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

Abstract

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen ((1)O2) in the facultative photosynthetic bacterium Rhodobacter sphaeroides. We have identified SorY as an sRNA, which is induced under several stress conditions and confers increased resistance against (1)O2. SorY by direct interaction affects the takP 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 R. sphaeroides enzymes for glycolysis, pentose phosphate pathway, Entner-Doudoroff pathway and gluconeogenesis are induced in response to (1)O2 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.

Keywords: Hfq; Rhodobacter sphaeroides; carbon metabolism; photooxidative stress; sRNA; singlet oxygen.

Figure 1.

Induction of the small RNA SorY under various stress conditions shown by northern blot analysis of total RNA isolated from R. sphaeroides. SorY fold changes (FC) are normalized to the 5S rRNA. The quantification was done with Quantity One Software (Bio-Rad). (A) The following reagents (and their final concentrations) were added to aerobically grown cultures at OD₆₀₀ of 0.4 and samples were taken immediately before (time 0) or 10 min after addition: 0.2 µM methylene blue in the presence of 800 Wm⁻² white light, 300 µM tBOOH, 1 mM H₂O₂ and 250 µM paraquat were added to aerobically grown cultures at OD 0.4 and samples were taken before and 10 min after the addition. (B) Semiaerobic cultures were shifted to 42°C at time 0 or the following reagents were added: 500 µM diamide, 500 mM NaCl, 10 µM CdCl₂, 2.5 % ethanol, 1 µg/mL polymyxin-B or 0.005 % SDS along with 1 mM EDTA. (C) Altered SorY transcript levels in the overexpression strains and the knockout mutant (2.4.1ΔSorY) before and after ¹O₂ stress in comparison to the wild-type strain harboring the empty vector (2.4.1pBBR). SorY transcription in plasmid born overexpression strains is either constitutive due to the use of a 16S rRNA promoter (2.4.1pBBRSorY) or inducible due to the RpoHI/II dependent promoter of SorY (2.4.1pBBRSorYi).

Figure 2.

(A) Validation of microarray results by real-time RT-PCR and expression changes in a SorY deletion strain for putative SorY target genes. Relative expression of strain 2.4.1pBBRSorY is shown in relation to expression in the control strain 2.4.1pBBR, whereas the relative expression of the SorY deletion strain is shown in relation to the parental wild-type strain. Real-time RT-PCR data represents the mean of 3 independent experiments. Error bars indicate the standard deviation. (B) Effect of SorY overexpression on takP mRNA stability. The half-life of the takP mRNA in the control strain pBBR and strain pBBRSorY was calculated based on real-time RT-PCR with total RNA isolated from cultures at several time points after the addition of rifampicin. Values are normalized to the 16S rRNA. The data represents the mean of 3 independent experiments. (C) Binding of takP and SorY in vitro. Band shift experiment with 150 fmol P-labeled takP (15 nM) fragment of 92 nucleotides length incubated with increasing concentrations of unlabeled SorY at 32°C for 20 min. Unlabeled sRNA SorY was added in a equimolar concentration (15 nM) to the labeled takP fragment or in 10 fold (150 nM) or 100 fold (1.5 µM) molar excess. As control 150 fmol of P-labeled takP fragment were incubated with water, the sRNA PcrZ (1.5 µM) or with an mRNA fragment of RSP_2591 (1.5 µM) in 100 fold excess.

Figure 3.

(A) SorY-takP seed region as predicted by IntaRNA. Single nucleotide exchanges of SorY and takP are indicated by arrows. Position +1 of the mRNA refers to the translational start site. (B) Relative β-galactosidase activity of the lacZ-based in vivo reporter system. Activity in the control strains containing the takP::lacZ, takP-17::lacZ or takP-20::lacZ reporter fusions and plasmid pBBR is set to 100% (open bar). Activities for all other combinations are calculated in relation to their respective control. Activity in the strain containing the reporter fusion and plasmid pBBRSorY is indicated by a solid bar. Gray bars represent strains overexpressing SorY with a single mutation at 3 different locations and the compensatory mutations of takP in the reporter plasmid. (C) Relative β-galactosidase activity of the in vivo reporter takP::lacZ in wild type R. sphaeroides compared to ΔSorY and Δhfq knockout strains. Activity in the control strain containing the reporter fusion is set to 100 % (open bar). For each strain, 3 independent biological experiments with technical duplicates were performed. Error bars indicate standard deviations.

Figure 4.

Effect of SorY expression levels on resistance against ¹O₂ stress visualized by inhibition zones. Comparison of strains with different SorY levels, in wild type, ΔtakP- or Δhfq- background. Zones of inhibition for the different strains are shown in relation to the zones determined for their respective control strain set to 100% (open bar). Assays were performed on media with malate (A) or lactate (B) as main carbon source. The asterisk highlights the parental wild type to the ΔtakP mutant. Error bars indicate the standard deviation of biological triplicates.

Figure 5.

To combat ¹O₂ stress the alternative sigma factor RpoE and subsequently RpoHII are activated in Rhodobacter sphaeroides. RpoHII induces expression of the depicted enzymes for pentose phosphate pathway (PPP), Entner–Doudoroff pathway (ED) and gluconeogenesis to generate the antioxidant NADPH and indirectly represses enzymes for the TCA cycle, which generates the prooxidant NADH. To favor this pathway shunt RpoHII also induces the sRNA SorY. SorY regulates TakP synthesis negatively to limit intermediate availability for the TCA cycle by decreasing malate influx into the cell. This assigns a so far unknown function to a sRNA in oxidative stress response. Abbreviations: G6P – Glucose 6-phosphate, F6P – Fructose 6-phosphate, F1,6P – Fructose1,6-bisphosphate, G3P – Glyceraldehyde 3-phosphate, 6PGL – Phosphoglucono-δ-lactone, 6PG – 6-Phosphogluconate, KDPG – 2-Keto-3-deoxy-6-phosphogluconate, Ru5P – Ribulose 5-P, SUC-CoA – Succinyl CoA, SUC – Succinate, RC – reaction center, LH – light harvesting complex,^a RpoHII regulon described by Nuss et al. (2009) or^b Dufour et al. (2012),^c RpoHII independent down- or^d upregulation due to ¹O₂.