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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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Pos19 influences GSH and ROS levels.(A) Measurement of total intracellular glutathione (GSH). GSH was measured using Ellmann’s reagent (DTNB) in cell samples from three R. sphaeroides strains: an empty vector control (pRK16S), a strain with a constitutive, plasmid-borne over-expression of Pos19 under the control of the 16S rRNA promoter (pRK16S::Pos19), and the Pos19 mutant (ΔPos19). The relative GSH amount is depicted in percent; the GSH amount of the empty vector control (pRK16S) was set to 100 percent. All results represent the mean of nine independent biological experiments with technical duplicates. Error bars reflect the standard error of mean. The level of significance compared to the control (pRK16S) is indicated († 0.05 < p < 0.1 and * p < 0.05). (B) Inhibition zone assay using 10 mM methylene blue (MB) in the light, 500 mM tBOOH, 1 M and 2 M hydrogen peroxide (H2O2), and 500 mM diamide. The diameter of the zones of inhibition was measured for the three R. sphaeroides strains described above for the GSH assay. The data represent the mean of three independent experiments with technical duplicates. The error bars represent the standard error of mean. (C) Effect of Pos19 on ROS levels. Determination of intracellular levels of ROS in the control strain (pRK16S) compared to Pos19 over-expression (pRK16S::Pos19) and mutant (ΔPos19). ROS generated by the cells were analyzed after reaction with 10 μM 2,7-dihydrodichlorofluorescein diacetate. The fluorescence intensity was normalized to the optical densities of the samples. The resulting values are presented in arbitrary units (AU). The data represent the mean of three independent experiments. The error bars indicate the standard error of mean. The level of significance compared to the control (pRK16S) is indicated (* p < 0.05).
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pone.0163425.g004: Pos19 influences GSH and ROS levels.(A) Measurement of total intracellular glutathione (GSH). GSH was measured using Ellmann’s reagent (DTNB) in cell samples from three R. sphaeroides strains: an empty vector control (pRK16S), a strain with a constitutive, plasmid-borne over-expression of Pos19 under the control of the 16S rRNA promoter (pRK16S::Pos19), and the Pos19 mutant (ΔPos19). The relative GSH amount is depicted in percent; the GSH amount of the empty vector control (pRK16S) was set to 100 percent. All results represent the mean of nine independent biological experiments with technical duplicates. Error bars reflect the standard error of mean. The level of significance compared to the control (pRK16S) is indicated († 0.05 < p < 0.1 and * p < 0.05). (B) Inhibition zone assay using 10 mM methylene blue (MB) in the light, 500 mM tBOOH, 1 M and 2 M hydrogen peroxide (H2O2), and 500 mM diamide. The diameter of the zones of inhibition was measured for the three R. sphaeroides strains described above for the GSH assay. The data represent the mean of three independent experiments with technical duplicates. The error bars represent the standard error of mean. (C) Effect of Pos19 on ROS levels. Determination of intracellular levels of ROS in the control strain (pRK16S) compared to Pos19 over-expression (pRK16S::Pos19) and mutant (ΔPos19). ROS generated by the cells were analyzed after reaction with 10 μM 2,7-dihydrodichlorofluorescein diacetate. The fluorescence intensity was normalized to the optical densities of the samples. The resulting values are presented in arbitrary units (AU). The data represent the mean of three independent experiments. The error bars indicate the standard error of mean. The level of significance compared to the control (pRK16S) is indicated (* p < 0.05).

Mentions: Since sulfur metabolism genes were affected upon Pos19 over-expression, we took a closer look at the intracellular levels of the sulfur-containing antioxidant GSH. A Pos19 deletion mutant (ΔPos19), a strain constitutively over-expressing Pos19 (pRK16S::Pos19), and a strain carrying the respective empty vector (pRK16S) were compared. GSH assays revealed a significantly reduced amount of GSH in the Pos19 over-expression (76%) and an increased amount of GSH (119%) in the Pos19 deletion mutant (Fig 4A). Regarding these changes in the antioxidant GSH, we expected an effect on oxidative stress resistance in the corresponding strains. Surprisingly, we found only very minor differences in resistance that were not consistent with GSH measurements (Fig 4B). However, ROS levels were significantly increased in the Pos19 mutant while not changed in the over-expression strain (Fig 4C).


Characteristics of Pos19 – A Small Coding RNA in the Oxidative Stress Response of Rhodobacter sphaeroides
Pos19 influences GSH and ROS levels.(A) Measurement of total intracellular glutathione (GSH). GSH was measured using Ellmann’s reagent (DTNB) in cell samples from three R. sphaeroides strains: an empty vector control (pRK16S), a strain with a constitutive, plasmid-borne over-expression of Pos19 under the control of the 16S rRNA promoter (pRK16S::Pos19), and the Pos19 mutant (ΔPos19). The relative GSH amount is depicted in percent; the GSH amount of the empty vector control (pRK16S) was set to 100 percent. All results represent the mean of nine independent biological experiments with technical duplicates. Error bars reflect the standard error of mean. The level of significance compared to the control (pRK16S) is indicated († 0.05 < p < 0.1 and * p < 0.05). (B) Inhibition zone assay using 10 mM methylene blue (MB) in the light, 500 mM tBOOH, 1 M and 2 M hydrogen peroxide (H2O2), and 500 mM diamide. The diameter of the zones of inhibition was measured for the three R. sphaeroides strains described above for the GSH assay. The data represent the mean of three independent experiments with technical duplicates. The error bars represent the standard error of mean. (C) Effect of Pos19 on ROS levels. Determination of intracellular levels of ROS in the control strain (pRK16S) compared to Pos19 over-expression (pRK16S::Pos19) and mutant (ΔPos19). ROS generated by the cells were analyzed after reaction with 10 μM 2,7-dihydrodichlorofluorescein diacetate. The fluorescence intensity was normalized to the optical densities of the samples. The resulting values are presented in arbitrary units (AU). The data represent the mean of three independent experiments. The error bars indicate the standard error of mean. The level of significance compared to the control (pRK16S) is indicated (* p < 0.05).
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pone.0163425.g004: Pos19 influences GSH and ROS levels.(A) Measurement of total intracellular glutathione (GSH). GSH was measured using Ellmann’s reagent (DTNB) in cell samples from three R. sphaeroides strains: an empty vector control (pRK16S), a strain with a constitutive, plasmid-borne over-expression of Pos19 under the control of the 16S rRNA promoter (pRK16S::Pos19), and the Pos19 mutant (ΔPos19). The relative GSH amount is depicted in percent; the GSH amount of the empty vector control (pRK16S) was set to 100 percent. All results represent the mean of nine independent biological experiments with technical duplicates. Error bars reflect the standard error of mean. The level of significance compared to the control (pRK16S) is indicated († 0.05 < p < 0.1 and * p < 0.05). (B) Inhibition zone assay using 10 mM methylene blue (MB) in the light, 500 mM tBOOH, 1 M and 2 M hydrogen peroxide (H2O2), and 500 mM diamide. The diameter of the zones of inhibition was measured for the three R. sphaeroides strains described above for the GSH assay. The data represent the mean of three independent experiments with technical duplicates. The error bars represent the standard error of mean. (C) Effect of Pos19 on ROS levels. Determination of intracellular levels of ROS in the control strain (pRK16S) compared to Pos19 over-expression (pRK16S::Pos19) and mutant (ΔPos19). ROS generated by the cells were analyzed after reaction with 10 μM 2,7-dihydrodichlorofluorescein diacetate. The fluorescence intensity was normalized to the optical densities of the samples. The resulting values are presented in arbitrary units (AU). The data represent the mean of three independent experiments. The error bars indicate the standard error of mean. The level of significance compared to the control (pRK16S) is indicated (* p < 0.05).
Mentions: Since sulfur metabolism genes were affected upon Pos19 over-expression, we took a closer look at the intracellular levels of the sulfur-containing antioxidant GSH. A Pos19 deletion mutant (ΔPos19), a strain constitutively over-expressing Pos19 (pRK16S::Pos19), and a strain carrying the respective empty vector (pRK16S) were compared. GSH assays revealed a significantly reduced amount of GSH in the Pos19 over-expression (76%) and an increased amount of GSH (119%) in the Pos19 deletion mutant (Fig 4A). Regarding these changes in the antioxidant GSH, we expected an effect on oxidative stress resistance in the corresponding strains. Surprisingly, we found only very minor differences in resistance that were not consistent with GSH measurements (Fig 4B). However, ROS levels were significantly increased in the Pos19 mutant while not changed in the over-expression strain (Fig 4C).

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