Data_Sheet_1_Comparative Genomics Reveals the Regulatory Complexity of Bifidobacterial Arabinose and Arabino-Oligosaccharide Utilization.PDF

<p>Members of the genus Bifidobacterium are common inhabitants of the human gastrointestinal tract. Previously it was shown that arabino-oligosaccharides (AOS) might act as prebiotics and stimulate the bifidobacterial growth in the gut. However, despite the rapid accumulation of genomic data, the precise mechanisms by which these sugars are utilized and associated transcription control still remain unclear. In the current study, we used a comparative genomic approach to reconstruct arabinose and AOS utilization pathways in over 40 bacterial species belonging to the Bifidobacteriaceae family. The results indicate that the gene repertoire involved in the catabolism of these sugars is highly diverse, and even phylogenetically close species may differ in their utilization capabilities. Using bioinformatics analysis we identified potential DNA-binding motifs and reconstructed putative regulons for the arabinose and AOS utilization genes in the Bifidobacteriaceae genomes. Six LacI-family transcriptional factors (named AbfR, AauR, AauU1, AauU2, BauR1 and BauR2) and a TetR-family regulator (XsaR) presumably act as local repressors for AOS utilization genes encoding various α- or β-L-arabinofuranosidases and predicted AOS transporters. The ROK-family regulator AraU and the LacI-family regulator AraQ control adjacent operons encoding putative arabinose transporters and catabolic enzymes, respectively. However, the AraQ regulator is universally present in all Bifidobacterium species including those lacking the arabinose catabolic genes araBDA, suggesting its control of other genes. Comparative genomic analyses of prospective AraQ-binding sites allowed the reconstruction of AraQ regulons and a proposed binary repression/activation mechanism. The conserved core of reconstructed AraQ regulons in bifidobacteria includes araBDA, as well as genes from the central glycolytic and fermentation pathways (pyk, eno, gap, tkt, tal, galM, ldh). The current study expands the range of genes involved in bifidobacterial arabinose/AOS utilization and demonstrates considerable variations in associated metabolic pathways and regulons. Detailed comparative and phylogenetic analyses allowed us to hypothesize how the identified reconstructed regulons evolved in bifidobacteria. Our findings may help to improve carbohydrate catabolic phenotype prediction and metabolic modeling, while it may also facilitate rational development of novel prebiotics.</p

Comparative Genomics of Transcriptional Regulation of Methionine Metabolism in Proteobacteria

<div><p>Methionine metabolism and uptake genes in Proteobacteria are controlled by a variety of RNA and DNA regulatory systems. We have applied comparative genomics to reconstruct regulons for three known transcription factors, MetJ, MetR, and SahR, and three known riboswitch motifs, SAH, SAM-SAH, and SAM_alpha, in ∼200 genomes from 22 taxonomic groups of Proteobacteria. We also identified two novel regulons: a SahR-like transcription factor SamR controlling various methionine biosynthesis genes in the Xanthomonadales group, and a potential RNA regulatory element with terminator-antiterminator mechanism controlling the <i>metX</i> or <i>metZ</i> genes in beta-proteobacteria. For each analyzed regulator we identified the core, taxon-specific and genome-specific regulon members. By analyzing the distribution of these regulators in bacterial genomes and by comparing their regulon contents we elucidated possible evolutionary scenarios for the regulation of the methionine metabolism genes in Proteobacteria.</p></div

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Transport of Magnesium by a Bacterial Nramp-Related Gene

<div><p>Magnesium is an essential divalent metal that serves many cellular functions. While most divalent cations are maintained at relatively low intracellular concentrations, magnesium is maintained at a higher level (∼0.5–2.0 mM). Three families of transport proteins were previously identified for magnesium import: CorA, MgtE, and MgtA/MgtB P-type ATPases. In the current study, we find that expression of a bacterial protein unrelated to these transporters can fully restore growth to a bacterial mutant that lacks known magnesium transporters, suggesting it is a new importer for magnesium. We demonstrate that this transport activity is likely to be specific rather than resulting from substrate promiscuity because the proteins are incapable of manganese import. This magnesium transport protein is distantly related to the Nramp family of proteins, which have been shown to transport divalent cations but have never been shown to recognize magnesium. We also find gene expression of the new magnesium transporter to be controlled by a magnesium-sensing riboswitch. Importantly, we find additional examples of riboswitch-regulated homologues, suggesting that they are a frequent occurrence in bacteria. Therefore, our aggregate data discover a new and perhaps broadly important path for magnesium import and highlight how identification of riboswitch RNAs can help shed light on new, and sometimes unexpected, functions of their downstream genes.</p></div

Distribution of regulatory interactions for core members of methionine regulons in Proteobacteria.

<p>The presence or absence of gene orthologs in at least one studied genome in a taxonomic group is shown by light green or gray background, respectively. Regulation of at least one gene ortholog within each taxonomic group is shown by colored circles and squares as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113714#pone-0113714-g001" target="_blank">Figure 1</a>.</p

Conservation of regulatory interactions in the reconstructed SAM_alpha riboswitch regulons.

<p>The core regulon, taxon- and genome-specific regulon members are highlighted and listed along with their average conservation scores and functional annotations in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113714#pone.0113714.s004" target="_blank">Table S2</a>.</p

Statistics of reconstructed methionine regulons in Proteobacteria.

<p>The table shows number of genomes containing the methionine regulons per a taxon.</p>a<p>Genomes are classified into 22 taxonomic groups by analyzing the phylogenetic species tree on the MicrobesOnline <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113714#pone.0113714-Dehal1" target="_blank">[40]</a>. The detailed list of analyzed genomes and taxonomic groups is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113714#pone.0113714.s003" target="_blank">Table S1</a>.</p>b<p>This column shows the number of analyzed genomes in each taxon.</p>c<p>This column combines the numbers of SahR and SamR regulons. SamR was identified only in <i>Xanthomonadales</i>, whereas SahR regulons are distributed across the remaining nine groups.</p>1<p>this family belongs to the <i>Alteromonadales</i> order.</p>2<p>this family belongs to the <i>Pseudomonadales</i> order.</p>3<p>this family belongs to the <i>Burkholderiales</i> order.</p><p>Statistics of reconstructed methionine regulons in Proteobacteria.</p

Heterologous expression of Ca_c0685 and Ca_c3329.

<p>The <i>ca_c0685</i> and <i>ca_c3329</i> genes were subcloned under IPTG-inducible control and integrated single-copy into the <i>B. subtilis amyE</i> gene. These expression cassettes were also integrated into various strains containing deletions of different divalent cation transporters. In all instances, expression of <i>ca_c0685</i> and <i>ca_c3329</i> was monitored by S1 mapping (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s001" target="_blank">Figure S1</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s002" target="_blank">S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s003" target="_blank">S3</a>). To investigate the effect of gene expression in these various strains, cells were cultured alongside control strains. Shown herein are bar graphs plots of stationary phase growth for these respective strains. (A) Growth after entry into stationary phase is shown for <i>B. subtilis</i> control strains, including a wild-type and a manganese transport-deficient strain, and transport-deficient strains complemented with IPTG-inducible control of <i>B. subtilis</i> MntH, Ca_c0685 or Ca_c3329. These strains were cultured in minimal medium with no added manganese in the presence of 0.5 mM IPTG. (B) Heterologous expression of <i>B. subtilis</i> MntH and MntABCD do not rescue a magnesium-transport deficient phenotype. Growth measurements immediately after entry into stationary phase are shown for <i>B. subtilis</i> control strains, including wild-type, a magnesium transport-deficient strain, and transport-deficient strains complemented with either IPTG-inducible MntABCD or MntH. Full growth curves are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s002" target="_blank">Figure S2</a>. These strains were cultured in rich medium in the presence of 0.5 mM IPTG. (C) Heterologous expression of Ca_c0685 and Ca_c3329 in a magnesium transport-deficient strain. Growth measurements immediately after entry into stationary phase (full growth curves are included in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s003" target="_blank">Figure S3</a>) are shown for <i>B. subtilis</i> strains, including wild-type, a magnesium transport-deficient control strain, and transport-deficient strains complemented with inducible Ca_c0685, Ca_c3329, or the magnesium transporter MgtE. The strains were cultured in rich medium in the presence of 0.5 mM IPTG and 2.5 mM magnesium. Expression of Ca_c0685 and Ca_c3329 both fully rescued growth in this medium. (D) In addition to the liquid culture growth experiments, 3 µl of each of these strains (∼1×10⁴/µl) was spotted onto solid medium containing a gradient of magnesium that ranged from 0 to 5 mM magnesium, respectively. These plates were incubated for 10 hours at 37°C before they were photographed.</p

Control of gene expression by a <i>C. acetobutylicum</i> M-box RNA.

<p>The <i>Ca_c0685</i> and <i>Ca_c3329</i> riboswitches were fused downstream of a constitutive promoter (<i>PrpsD</i>) and upstream of the yellow fluorescent reporter gene (<i>yfp</i>) to determine whether they could control heterologous gene expression in a divalent cation-dependent manner. Control strains either lacking the <i>yfp</i> reporter construct or containing a constitutive <i>PrpsD-yfp</i> fusion were included in this study. Cells were cultured to mid-logarithmic growth phase in 2xYT rich medium supplemented with 50 µM MgCl₂ (-), then incubated with 2 mM chelating agent, EDTA, or incubated in the presence of excess magnesium (2 mM) for one hour. Total RNA was assessed by staining of <i>rRNA</i> bands. Abundance of the <i>yfp</i> gene and of a zinc-responsive control transcript were monitored by S1 mapping. Radiolabeled DNA probes (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004429#pgen.1004429.s008" target="_blank">Table S2</a>) were used for S1 mapping of the <i>yfp</i> transcript.</p

Alternative secondary structures of candidate metXZ RNA element.

<p>Regions 1–6 shown in yellow boxes are conserved sequences found in the multiple alignment of leader regions of 34 <i>metX</i> and <i>metZ</i> genes from β-proteobacteria (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113714#pone.0113714.s002" target="_blank">Figure S2</a>). In the consensus RNA sequence, N denotes any nucleotide, and M stands for A or C. Possible secondary structures formed by the interaction between the conserved regions are shown by yellow lines.</p