP Family of Transcriptional Regulators

<p>The third column contains sequences of helix-turn-helix motifs in the proteins. Two specificity-determining positions correlated with DNA motifs are colored (R₁₈₀ and E₁₈₁ in proteins correlate with G₃ and A₆ in DNA sites, respectively). The fourth column includes sequence logos for presumably homogeneous and large site sets and sequence consensi for small sets of DNA sites and for well-established motifs of other factors (FNR, CRP, CooA, NtcA, ArcR). The last column indicates the name of a search profile constructed in this study.</p

Genomic Organization of Genes Regulated by NsrR, NorR, and DNR in γ-Proteobacteria

<p>Magenta, green, and blue circles denote candidate NsrR, NorR, and DNR sites, respectively. Candidate σ⁵⁴ promoters associated with NorR sites are shown by angle arrows. Experimentally known sites of NorR and DNR are marked by “s.” Additional sites of the NarP and FNR factors are indicated by purple squares and black triangles, respectively.</p

The Bacterial Inorganic Nitrogen Cycle

<p>The ammonification, denitrification, detoxification, nitrogen fixation, and nitrification pathways are shown by colored solid lines with genes names involved in the pathway. The dashed black line shows possible non-enzymatic interconversions of nitrogen oxides. The dotted line shows additional formation of NO and hydroxylamine during nitrite ammonification.</p

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