The sRNA SorY confers resistance during photooxidative stress by affecting a metabolite transporter in <i>Rhodobacter sphaeroides</i>

<div><p>Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll <i>a</i> mediated formation of singlet oxygen (¹O₂) in the facultative photosynthetic bacterium <i>Rhodobacter sphaeroides</i>. We have identified SorY as an sRNA, which is induced under several stress conditions and confers increased resistance against ¹O₂. SorY by direct interaction affects the <i>takP</i> mRNA, encoding a TRAP-T transporter. We present a model in which SorY reduces the metabolite flux into the tricarboxylic acid cycle (TCA cycle) by reducing malate import through TakP. It was previously shown that oxidative stress in bacteria leads to switch from glycolysis to the pentose phosphate pathway and to reduced activity of the TCA cycle. As a consequence the production of the prooxidant NADH is reduced and production of the protective NADPH is enhanced. In <i>R. sphaeroides</i> enzymes for glycolysis, pentose phosphate pathway, Entner–Doudoroff pathway and gluconeogenesis are induced in response to ¹O₂ by the alternative sigma factor RpoHII. The same is true for the sRNA SorY. By limiting malate import SorY thus contributes to the balance of the metabolic fluxes under photooxidative stress conditions. This assigns a so far unknown function to an sRNA in oxidative stress response.</p></div

Validation of microarray data by real-time RT-PCR.

<p>Relative gene expression (A) in 2.4.1Δ<i>irr</i> under normal iron conditions compared to the wild type under normal iron conditions and (B) in 2.4.1Δ<i>irr</i> under iron limitation compared to normal iron conditions (light gray bars) and in wild type under iron limitation compared to normal iron conditions (dark gray bars). Values were normalized to <i>rpoZ</i> and to the respective control treatment. The data represent the mean of at least three independent experiments and error bars indicate standard deviation. Numbers in parentheses show the fold change of the respective genes as determined by microarray analysis.</p

Relative expression levels of selected genes as determined by real time RT-PCR.

<p>Real time RT-PCR was performed for selected genes of the stress response (A) and genes of RNA processing and degradation (B). Cells were treated by blue light illumination or ¹O₂ exposure and RNA was isolated as described. To visualize gene induction or repression RNA was also isolated of untreated cells. White bars depict the <i>R. sphaeroides</i> wild type after 60 min blue light treatment compared to untreated cells. Grey bars show the results for the <i>cryB</i> deletion mutant under the same conditions. White, striped bars correspond to wild type treated by 20 min ¹O₂ compared to unstressed cells. Grey, striped bars show the results for the <i>cryB</i> deletion under photooxidative stress. Gene annotations are the same as listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033791#pone-0033791-t001" target="_blank">Table 1</a> or <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033791#pone-0033791-g002" target="_blank">Figure 2</a>.</p

Growth curves of the <i>R. sphaeroides</i> wild type (black) and the 2.4.1Δ<i>irr</i> mutant (gray) under normal iron (continuous line) and under iron limitation (dashed line) conditions are shown.

<p>The optical density at 660 nm (OD₆₆₀) of microaerobically grown <i>R. sphaeroides</i> cultures was determined over time. The data represent the mean of at least three independent experiments and error bars indicate standard error of the mean.</p

Additional file 1: of The PhyR homolog RSP_1274 of Rhodobacter sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity

Strains and plasmids (Table S1), Oligodeoxynucleotides (Table S2) used in this study. (PDF 163 kb

Expression of functionally related genes of <i>R. sphaeroides</i> 2.4.1Δ<i>cryB</i> compared to wild type under various conditions.

<p>Significant changes of the microarray data are in bold. Numbers in brackets failed to reach the set A-value criteria.</p

Relative expression of <i>katE</i> (RSP_2779) in <i>R. sphaeroides</i> wild type and 2.4.1Δ<i>irr</i> mutant.

<p>(A) Real-time RT-PCR was used to investigate the relative expression of <i>katE</i> in 2.4.1Δ<i>irr</i> mutant 0 min (light gray bar) and 20 min (dark gray bar) after exposure to 1 mM H₂O₂ compared to the wild type. (B) Relative <i>katE</i> expression 20 min of 1 mM H₂O₂ in the <i>R. sphaeroides</i> wild type (white bar) and the <i>irr</i> deletion mutant (black bar). Values were normalized to <i>rpoZ</i> and to the control at time point 0. The data represent the mean of three independent experiments and error bars indicate standard deviation. Levels of significance are indicated as follows: *<i>P</i>≤0.01; **<i>P</i>≤0.05.</p

Effects of CryB on PrrA-regulated genes.

<p>Real time RT-PCR experiments of selected genes with predicted PrrA binding sites show that CryB has no general effect on genes regulated by the PrrB/PrrA system under the tested conditions. White bars correspond to the ratio of expression of the CryB mutant compared to wild type after blue light illumination. Grey bars depict the expression ratios after ¹O₂ treatment. Numbers correspond to <i>R. sphaeroides</i> gene annotations. RSP_2879, hypothetical protein, <i>cox</i> operon; RSP_2234, predicted DNA-binding protein; RSP_2395, BCCP, cytochrome c peroxidase; RSP_3496, zinc carboxypeptidase A metalloprotease; RSP_3706, hypothetical protein.</p

Determination of 5′ ends of <i>mbfA</i> (RSP_0850) (A) and <i>ccpA</i> (RSP_2395) (B) mRNA by 5′ rapid amplification of cDNA ends (RACE).

<p>Separation of 5′-RACE products <i>mbfA</i> and <i>ccpA</i> obtained from RNA extracts of the wild type strain under normal iron conditions. PCR products obtained after second PCR (nested) were separated on a 10% polyacrylamid gel and stained with ethidium bromide. Determined 5′ ends are indicated by an arrow. The putative translational start is indicated by an asterisk. The Irr-box (ICE, iron control element) is marked by a frame.</p

Stability determination of <i>puc</i> and <i>puf</i> mRNA.

<p><i>puc</i> and <i>puf</i> mRNA encode structural proteins of the photosynthetic apparatus. (A) After addition of rifampicin at indicated time points, RNA was isolated and Northern blots were hybridized with <i>puc</i>- or <i>puf</i>-specific probes and re-hybridized with 14S rRNA-specific probes, serving as internal loading control. (B) Graphical analysis was used to determine the mRNA turn-over by normalizing <i>puc</i> and <i>puf</i> band intensities to the loading control and plotting against the time. Squares correspond to percentage of 0.5 kb <i>pucBA</i> mRNA, triangles display percentage of 0.5 kb <i>pufBA</i> mRNA and circles show percentage of 2.7 kb <i>pufBALMX</i> mRNA. Depicted are examples of <i>Rhodobacter sphaeroides</i> wild type (solid line) and the <i>cryB</i> deletion mutant (dashed line). (C) No significant changes in the turn-over of the mRNAs can be detected for <i>R. sphaeroides</i> wild type (WT) and the <i>cryB</i> deletion mutant.</p