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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.

No MeSH data available.


Related in: MedlinePlus

Pos19 is a coding sRNA.(A) Detailed illustration of the pos19 gene. Relevant features are shown in bold. The -35 and -10 motifs of the RpoE-dependent promoter are marked. The 219 nt long sRNA (shaded in grey) contains two potential ORFs with lengths of 150 (Start1) and 135 nt (Start2). The Rho-independent terminator (italic letters) was predicted by TransTermHP [27] and correlates with the 3’ end mapped by 3’ RACE. The Shine-Dalgarno (SD) sequence, the two start codons as well as one internal codon (Codon16) were mutated according to the chart below the sequence. (B) In vivo translation of the Pos19-sORF was monitored by translational lacZ fusions. A 300 bp fragment containing the RpoE-dependent promoter and the first 178 bp of the pos19 gene (including codons 1–48) was fused to the promoter-less lacZ gene on reporter plasmid pPHU236. The wild-type (wt) sORF was subsequently mutated as shown in (A). The fusion plasmids and control plasmid pPHU236 were transferred to R. sphaeroides ΔPos19 or strain TF18, which lacks the rpoE-chrR locus. The corresponding strains were subjected to 1O2 stress for 60 min with samples collected at the indicated time points. ß-galactosidase activities were measured from biological triplicates with technical duplicates. Error bars represent the standard error of the mean. (C) In vivo translation of the Pos19-sORF was monitored by translational eCFP fusion. The Pos19 peptide, C-terminally fused to eCFP (Pos19up+ORF), was detected with a polyclonal anti-GFP antibody on a Western blot. Cultures were stressed as described for Fig 1 and samples were withdrawn at the indicated time points. The amount of total protein extracts is shown in brackets. Protein from a strain carrying the pBE_eCFP::eCFP with 16S rRNA promoter (16S) was used as control. The size difference between control and Pos19 fusion of around 4 kDa corresponds to molecular weight of the predicted Pos19 peptide.
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pone.0163425.g002: Pos19 is a coding sRNA.(A) Detailed illustration of the pos19 gene. Relevant features are shown in bold. The -35 and -10 motifs of the RpoE-dependent promoter are marked. The 219 nt long sRNA (shaded in grey) contains two potential ORFs with lengths of 150 (Start1) and 135 nt (Start2). The Rho-independent terminator (italic letters) was predicted by TransTermHP [27] and correlates with the 3’ end mapped by 3’ RACE. The Shine-Dalgarno (SD) sequence, the two start codons as well as one internal codon (Codon16) were mutated according to the chart below the sequence. (B) In vivo translation of the Pos19-sORF was monitored by translational lacZ fusions. A 300 bp fragment containing the RpoE-dependent promoter and the first 178 bp of the pos19 gene (including codons 1–48) was fused to the promoter-less lacZ gene on reporter plasmid pPHU236. The wild-type (wt) sORF was subsequently mutated as shown in (A). The fusion plasmids and control plasmid pPHU236 were transferred to R. sphaeroides ΔPos19 or strain TF18, which lacks the rpoE-chrR locus. The corresponding strains were subjected to 1O2 stress for 60 min with samples collected at the indicated time points. ß-galactosidase activities were measured from biological triplicates with technical duplicates. Error bars represent the standard error of the mean. (C) In vivo translation of the Pos19-sORF was monitored by translational eCFP fusion. The Pos19 peptide, C-terminally fused to eCFP (Pos19up+ORF), was detected with a polyclonal anti-GFP antibody on a Western blot. Cultures were stressed as described for Fig 1 and samples were withdrawn at the indicated time points. The amount of total protein extracts is shown in brackets. Protein from a strain carrying the pBE_eCFP::eCFP with 16S rRNA promoter (16S) was used as control. The size difference between control and Pos19 fusion of around 4 kDa corresponds to molecular weight of the predicted Pos19 peptide.

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 a coding sRNA.(A) Detailed illustration of the pos19 gene. Relevant features are shown in bold. The -35 and -10 motifs of the RpoE-dependent promoter are marked. The 219 nt long sRNA (shaded in grey) contains two potential ORFs with lengths of 150 (Start1) and 135 nt (Start2). The Rho-independent terminator (italic letters) was predicted by TransTermHP [27] and correlates with the 3’ end mapped by 3’ RACE. The Shine-Dalgarno (SD) sequence, the two start codons as well as one internal codon (Codon16) were mutated according to the chart below the sequence. (B) In vivo translation of the Pos19-sORF was monitored by translational lacZ fusions. A 300 bp fragment containing the RpoE-dependent promoter and the first 178 bp of the pos19 gene (including codons 1–48) was fused to the promoter-less lacZ gene on reporter plasmid pPHU236. The wild-type (wt) sORF was subsequently mutated as shown in (A). The fusion plasmids and control plasmid pPHU236 were transferred to R. sphaeroides ΔPos19 or strain TF18, which lacks the rpoE-chrR locus. The corresponding strains were subjected to 1O2 stress for 60 min with samples collected at the indicated time points. ß-galactosidase activities were measured from biological triplicates with technical duplicates. Error bars represent the standard error of the mean. (C) In vivo translation of the Pos19-sORF was monitored by translational eCFP fusion. The Pos19 peptide, C-terminally fused to eCFP (Pos19up+ORF), was detected with a polyclonal anti-GFP antibody on a Western blot. Cultures were stressed as described for Fig 1 and samples were withdrawn at the indicated time points. The amount of total protein extracts is shown in brackets. Protein from a strain carrying the pBE_eCFP::eCFP with 16S rRNA promoter (16S) was used as control. The size difference between control and Pos19 fusion of around 4 kDa corresponds to molecular weight of the predicted Pos19 peptide.
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pone.0163425.g002: Pos19 is a coding sRNA.(A) Detailed illustration of the pos19 gene. Relevant features are shown in bold. The -35 and -10 motifs of the RpoE-dependent promoter are marked. The 219 nt long sRNA (shaded in grey) contains two potential ORFs with lengths of 150 (Start1) and 135 nt (Start2). The Rho-independent terminator (italic letters) was predicted by TransTermHP [27] and correlates with the 3’ end mapped by 3’ RACE. The Shine-Dalgarno (SD) sequence, the two start codons as well as one internal codon (Codon16) were mutated according to the chart below the sequence. (B) In vivo translation of the Pos19-sORF was monitored by translational lacZ fusions. A 300 bp fragment containing the RpoE-dependent promoter and the first 178 bp of the pos19 gene (including codons 1–48) was fused to the promoter-less lacZ gene on reporter plasmid pPHU236. The wild-type (wt) sORF was subsequently mutated as shown in (A). The fusion plasmids and control plasmid pPHU236 were transferred to R. sphaeroides ΔPos19 or strain TF18, which lacks the rpoE-chrR locus. The corresponding strains were subjected to 1O2 stress for 60 min with samples collected at the indicated time points. ß-galactosidase activities were measured from biological triplicates with technical duplicates. Error bars represent the standard error of the mean. (C) In vivo translation of the Pos19-sORF was monitored by translational eCFP fusion. The Pos19 peptide, C-terminally fused to eCFP (Pos19up+ORF), was detected with a polyclonal anti-GFP antibody on a Western blot. Cultures were stressed as described for Fig 1 and samples were withdrawn at the indicated time points. The amount of total protein extracts is shown in brackets. Protein from a strain carrying the pBE_eCFP::eCFP with 16S rRNA promoter (16S) was used as control. The size difference between control and Pos19 fusion of around 4 kDa corresponds to molecular weight of the predicted Pos19 peptide.
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