Comparative transcriptomics in : a global view of environmental modulation of gene expression-4

<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptomics in : a global view of environmental modulation of gene expression"</p><p>http://www.biomedcentral.com/1471-2180/7/96</p><p>BMC Microbiology 2007;7():96-96.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2231364.</p><p></p>7 contain 1.0, 0.7, 0.4, 0.1 and 0 μg of the recombinant Fur protein. In (A) – (I), an arrow and an asterisk indicate the probe (free) and the Fur-probe complex (bound), respectively

Comparative transcriptomics in : a global view of environmental modulation of gene expression-0

<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptomics in : a global view of environmental modulation of gene expression"</p><p>http://www.biomedcentral.com/1471-2180/7/96</p><p>BMC Microbiology 2007;7():96-96.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2231364.</p><p></p>s show the transcriptional changes of the virulence genes, where columns represent different microarray experiments, and rows represent genes. Color intensities denote logratios as follows: green, negative; black, zero; red, positive; gray, missing data

Comparative transcriptomics in : a global view of environmental modulation of gene expression-3

<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptomics in : a global view of environmental modulation of gene expression"</p><p>http://www.biomedcentral.com/1471-2180/7/96</p><p>BMC Microbiology 2007;7():96-96.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2231364.</p><p></p>R-like box; and (c) Fnr-like box. The underlined number is the maximum possible score with PSSM. For the sequence logo, the height of each letter indicates the relative frequency of that base at that position, while the height of each stack of letters corresponds to the sequence conservation at that position

Comparative transcriptomics in : a global view of environmental modulation of gene expression-2

<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptomics in : a global view of environmental modulation of gene expression"</p><p>http://www.biomedcentral.com/1471-2180/7/96</p><p>BMC Microbiology 2007;7():96-96.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2231364.</p><p></p>n in Table 4, while rows from up to down represent genes and their corresponding gene names were listed in the order (left to right and up to down). The black vertical lines are used to define the range of clusters of co-expressed genes. Red represents up-regulation and green represents down-regulation of the corresponding genes

Comparative transcriptomics in : a global view of environmental modulation of gene expression-5

<p><b>Copyright information:</b></p><p>Taken from "Comparative transcriptomics in : a global view of environmental modulation of gene expression"</p><p>http://www.biomedcentral.com/1471-2180/7/96</p><p>BMC Microbiology 2007;7():96-96.</p><p>Published online 29 Oct 2007</p><p>PMCID:PMC2231364.</p><p></p>s show the transcriptional changes of the virulence genes, where columns represent different microarray experiments, and rows represent genes. Color intensities denote logratios as follows: green, negative; black, zero; red, positive; gray, missing data

Molecular Characterization of Direct Target Genes and <em>cis</em>-Acting Consensus Recognized by Quorum-Sensing Regulator AphA in <em>Vibrio parahaemolyticus</em>

<div><h3>Background</h3><p>AphA is the master quorum-sensing (QS) regulator operating at low cell density in vibrios. Molecular regulation of target genes by AphA has been characterized in <em>Vibrio harveyi</em> and <em>V. cholerae</em>, but it is still poorly understood in <em>V. parahaemolyticus</em>.</p> <h3>Methodology/Principal Findings</h3><p>The AphA proteins are extremely conserved in <em>V. parahaemolyticus, Vibrio</em> sp. Ex25, <em>Vibrio</em> sp. EJY3, <em>V. harveyi, V. vulnificus, V. splendidus, V. anguillarum, V. cholerae,</em> and <em>V. furnissii.</em> The above nine AphA orthologs appear to recognize conserved <em>cis</em>-acting DNA signals which can be represented by two consensus constructs, a 20 bp box sequence and a position frequency matrix. <em>V. parahaemolyticus</em> AphA represses the transcription of <em>ahpA, qrr4</em>, and <em>opaR</em> through direct AphA-target promoter DNA association, while it inhibits the <em>qrr2-3</em> transcription in an indirect manner. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and AphA-binding sites (containing corresponding AphA box-like sequences) were determined for the three direct AphA targets <em>ahpA, qrr4,</em> and <em>opaR</em> in <em>V. parahaemolyticus</em>.</p> <h3>Conclusions/Significance</h3><p>AphA-mediated repression of <em>ahpA</em>, <em>qrr2-4</em>, and <em>opaR</em> was characterized in <em>V. parahaemolyticus</em> by using multiple biochemical and molecular experiments. The computational promoter analysis indicated the conserved mechanism of transcriptional regulation of QS regulator-encoding genes <em>ahpA</em>, <em>qrr4</em>, and <em>opaR</em> in vibrios.</p> </div

Phylogenetic tree of AphA orthologs.

<p>The protein sequences were derived from V. parahaemolyticus RIMD 2210633 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Makino1" target="_blank">[14]</a>, Vibrio sp. Ex25 (Accession numbers CP001805.1, and CP001806.1), Vibrio sp. EJY3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Roh1" target="_blank">[48]</a>, V. harveyi ATCC BAA-1116 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Lin3" target="_blank">[49]</a>, V. vulnificus YJ016 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Chen1" target="_blank">[50]</a>, V. splendidus LGP32 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-LeRoux1" target="_blank">[51]</a>, V. anguillarum 775 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Naka1" target="_blank">[52]</a>, V. cholerae N16961 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Heidelberg1" target="_blank">[53]</a>, V. furnissii NCTC 11218 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Lux1" target="_blank">[54]</a>, and P. profundum SS9 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Vezzi1" target="_blank">[25]</a>. The a.a. sequences were aligned by the CLUSTALW <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Thompson1" target="_blank">[55]</a> web server at <a href="http://align.genome.jp/" target="_blank">http://align.genome.jp/</a>. The aligned sequences were then used to construct an unrooted neighbor-joining tree using MEGA version 5.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Tamura1" target="_blank">[56]</a> with a bootstrap iteration number of 1000. Shown on branch points of phylogenic tree were bootstrap values (%).</p

Bacterial growth curves.

<p>A two-round design to prepare the bacterial seed culture was employed: firstly, the glyceric stock of bacterial cells was inoculated into 3 ml of MR broth for growing at 30°C with shaking at 200 rpm for 12 to 14 h to enter the stationary growth phase; secondly, the resulting cell culture was 50-fold diluted into 15 ml of fresh MR broth, and allowed to grow under the above conditions to reach an OD₆₀₀ value of about 1.0 to 1.2. The seed culture was then 1000-fold diluted into 30 ml of fresh MR broth for the further growth under the above conditions, and the OD₆₀₀ values were monitored for each culture with an 1 h interval.</p

Organization of promoter-proximal DNA regions.

<p>DNA sequences were derived from V. parahaemolyticus RIMD 2210633 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Makino1" target="_blank">[14]</a>, Vibrio sp. Ex25 (Accession numbers CP001805.1, and CP001806.1), Vibrio sp. EJY3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Roh1" target="_blank">[48]</a>, V. harveyi ATCC BAA-1116 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Lin3" target="_blank">[49]</a>, V. vulnificus YJ016 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Chen1" target="_blank">[50]</a>, V. splendidus LGP32 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-LeRoux1" target="_blank">[51]</a>, V. anguillarum 775 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Naka1" target="_blank">[52]</a>, V. cholerae N16961 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Heidelberg1" target="_blank">[53]</a>, and V. furnissii NCTC 11218 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Lux1" target="_blank">[54]</a>. Shown were translation and transcription starts, SD sequences, AphA or OpaR box-like sequences, and –0/−12 and –35/−24 core promoter elements. The prediction of OpaR box-like sequences were described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone.0044210-Zhang1" target="_blank">[15]</a>.</p

Repression of opaR by AphA.

<p>For LacZ fusion (a), the promoter-proximal DNA fragment of opaR was cloned into pHRP309, and then transformed into WT or ΔaphA to determine the β-galactosidase activity (Miller units) in cellular extracts. For primer extension (b), an oligonucleotide primer was designed to be complementary to the RNA transcript of opaR. For EMSA (c) and DNase I footprinting (d), the promoter-proximal DNA fragment of opaR was incubated with increasing amounts of purified His-AphA protein. The experiments were done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044210#pone-0044210-g006" target="_blank">Fig 6</a>. The transcription start site of opaR was underlined in the DNA sequence. Lanes G, A, T, and C represented Sanger sequencing reactions. The footprint regions were indicated with vertical bars. The negative or positive numbers indicated nucleotide positions upstream or downstream of opaR, respectively.</p