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Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides

View Article: PubMed Central - PubMed

ABSTRACT

The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides.

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Related in: MedlinePlus

Pos19 is induced by singlet oxygen and peroxides.(A) Graphical representation of the Pos19 locus in R. sphaeroides wild-type strain 2.4.1. The pos19 gene is located on chromosome 2 between cxp and fadB. The conserved RpoE-dependent promoter (PRpoE) and the Rho-independent terminator (lollipop structure) are indicated. (B) Northern blot for stress-dependent Pos19 induction. R. sphaeroides wild-type 2.4.1 cultures were treated with stress-generating chemicals and samples collected at time points 0 and 7 min. Singlet oxygen (1O2) was generated by the addition of 0.2 μM methylene blue in the presence of high light intensities (800 W m-2). Hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (tBOOH) were added in final concentrations of 1 mM and 300 μM, respectively. 250 μM of paraquat (PQ) were used for the generation of superoxide radicals (O2‒). 5S rRNA was probed as loading control. (C) Northern blot for RpoE-dependent Pos19 expression. The R. sphaeroides wild-type (wt), the Pos19 over-expression (pPos19), RpoHI and RpoHII mutant strains, as well as a strain lacking the rpoE-chrR locus (TF18), were treated with the indicated stress-generating chemicals as described above. Samples were collected at the indicated time points. 5S rRNA was probed as loading control.
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pone.0163425.g001: Pos19 is induced by singlet oxygen and peroxides.(A) Graphical representation of the Pos19 locus in R. sphaeroides wild-type strain 2.4.1. The pos19 gene is located on chromosome 2 between cxp and fadB. The conserved RpoE-dependent promoter (PRpoE) and the Rho-independent terminator (lollipop structure) are indicated. (B) Northern blot for stress-dependent Pos19 induction. R. sphaeroides wild-type 2.4.1 cultures were treated with stress-generating chemicals and samples collected at time points 0 and 7 min. Singlet oxygen (1O2) was generated by the addition of 0.2 μM methylene blue in the presence of high light intensities (800 W m-2). Hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (tBOOH) were added in final concentrations of 1 mM and 300 μM, respectively. 250 μM of paraquat (PQ) were used for the generation of superoxide radicals (O2‒). 5S rRNA was probed as loading control. (C) Northern blot for RpoE-dependent Pos19 expression. The R. sphaeroides wild-type (wt), the Pos19 over-expression (pPos19), RpoHI and RpoHII mutant strains, as well as a strain lacking the rpoE-chrR locus (TF18), were treated with the indicated stress-generating chemicals as described above. Samples were collected at the indicated time points. 5S rRNA was probed as loading control.

Mentions: Pos19 was first identified as RSs0019 in a global screen for sRNAs in R. sphaeroides by differential RNA-sequencing (dRNA-seq) [16]. The primary 5’ end of Pos19 was unambiguously determined by the dRNA-seq approach. In addition, we mapped the 3’ end by 3’ RACE, which validated a size of 219 nt. The detected 3’ end correlates with a predicted Rho-independent terminator (Figs 1A and 2A). Interestingly, the terminating structure misses the typical 3’ polyU stretch that is found in many bacterial sRNAs. It was already demonstrated that 1O2, but not superoxide radicals (O2‒), induce Pos19 and that the alternative sigma factor RpoE is responsible for this induction [16]. Pos19 is preceded by a perfectly conserved RpoE-dependent promoter (TGATCC(N15)GCGTA; Figs 1A and 2A), which can be targeted by RpoE when the inhibitory interaction with its cognate anti-sigma factor ChrR is released upon 1O2 stress. Additional stress factors lead to RpoE activation: peroxides like hydrogen peroxide (H2O2) and organic hydroperoxides act as signals for RpoE activation [26,7]. In line with these observations, Pos19 is clearly induced after treatment with H2O2 and the organic hydroperoxide tBOOH (tert-butyl hydroperoxide) in an RpoE-dependent manner but independently of RpoHI and RpoHII (Fig 1B and 1C). In strain TF18, lacking the rpoE-chrR locus, there is no induction of Pos19 under the tested stress conditions, while in the RpoHI and RpoHII mutant strains the expression of Pos19 is not altered upon 1O2 stress compared to the wild-type.


Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides
Pos19 is induced by singlet oxygen and peroxides.(A) Graphical representation of the Pos19 locus in R. sphaeroides wild-type strain 2.4.1. The pos19 gene is located on chromosome 2 between cxp and fadB. The conserved RpoE-dependent promoter (PRpoE) and the Rho-independent terminator (lollipop structure) are indicated. (B) Northern blot for stress-dependent Pos19 induction. R. sphaeroides wild-type 2.4.1 cultures were treated with stress-generating chemicals and samples collected at time points 0 and 7 min. Singlet oxygen (1O2) was generated by the addition of 0.2 μM methylene blue in the presence of high light intensities (800 W m-2). Hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (tBOOH) were added in final concentrations of 1 mM and 300 μM, respectively. 250 μM of paraquat (PQ) were used for the generation of superoxide radicals (O2‒). 5S rRNA was probed as loading control. (C) Northern blot for RpoE-dependent Pos19 expression. The R. sphaeroides wild-type (wt), the Pos19 over-expression (pPos19), RpoHI and RpoHII mutant strains, as well as a strain lacking the rpoE-chrR locus (TF18), were treated with the indicated stress-generating chemicals as described above. Samples were collected at the indicated time points. 5S rRNA was probed as loading control.
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pone.0163425.g001: Pos19 is induced by singlet oxygen and peroxides.(A) Graphical representation of the Pos19 locus in R. sphaeroides wild-type strain 2.4.1. The pos19 gene is located on chromosome 2 between cxp and fadB. The conserved RpoE-dependent promoter (PRpoE) and the Rho-independent terminator (lollipop structure) are indicated. (B) Northern blot for stress-dependent Pos19 induction. R. sphaeroides wild-type 2.4.1 cultures were treated with stress-generating chemicals and samples collected at time points 0 and 7 min. Singlet oxygen (1O2) was generated by the addition of 0.2 μM methylene blue in the presence of high light intensities (800 W m-2). Hydrogen peroxide (H2O2) and tert-butyl hydroperoxide (tBOOH) were added in final concentrations of 1 mM and 300 μM, respectively. 250 μM of paraquat (PQ) were used for the generation of superoxide radicals (O2‒). 5S rRNA was probed as loading control. (C) Northern blot for RpoE-dependent Pos19 expression. The R. sphaeroides wild-type (wt), the Pos19 over-expression (pPos19), RpoHI and RpoHII mutant strains, as well as a strain lacking the rpoE-chrR locus (TF18), were treated with the indicated stress-generating chemicals as described above. Samples were collected at the indicated time points. 5S rRNA was probed as loading control.
Mentions: Pos19 was first identified as RSs0019 in a global screen for sRNAs in R. sphaeroides by differential RNA-sequencing (dRNA-seq) [16]. The primary 5’ end of Pos19 was unambiguously determined by the dRNA-seq approach. In addition, we mapped the 3’ end by 3’ RACE, which validated a size of 219 nt. The detected 3’ end correlates with a predicted Rho-independent terminator (Figs 1A and 2A). Interestingly, the terminating structure misses the typical 3’ polyU stretch that is found in many bacterial sRNAs. It was already demonstrated that 1O2, but not superoxide radicals (O2‒), induce Pos19 and that the alternative sigma factor RpoE is responsible for this induction [16]. Pos19 is preceded by a perfectly conserved RpoE-dependent promoter (TGATCC(N15)GCGTA; Figs 1A and 2A), which can be targeted by RpoE when the inhibitory interaction with its cognate anti-sigma factor ChrR is released upon 1O2 stress. Additional stress factors lead to RpoE activation: peroxides like hydrogen peroxide (H2O2) and organic hydroperoxides act as signals for RpoE activation [26,7]. In line with these observations, Pos19 is clearly induced after treatment with H2O2 and the organic hydroperoxide tBOOH (tert-butyl hydroperoxide) in an RpoE-dependent manner but independently of RpoHI and RpoHII (Fig 1B and 1C). In strain TF18, lacking the rpoE-chrR locus, there is no induction of Pos19 under the tested stress conditions, while in the RpoHI and RpoHII mutant strains the expression of Pos19 is not altered upon 1O2 stress compared to the wild-type.

View Article: PubMed Central - PubMed

ABSTRACT

The phototrophic bacterium Rhodobacter sphaeroides induces several small RNAs (sRNAs) when singlet oxygen (1O2) levels are elevated, a situation also referred to as photo-oxidative stress. An RNA-seq study identified the RSs0019 sRNA, which is renamed Pos19 (photo-oxidative stress induced sRNA 19). Pos19 is part of the RpoE regulon and consequently induced upon 1O2 and peroxide stress. The 219 nt long Pos19 transcript contains a small open reading frame (sORF) of 150 nt, which is translated in vivo. Over-expression of Pos19 results in reduced mRNA levels for several genes, of which numerous are involved in sulfur metabolism. The negative effect on the potential targets is maintained even when translation of the sORF is abolished, arguing that regulation is entailed by the sRNA itself. Reporter studies further revealed that regulation of the most affected mRNA, namely RSP_0557, by Pos19 is Hfq-dependent. Direct binding of Pos19 to Hfq was shown by co-immunoprecipitation. Physiological experiments indicated Pos19 to be involved in the balance of glutathione biosynthesis. Moreover, a lack of Pos19 leads to elevated reactive oxygen species levels. Taken together our data identify the sRNA Pos19 as a coding sRNA with a distinct expression pattern and potential role under oxidative stress in the phototrophic bacterium R. sphaeroides.

No MeSH data available.


Related in: MedlinePlus