Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and -1

<p><b>Copyright information:</b></p><p>Taken from "Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and "</p><p>http://www.biomedcentral.com/1471-2180/7/69</p><p>BMC Microbiology 2007;7():69-69.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1950310.</p><p></p>, and phospholipid detection, are described in Methods. (A) Wild-type cells labeled at 30°C and 42°C. (B) -cells (strain MY112) labeled at 30°C and 42°C. (C) cells (strain MY103) labeled at 30°C and 42°C. (D) Pspac-cells (strain MY111) labeled in the presence and absence of IPTG. Abbreviations: 1-acyl-G3P, 1-acylglycerol-phosphate; PA, phosphatidic acid; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; CA, cardiolipin

Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and -0

<p><b>Copyright information:</b></p><p>Taken from "Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and "</p><p>http://www.biomedcentral.com/1471-2180/7/69</p><p>BMC Microbiology 2007;7():69-69.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1950310.</p><p></p>re grown in LB, LB with 0.1% (v/v) glycerol, and LB with 0.1% (w/v) glycerol-3-phosphate, at 30°C and 45°C

Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and -2

<p><b>Copyright information:</b></p><p>Taken from "Involvement of the YneS/YgiH and PlsX proteins in phospholipid biosynthesis in both and "</p><p>http://www.biomedcentral.com/1471-2180/7/69</p><p>BMC Microbiology 2007;7():69-69.</p><p>Published online 24 Jul 2007</p><p>PMCID:PMC1950310.</p><p></p>llin, with or without IAA (100 μg/ml), at 37°C. (A) a; MEC199 (Pw-), b; MEC201 (Pw-Δ), c; MEC212 (Pw-ΔpSTV29 pMW118), d; MEC214 (Pw-ΔpSTV29 pMW118), e; MEC218 (Pw-ΔpSTV29pMW118), and f; MEC220 (Pw-ΔpSTV29pMW118). (B) a; MEC308 (Pw-), b; MEC309 (Pw-), c; MEC306 (Pw-), and d; MEC307 (Pw-). (C) strains harboring pUC18S or pUC18S were grown in LB medium containing 50 μg/ml spectinomycin, with or without IAA (50 μg/ml), at 30°C. a; MEC323 (Pw-ΔΔpUC18S), b; MEC324 (Pw-ΔΔpUC18S ), c; MEC325 (Pw-pUC18S), d; MEC326 (Pw-pUC18S ), e; MEC327 (Pw-pUC18S), e; MEC328 (Pw-pUC18S )

Characterization of the YdeO Regulon in <i>Escherichia coli</i>

<div><p>Enterobacteria are able to survive under stressful conditions within animals, such as acidic conditions in the stomach, bile salts during transfer to the intestine and anaerobic conditions within the intestine. The glutamate-dependent (GAD) system plays a major role in acid resistance in <i>Escherichia coli</i>, and expression of the GAD system is controlled by the regulatory cascade consisting of EvgAS > YdeO > GadE. To understand the YdeO regulon <i>in vivo</i>, we used ChIP-chip to interrogate the <i>E. coli</i> genome for candidate YdeO binding sites. All of the seven operons identified by ChIP-chip as being potentially regulated by YdeO were confirmed as being under the direct control of YdeO using RT-qPCR, EMSA, DNaseI-footprinting and reporter assays. Within this YdeO regulon, we identified four stress-response transcription factors, DctR, NhaR, GadE, and GadW and enzymes for anaerobic respiration. Both GadE and GadW are involved in regulation of the GAD system and NhaR is an activator for the sodium/proton antiporter gene. In conjunction with co-transcribed Slp, DctR is involved in protection against metabolic endoproducts under acidic conditions. Taken all together, we suggest that YdeO is a key regulator of <i>E. coli</i> survival in both acidic and anaerobic conditions.</p></div

Genome-wide regulation of the <i>Escherichia coli</i> YdeO protein.

<p>Location of YdeO binding sites. The panel shows detailed YdeO binding data from ChIP-chip experiments at the <i>nhaR</i> (A), <i>hyaA</i> (B), <i>appC</i> (C), <i>yiiS</i> (D), <i>gadW</i> (E), <i>gadE</i> (F), and <i>slp</i> (G) genomic loci. The box indicates the YdeO-binding site.</p

Genes up-regulated by YdeO expression.

a<p>Transcriptional unit is represented according to the Regulon DB (<a href="http://regulondb.ccg.unam.mx/" target="_blank">http://regulondb.ccg.unam.mx/</a>).</p>b<p>The processed intensity was calculated by Agilent Future Extraction. More than 0.5 of log ratio in WT.</p>c<p>Experiment was independently performed twice (each ratio is shown as 1st and 2nd).</p><p>Genes up-regulated by YdeO expression.</p

The binding of YdeO on target promoters.

<p>[A] The binding of YdeO to the target DNA, <i>nhaR</i> (a), <i>hyaA</i> (b), <i>appC</i> (c), <i>yiiS</i> (d), <i>gadW</i> (e), <i>gadE</i> (f), and <i>slp</i> (g). Probes were amplified by PCR using constructed reporter plasmids as templates and a pair of primers as the following; pLUXnhaR and a pair of NHAR-lux-F and Lux-R-FITC for <i>nhaR</i> probe; pHYAA-L and a pair of HYAA-LF and lacZ-30R-FITC for <i>hyaA</i> probe; pAPPC-L and a pair of APPC-LF and lacZ-30R-FITC for <i>appC</i> probe; pYY0503 and a pair of YIIS-LF and lacZ-30R-FITC for <i>yiiS</i> probe; pLUXgadWp and a pair of GADW-F-2 and Lux-R-FITC for <i>gadW</i> probe; pLUXgadEp and a pair of GADE-SCL-F-2 and Lux-R-FITC for <i>gadE</i> probe; and pLUXslpp and a pair of SLP-F-2 and Lux-R-FITC for <i>slp</i> probe; (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111962#pone.0111962.s001" target="_blank">Table S1</a>). Each FITC-labeled probe (1 pmol) was incubated with YdeO protein (1, 5, 10, 25, or 35 pmol) and then DNA-YdeO complex was analyzed by native PAGE. Solid and dot lines indicate the migration of free DNA probe and DNA-YdeO complex, respectively. [B] The YdeO-binding site on <i>gadW</i> promoter. FITC-labeled probe (1 pmol) was incubated with 0 (lane 1), 0.5 (lane 2), 1.0 (lane 3), 5.0 (lane 4), or 15 (lane 5) pmol YdeO protein and then digested by DNase I. Sanger ladders are synthesized using Lux-R-FITC primer and pLUXgadWp plasmid as a template. A bar indicates the major region protected from DNase I digestion. The numbers represent the position from the transcription start site of <i>gadW</i>p1 promoter. [C] The sequence of YdeO-binding on <i>gadW</i> promoter. Black and gray bars indicate the major and minor YdeO-binding regions, respectively, as shown in [B]. The initiation codon of <i>gadW</i> coding is represented as a bold triplet. The numbers represent the position from the transcription start site of <i>gadW</i>p1 promoter.</p

YdeO > GadE cascade regulation in <i>E. coli</i>.

<p>This shows the clustering pattern of expression of genes induced by the <i>ydeO</i>-expression in the parent (KP7600) and <i>gadE</i>-deficient mutant (JD25278) by Cluster 3.0 (<a href="http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm" target="_blank">http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm</a>).</p

Induction of NhaR, GadE, and GadW by YdeO in <i>E. coli</i>.

<p>YY5002 (<i>gadE</i>-<i>3xflag</i>) (a), YY5003 (<i>gadW</i>-<i>3xflag</i>) (b), and BW25113, (c) harboring pTrc99A (−) or pYdeO (+), were grown in LB medium until logarithmic phase. After centrifugation the lysate solution was prepared in lysis buffer containing 8 M urea by sonication. The lysates were subjected to western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111962#s2" target="_blank">Materials and Methods</a>. Anti-FLAG (SIGMA) and anti-NhaR (Lab preparation) were used for detection of GadE-3xFLAG/GadW-3xFLAG, and NhaR, respectively [A]. The amounts of GadE-3xFLAG, GadW-3xFLAg, and NhaR were represented as the ratio of level of RNA polymerase-α subunit, detected by anti-α (Neoclone) [B].</p

Reporter assays for transcriptional regulation by YdeO.

<p>[A] YdeO-expression induces the expression of target promoters. YY0201/pLUXnhaR (<i>nhaR-lux</i>), HYAA-JL (<i>hyaA-lacZ</i>), YY1101 (<i>yiiS-lacZ</i>), YY0201/pLUXgadWp (gadW-lux), YY0201/pLUXgadE (<i>gadE-lux</i>), and YY0201/pLUXslpp (<i>slp-lux</i>), and were transformed with pTrc99A (vector, white bar) and pYdeO (<i>ydeO</i>, black bar). Transformants grew until logarithmic phase and β-galactosidase and luciferase activities of cultures were measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111962#s2" target="_blank">Materials and methods</a>. The data show the average of independent eight experiments with standard deviation as the ratio of a vector-transformant. [B] APPC-JL (<i>appC-lacZ</i>) was transformed with pTrc99A (vector) or pYdeO (<i>ydeO</i>). Transformants grew until logarithmic phase and β-galactosidase was measured as decribed in [A]. The data show the average of independent eight experiments with standard deviation as the Miller unit. [C] The <i>ydeO</i> expression induced under anaerobic conditions. The activity of <i>ydeO</i> promoter was measured in YY0101 growing in LB medium with pH 7.2 and 5.5 under aerobic (+) and anaerobic (−) conditions at logarithmic phase.</p