Regulation of a polyamine transporter by the conserved 3' UTR-derived sRNA SorX confers resistance to singlet oxygen and organic hydroperoxides in Rhodobacter sphaeroides

Abstract

Singlet oxygen is generated by bacteriochlorophylls when light and oxygen are simultaneously present in Rhodobacter sphaeroides. Singlet oxygen triggers a specific response that is partly regulated by the alternative sigma factor RpoHI/HII. The sRNA RSs2461 has previously been identified as an RpoHI/HII-dependent sRNA and is derived from the 3' UTR of the mRNA for an OmpR-type transcriptional regulator. Similar to the RpoHI/HII-dependent CcsR and SorY sRNAs, RSs2461 affects the resistance of R. sphaeroides against singlet oxygen and was therefore renamed here SorX. Furthermore, SorX has a strong impact on resistance against organic hydroperoxides that usually occur as secondary damages downstream of singlet oxygen. The 75-nt SorX 3' fragment, which is generated by RNase E cleavage and highly conserved among related species, represents the functional entity. A target search identified potA mRNA, which encodes a subunit of a polyamine transporter, as a direct SorX target and stress resistance via SorX could be linked to potA. The PotABCD transporter is an uptake system for spermidine in E. coli. While spermidine is generally described as beneficial during oxidative stress, we observed significantly increased sensitivity of R. sphaeroides to organic hydroperoxides in the presence of spermidine. We therefore propose that the diminished import of spermidine, due to down-regulation of potA by SorX, counteracts oxidative stress. Together with results from other studies this underlines the importance of regulated transport to bacterial stress defense.

Keywords: 3′ UTR-derived; Hfq; Rhodobacter sphaeroides; organic hydroperoxide; polyamine transporter; potA; singlet oxygen; small RNA.

Figure 1.

The SorX 3′ fragment is generated by RNase E cleavage and conserved among Rhodobacteraceae. (A) The genetic context of the sorX gene in R. sphaeroides 2.4.1. SorX is transcribed together with the upstream gene RSP_0847 from an RpoHI/HII promoter and the 116-nt long pre-SorX is further processed into a more abundant 75-nt SorX fragment. The lollipop indicates a Rho-independent terminator. (B) Secondary structure prediction of SorX using RNAfold from the ViennaRNA web server. (C) 5′ RACE analysis of SorX. Total RNA, treated with TAP (+) or untreated (−), was subjected to 5′-adapter ligation and RT-PCR, using one gene-specific and one adapter-specific primer. As a negative control, the RT step was omitted (-RT). The pre-SorX and SorX transcripts produce RT-PCR products of 115 (asterisk) and 74 bp, respectively. RT-PCR products were analyzed on 10% polyacrylamide gels. (D) Northern blot analysis of SorX in R. sphaeroides wild type 2.4.1 and strain 2.4.1rne^E.c.(ts) that expresses a temperature-sensitive RNase E variant (rne-3071^ts mutation) from E. coli. The strains were analyzed at different temperatures (32°C to 42°C). The upper band is an unspecific signal (see Fig. S2). 5.8S rRNA signals are displayed as a loading control. (E) Synteny analysis of the sorX locus within the Rhodobacteraceae family. Light gray arrows represent the gene for a 2-component transcriptional regulator that is present upstream of sorX. The dashed line indicates the RNase E-dependent processing site of SorX in R. sphaeroides 2.4.1. The gene for an Exodeoxyribonuclease III (dark gray arrows) can be found downstream of sorX in several cases.

Figure 2.

Various stress conditions induce SorX expression. R. sphaeroides wild type 2.4.1 was grown under aerobic conditions to apply singlet oxygen (¹O₂), superoxide (O₂^–), organic hydroperoxide (tBOOH), and hydrogen peroxide (H₂O₂) stress. Growth under microaerobic condition was conducted to induce heat stress (42°C) or to treat the cells with CdCl₂, SDS/EDTA, and diamide. Total RNA isolated at the indicated time points was analyzed on Northern blots. Results for the 75-nt SorX 3′ fragment are shown. Band intensities for SorX were normalized to the 5S rRNA. Fold changes and standard deviations were calculated from biological triplicates.

Figure 3.

Effects of SorX deletion or overexpression on resistance to singlet oxygen and organic hydroperoxides monitored by zone inhibition assays. Zones of inhibition for each strain were calculated relative to their respective control strain (100%). 1 M tBOOH or 50 mM methylene blue with light were used to generate organic hydroperoxide or singlet oxygen stress, respectively. (A) SorX deletion strain (2.4.1ΔSorXpRK4352), SorX overexpression strain (2.4.1pRKSorX¹⁴⁴) and complemented SorX deletion strain (2.4.1ΔSorXpRKSorX¹⁴⁴) were compared to wild type 2.4.1 carrying an empty vector control (2.4.1pRK4352). (B) The SorX deletion was complemented either with full-length SorX (2.4.1ΔSorXpBBRSorX¹⁴⁴) or the 76-nt 3′ part of SorX (2.4.1ΔSorXpBBRSorX⁷⁶). The corresponding strains were compared to wild type 2.4.1 (2.4.1pBBR4352) and the SorX deletion strain (2.4.1ΔSorXpBBR4352), each carrying an empty vector control. The error bars indicate the standard deviation from the mean of biological triplicates.

Figure 4.

SorX affects transcription of 2 other sRNAs. (A) Analysis of the SorX overexpression strain 2.4.1pRKSorX¹⁴⁴ by microarrays. The Volcano plot depicts log₂ ratios and p-values (negative log10) for the analyzed genes. Vertical lines indicate log₂ ratios of ≥ 0.6 and ≤ −0.6. The horizontal line represents a p-value threshold of 0.05. Genes discussed in this study are marked. (B) Expression of CcsR1 and SorY depends on SorX. Total RNA was isolated from each strain before and 7 min after adding 100 mM tBOOH and used for Northern blot analysis. 5.8S rRNA was hybridized as a loading control. (C) Effect of SorX on intracellular glutathione (GSH) level. The error bars indicate the standard deviation from the mean of 2 biological replicates with 3 technical replicates. Asterisks indicate a statistically significant change (p ≤ 0.05) compared to the wild type harboring empty plasmid pRK4352 as control. (D, E) Effect of SorX on the promoter activity of RSP_6037 (D) and SorY (E). The promoter regions were transcriptionally fused to ecfp to determine promoter activities. Fluorescence intensities were calculated relative to wild type 2.4.1 carrying the empty vector pRK4352 (set to 100%). The error bars indicate the standard deviation from the mean of biological triplicates with 3 technical replicates.

Figure 5.

SorX affects mRNA level and stability of potA. (A) Effect of SorX on potA mRNA levels as quantified by real time RT-PCR. Total RNA was isolated from wild type 2.4.1 and the SorX deletion strain (2.4.1ΔSorX) before and 7 min after adding 100 mM tBOOH. The relative transcript level (RTL) is given as log₂ fold change. (B) Effect of SorX on potA mRNA stability. The levels of mRNA at different time points after addition of rifampicin were quantified by real time RT-PCR (expression values were normalized to 16S rRNA) and the half-life was calculated. The error bars indicate the standard deviation from the mean of biological triplicates with 2 technical replicates.

Figure 6.

Direct interaction between SorX and potA mRNA. (A) Interaction of SorX-potA as predicted by IntaRNA. Position +1 of the mRNA represents the translational start site. The seed region in potA is from −21 to −5 with respect to the start codon. A triple mutation (M3) in SorX (CUC → GAG) and a compensatory mutation in potA (GAG → CUC) are marked by asterisks. (B) Relative ß-galactosidase activities for lacZ fusions with wild-type potA or mutated (M3) potA. Wild-type SorX or mutated SorX were overexpressed in the SorX deletion background (2.4.1ΔSorXpBBRSorX¹⁴⁴, 2.4.1ΔSorXpBBRSorX¹⁴⁴-M3). A SorX deletion strain harboring the empty vector pBBR4352 served as a control. ß-galactosidase activities were calculated relative to the corresponding control (set to 100%). (C, D) Relative ß-galactosidase activities for a lacZ fusion with potA. Full-length SorX (2.4.1ΔSorXpBBRSorX¹⁴⁴) and the 76-nt SorX 3′ fragment (2.4.1ΔSorXpBBRSorX⁷⁶) were overexpressed in the SorX deletion strain (C) or in an hfq deletion strain (D). ß-galactosidase activities in an empty vector control strain were set to 100%. The error bars indicate the standard deviation from the mean of biological triplicates with 2 technical replicates.

Figure 7.

Effect of SorX overexpression in the SorX deletion background (A) or the hfq deletion strain (B) on resistance to organic hydroperoxides as determined by zone inhibition assays. Strains harboring the empty vector pBBR4352 served as controls (set to 100%). 1 M tBOOH was applied for the assay. Asterisks indicate a statistically significant change in inhibition zones (p ≤ 0.05) compared to their respective control strains (C) Effect of potA deletion on resistance to singlet oxygen and organic hydroperoxides monitored by zone inhibition assays. 1 M tBOOH or 50 mM methylene blue with light were used. Asterisks indicate a statistically significant change in inhibition zones (p ≤ 0.05) compared to wild type 2.4.1 (D) Effect of SorX overexpression in the potA deletion strain on resistance to singlet oxygen and organic hydroperoxides. The error bars indicate the standard deviation from the mean of biological triplicates.

Figure 8.

(A) Effect of spermidine on cellular reactive oxygen species levels. Cultures were grown under aerobic condition and harvested at an OD₆₆₀ of 0.2 to quantify cellular reactive oxygen species (ROS) levels using the oxidation-sensitive fluorescent probe 2,7-dihydrodichlorofluorescein diacetate. The ROS level in wild type 2.4.1 without spermidine served as a control and was set to 100%. All other ROS levels were calculated relative to the control. (B) Effect of spermidine on resistance to organic hydroperoxides monitored by zone inhibition assays. The inhibition zone diameter from wild type 2.4.1 without spermidine served as a control and was set to 100%. (C) Effect of spermidine on intracellular GSH levels. The GSH level in wild type 2.4.1 without spermidine served as a control and was set to 100%. The error bars indicate the standard deviation from the mean of biological triplicates with technical replicates. Asterisks indicate a statistically significant change (p ≤ 0.05). Cultures were untreated (white bars) or treated with 10 mM spermidine (gray bars).