RNase III participates in control of quorum sensing, pigmentation and oxidative stress resistance in Rhodobacter sphaeroides.

RNase III is a dsRNA-specific endoribonuclease, highly conserved in bacteria and eukarya. In this study, we analysed the effects of inactivation of RNase III on the transcriptome and the phenotype of the facultative phototrophic α-proteobacterium Rhodobacter sphaeroides. RNA-seq revealed an unexpectedly high amount of genes with increased expression located directly downstream to the rRNA operons. Chromosomal insertion of additional transcription terminators restored wild type-like expression of the downstream genes, indicating that RNase III may modulate the rRNA transcription termination in R. sphaeroides. Furthermore, we identified RNase III as a major regulator of quorum-sensing autoinducer synthesis in R. sphaeroides. It negatively controls the expression of the autoinducer synthase CerI by reducing cerI mRNA stability. In addition, RNase III inactivation caused altered resistance against oxidative stress and impaired formation of photosynthetically active pigment-protein complexes. We also observed an increase in the CcsR small RNAs that were previously shown to promote resistance to oxidative stress. Taken together, our data present interesting insights into RNase III-mediated regulation and expand the knowledge on the function of this important enzyme in bacteria.

Ribonuclease E strongly impacts bacterial adaptation to different growth conditions.

Adaptation of bacteria to changes in their environment is often accomplished by changes of the transcriptome. While we learned a lot on the impact of transcriptional regulation in bacterial adaptation over the last decades, much less is known on the role of ribonucleases. This study demonstrates an important function of the endoribonuclease RNase E in the adaptation to different growth conditions. It was shown previously that RNase E activity does not influence the doubling time of the facultative phototroph Rhodobacter sphaeroides during chemotrophic growth, however, it has a strong impact on phototrophic growth. To better understand the impact of RNase E on phototrophic growth, we now quantified gene expression by RNA-seq and mapped 5' ends during chemotrophic growth under high oxygen or low oxygen levels and during phototrophic growth in the wild type and a mutant expressing a thermosensitive RNase E. Based on the RNase E-dependent expression pattern, the RNAs could be grouped into different classes. A strong effect of RNase E on levels of RNAs for photosynthesis genes was observed, in agreement with poor growth under photosynthetic conditions. RNase E cleavage sites and 5' ends enriched in the rnets mutant were differently distributed among the gene classes. Furthermore, RNase E affects the level of RNAs for important transcription factors thus indirectly affecting the expression of their regulons. As a consequence, RNase E has an important role in the adaptation of R. sphaeroides to different growth conditions. [Figure: see text].

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover.

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3' UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.

sRNA-mediated RNA processing regulates bacterial cell division.

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.

The Small RNA-Binding Protein CcaF1 Promotes Formation of Photosynthetic Complexes in Rhodobacter sphaeroides.

In natural habitats, bacteria frequently need to adapt to changing environmental conditions. Regulation of transcription plays an important role in this process. However, riboregulation also contributes substantially to adaptation. Riboregulation often acts at the level of mRNA stability, which is determined by sRNAs, RNases, and RNA-binding proteins. We previously identified the small RNA-binding protein CcaF1, which is involved in sRNA maturation and RNA turnover in Rhodobacter sphaeroides. Rhodobacter is a facultative phototroph that can perform aerobic and anaerobic respiration, fermentation, and anoxygenic photosynthesis. Oxygen concentration and light conditions decide the pathway for ATP production. Here, we show that CcaF1 promotes the formation of photosynthetic complexes by increasing levels of mRNAs for pigment synthesis and for some pigment-binding proteins. Levels of mRNAs for transcriptional regulators of photosynthesis genes are not affected by CcaF1. RIP-Seq analysis compares the binding of CcaF1 to RNAs during microaerobic and photosynthetic growth. The stability of the pufBA mRNA for proteins of the light-harvesting I complex is increased by CcaF1 during phototrophic growth but decreased during microaerobic growth. This research underlines the importance of RNA-binding proteins in adaptation to different environments and demonstrates that an RNA-binding protein can differentially affect its binding partners in dependence upon growth conditions.

A Small RNA, UdsC, Interacts with the RpoHII mRNA and Affects the Motility and Stress Resistance of Rhodobacter sphaeroides.

sRNAs have an important role in the regulation of bacterial gene expression. The sRNA, UdsC, of Rhodobacter sphaeroides is derived from the 3' UTR of the RSP_7527 mRNA, which encodes a hypothetical protein. Here, we showed the effect of UdsC on the resistance of Rhodobacter sphaeroides to hydrogen peroxide and on its motility. In vitro binding assays supported the direct interaction of UdsC with the 5' UTR of the rpoHII mRNA. RpoHII is an alternative sigma factor with an important role in stress responses in R. sphaeroides, including its response to hydrogen peroxide. We also demonstrated that RpoHII controls the expression of the torF gene, which encodes an important regulator of motility genes. This strongly suggested that the observed effect of UdsC on TorF expression is indirect and mediated by RpoHII.

Impact of PNPase on the transcriptome of Rhodobacter sphaeroides and its cooperation with RNase III and RNase E.

BACKGROUND: The polynucleotide phosphorylase (PNPase) is conserved among both Gram-positive and Gram-negative bacteria. As a core part of the Escherichia coli degradosome, PNPase is involved in maintaining proper RNA levels within the bacterial cell. It plays a major role in RNA homeostasis and decay by acting as a 3'-to-5' exoribonuclease. Furthermore, PNPase can catalyze the reverse reaction by elongating RNA molecules in 5'-to-3' end direction which has a destabilizing effect on the prolonged RNA molecule. RNA degradation is often initiated by an endonucleolytic cleavage, followed by exoribonucleolytic decay from the new 3' end. RESULTS: The PNPase mutant from the facultative phototrophic Rhodobacter sphaeroides exhibits several phenotypical characteristics, including diminished adaption to low temperature, reduced resistance to organic peroxide induced stress and altered growth behavior. The transcriptome composition differs in the pnp mutant strain, resulting in a decreased abundance of most tRNAs and rRNAs. In addition, PNPase has a major influence on the half-lives of several regulatory sRNAs and can have both a stabilizing or a destabilizing effect. Moreover, we globally identified and compared differential RNA 3' ends in RNA NGS sequencing data obtained from PNPase, RNase E and RNase III mutants for the first time in a Gram-negative organism. The genome wide RNA 3' end analysis revealed that 885 3' ends are degraded by PNPase. A fair percentage of these RNA 3' ends was also identified at the same genomic position in RNase E or RNase III mutant strains. CONCLUSION: The PNPase has a major influence on RNA processing and maturation and thus modulates the transcriptome of R. sphaeroides. This includes sRNAs, emphasizing the role of PNPase in cellular homeostasis and its importance in regulatory networks. The global 3' end analysis indicates a sequential RNA processing: 5.9% of all RNase E-dependent and 9.7% of all RNase III-dependent RNA 3' ends are subsequently degraded by PNPase. Moreover, we provide a modular pipeline which greatly facilitates the identification of RNA 5'/3' ends. It is publicly available on GitHub and is distributed under ICS license.

A major checkpoint for protein expression in Rhodobacter sphaeroides during heat stress response occurs at the level of translation.

Temperature above the physiological optimum is a stress condition frequently faced by bacteria in their natural environments. Here, we were interested in the correlation between levels of RNA and protein under heat stress. Changes in RNA and protein levels were documented in cultures of Rhodobacter sphaeroides using RNA sequencing, quantitative mass spectrometry, western blot analysis, in vivo [35 S] methionine-labelling and plasmid-borne reporter fusions. Changes in the transcriptome were extensive. Strikingly, the proteome remained unchanged except for very few proteins. Examples include a heat shock protein, a DUF1127 protein of unknown function and sigma factor proteins from leaderless transcripts. Insight from this study indicates that R. sphaeroides responds to heat stress by producing a broad range of transcripts while simultaneously preventing translation from nearly all of them, and that this selective production of protein depends on the untranslated region of the transcript. We conclude that measurements of transcript abundance are insufficient to understand gene regulation. Rather, translation can be an important checkpoint for protein expression under certain environmental conditions. Furthermore, during heat shock, regulation at the level of transcription might represent preparation for survival in an unpredictable environment while regulation at translation ensures production of only a few proteins.

Interplay between formation of photosynthetic complexes and expression of genes for iron-sulfur cluster assembly in Rhodobacter sphaeroides?

Formation of photosynthetic complexes leads to a higher demand for Fe-S clusters. We hypothesized that in the facultative phototrophic alpha-proteobacterium Rhodobacter sphaeroides expression of the isc-suf operon for Fe-S cluster formation may be increased under conditions that promote formation of photosynthetic complexes and that, vice versa, lack of the IscR regulator may also affect photosynthesis gene expression. To test this hypothesis, we monitored the activities of the isc-suf sense and anti-sense promoters under different growth conditions and in mutants which are impaired in formation of photosynthetic complexes. We also tested expression of photosynthesis genes in a mutant lacking the IscR regulator. Our results are not in agreement with a co-regulation of the Isc-Suf system and the photosynthetic apparatus at level of transcription. We provide evidence that, coordination of the systems occurs at post-transcriptional levels. Increased levels of isc-suf mRNAs under conditions promoting formation of photosynthetic complexes are due to higher RNA stability.

Antisense RNA asPcrL regulates expression of photosynthesis genes in Rhodobacter sphaeroides by promoting RNase III-dependent turn-over of puf mRNA.

Anoxygenic photosynthesis is an important pathway for Rhodobacter sphaeroides to produce ATP under oxygen-limiting conditions. The expression of its photosynthesis genes is tightly regulated at transcriptional and post-transcriptional levels in response to light and oxygen signals, to avoid photooxidative stress by the simultaneous presence of pigments, light and oxygen. The puf operon encodes pigment-binding proteins of the light-harvesting complex I (genes pufB and pufA), of the reaction centre (genes pufL and pufM), a scaffold protein (gene pufX) and includes the gene for sRNA PcrX. Segmental differences in the stability of the pufBALMX-pcrX mRNA contribute to the stoichiometry of LHI to RC complexes. With asPcrL we identified the third sRNA and the first antisense RNA that is involved in balancing photosynthesis gene expression in R. sphaeroides. asPcrL influences the stability of the pufBALMX-pcrX mRNA but not of the pufBA mRNA and consequently the stoichiometry of photosynthetic complexes. By base pairing to the pufL region asPcrL promotes RNase III-dependent degradation of the pufBALMX-prcX mRNA. Since asPcrL is activated by the same protein regulators as the puf operon including PcrX it is part of an incoherent feed-forward loop that fine-tunes photosynthesis gene expression.[Figure: see text].

Maturation of UTR-Derived sRNAs Is Modulated during Adaptation to Different Growth Conditions.

Small regulatory RNAs play a major role in bacterial gene regulation by binding their target mRNAs, which mostly influences the stability or translation of the target. Expression levels of sRNAs are often regulated by their own promoters, but recent reports have highlighted the presence and importance of sRNAs that are derived from mRNA 3' untranslated regions (UTRs). In this study, we investigated the maturation of 5' and 3' UTR-derived sRNAs on a global scale in the facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides. Including some already known UTR-derived sRNAs like UpsM or CcsR1-4, 14 sRNAs are predicted to be located in 5 UTRs and 16 in 3' UTRs. The involvement of different ribonucleases during maturation was predicted by a differential RNA 5'/3' end analysis based on RNA next generation sequencing (NGS) data from the respective deletion strains. The results were validated in vivo and underline the importance of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) during processing and maturation. The abundances of some UTR-derived sRNAs changed when cultures were exposed to external stress conditions, such as oxidative stress and also during different growth phases. Promoter fusions revealed that this effect cannot be solely attributed to an altered transcription rate. Moreover, the RNase E dependent cleavage of several UTR-derived sRNAs varied significantly during the early stationary phase and under iron depletion conditions. We conclude that an alteration of ribonucleolytic processing influences the levels of UTR-derived sRNAs, and may thus indirectly affect their mRNA targets.

A Complex Network of Sigma Factors and sRNA StsR Regulates Stress Responses in R. sphaeroides.

Adaptation of bacteria to a changing environment is often accompanied by remodeling of the transcriptome. In the facultative phototroph Rhodobacter sphaeroides the alternative sigma factors RpoE, RpoHI and RpoHII play an important role in a variety of stress responses, including heat, oxidative stress and nutrient limitation. Photooxidative stress caused by the simultaneous presence of chlorophylls, light and oxygen is a special challenge for phototrophic organisms. Like alternative sigma factors, several non-coding sRNAs have important roles in the defense against photooxidative stress. RNAseq-based transcriptome data pointed to an influence of the stationary phase-induced StsR sRNA on levels of mRNAs and sRNAs with a role in the photooxidative stress response. Furthermore, StsR also affects expression of photosynthesis genes and of genes for regulators of photosynthesis genes. In vivo and in vitro interaction studies revealed that StsR, that is under control of the RpoHI and RpoHII sigma factors, targets rpoE mRNA and affects its abundance by altering its stability. RpoE regulates expression of the rpoHII gene and, consequently, expression of stsR. These data provide new insights into a complex regulatory network of protein regulators and sRNAs involved in defense against photooxidative stress and the regulation of photosynthesis genes.

iCLIP analysis of RNA substrates of the archaeal exosome.

BACKGROUND: The archaeal exosome is an exoribonucleolytic multiprotein complex, which degrades single-stranded RNA in 3' to 5' direction phosphorolytically. In a reverse reaction, it can add A-rich tails to the 3'-end of RNA. The catalytic center of the exosome is in the aRrp41 subunit of its hexameric core. Its RNA-binding subunits aRrp4 and aDnaG confer poly(A) preference to the complex. The archaeal exosome was intensely characterized in vitro, but still little is known about its interaction with natural substrates in the cell, particularly because analysis of the transcriptome-wide interaction of an exoribonuclease with RNA is challenging. RESULTS: To determine binding sites of the exosome to RNA on a global scale, we performed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) analysis with antibodies directed against aRrp4 and aRrp41 of the chrenarchaeon Sulfolobus solfataricus. A relatively high proportion (17-19%) of the obtained cDNA reads could not be mapped to the genome. Instead, they corresponded to adenine-rich RNA tails, which are post-transcriptionally synthesized by the exosome, and to circular RNAs (circRNAs). We identified novel circRNAs corresponding to 5' parts of two homologous, transposase-related mRNAs. To detect preferred substrates of the exosome, the iCLIP reads were compared to the transcript abundance using RNA-Seq data. Among the strongly enriched exosome substrates were RNAs antisense to tRNAs, overlapping 3'-UTRs and RNAs containing poly(A) stretches. The majority of the read counts and crosslink sites mapped in mRNAs. Furthermore, unexpected crosslink sites clustering at 5'-ends of RNAs was detected. CONCLUSIONS: In this study, RNA targets of an exoribonuclease were analyzed by iCLIP. The data documents the role of the archaeal exosome as an exoribonuclease and RNA-tailing enzyme interacting with all RNA classes, and underlines its role in mRNA turnover, which is important for adaptation of prokaryotic cells to changing environmental conditions. The clustering of crosslink sites near 5'-ends of genes suggests simultaneous binding of both RNA ends by the S. solfataricus exosome. This may serve to prevent translation of mRNAs dedicated to degradation in 3'-5' direction.

Rapid Biophysical Characterization and NMR Spectroscopy Structural Analysis of Small Proteins from Bacteria and Archaea.

Proteins encoded by small open reading frames (sORFs) have a widespread occurrence in diverse microorganisms and can be of high functional importance. However, due to annotation biases and their technically challenging direct detection, these small proteins have been overlooked for a long time and were only recently rediscovered. The currently rapidly growing number of such proteins requires efficient methods to investigate their structure-function relationship. Herein, a method is presented for fast determination of the conformational properties of small proteins. Their small size makes them perfectly amenable for solution-state NMR spectroscopy. NMR spectroscopy can provide detailed information about their conformational states (folded, partially folded, and unstructured). In the context of the priority program on small proteins funded by the German research foundation (SPP2002), 27 small proteins from 9 different bacterial and archaeal organisms have been investigated. It is found that most of these small proteins are unstructured or partially folded. Bioinformatics tools predict that some of these unstructured proteins can potentially fold upon complex formation. A protocol for fast NMR spectroscopy structure elucidation is described for the small proteins that adopt a persistently folded structure by implementation of new NMR technologies, including automated resonance assignment and nonuniform sampling in combination with targeted acquisition.

Comparative analyses of the variation of the transcriptome and proteome of Rhodobacter sphaeroides throughout growth.

BACKGROUND: In natural environments, bacteria must frequently cope with extremely scarce nutrients. Most studies focus on bacterial growth in nutrient replete conditions, while less is known about the stationary phase. Here, we are interested in global gene expression throughout all growth phases, including the adjustment to deep stationary phase. RESULTS: We monitored both the transcriptome and the proteome in cultures of the alphaproteobacterium Rhodobacter sphaeroides, beginning with the transition to stationary phase and at different points of the stationary phase and finally during exit from stationary phase (outgrowth) following dilution with fresh medium. Correlation between the transcriptomic and proteomic changes was very low throughout the growth phases. Surprisingly, even in deep stationary phase, the abundance of many proteins continued to adjust, while the transcriptome analysis revealed fewer adjustments. This pattern was reversed during the first 90 min of outgrowth, although this depended upon the duration of the stationary phase. We provide a detailed analysis of proteomic changes based on the clustering of orthologous groups (COGs), and compare these with the transcriptome. CONCLUSIONS: The low correlation between transcriptome and proteome supports the view that post-transcriptional processes play a major role in the adaptation to growth conditions. Our data revealed that many proteins with functions in transcription, energy production and conversion and the metabolism and transport of amino acids, carbohydrates, lipids, and secondary metabolites continually increased in deep stationary phase. Based on these findings, we conclude that the bacterium responds to sudden changes in environmental conditions by a radical and rapid reprogramming of the transcriptome in the first 90 min, while the proteome changes were modest. In response to gradually deteriorating conditions, however, the transcriptome remains mostly at a steady state while the bacterium continues to adjust its proteome. Even long after the population has entered stationary phase, cells are still actively adjusting their proteomes.

PcrX, an sRNA derived from the 3'- UTR of the Rhodobacter sphaeroides puf operon modulates expression of puf genes encoding proteins of the bacterial photosynthetic apparatus.

Facultative phototrophic bacteria like Rhodobacter sphaeroides can produce ATP by anoxygenic photosynthesis, which is of advantage under conditions with limiting oxygen. However, the simultaneous presence of pigments, light and oxygen leads to the generation of harmful singlet oxygen. In order to avoid this stress situation, the formation of photosynthetic complexes is tightly regulated by light and oxygen signals. In a complex regulatory network several regulatory proteins and the small non-coding RNA PcrZ contribute to the balanced expression of photosynthesis genes. With PcrX this study identifies a second sRNA that is part of this network. The puf operon encodes pigment binding proteins of the light-harvesting I complex (PufBA) and of the reaction center (PufLM), a protein regulating porphyrin flux (PufQ), and a scaffolding protein (PufX). The PcrX sRNA is derived from the 3' UTR of the puf operon mRNA by RNase E-mediated cleavage. It targets the pufX mRNA segment, reduces the half-life of the pufBALMX mRNA and as a consequence affects the level of photosynthetic complexes. By its action PcrX counteracts the increased expression of photosynthesis genes that is mediated by protein regulators and is thus involved in balancing the formation of photosynthetic complexes in response to external stimuli.

Adaptation of the Alphaproteobacterium Rhodobacter sphaeroides to stationary phase.

Exhaustion of nutritional resources stimulates bacterial populations to adapt their growth behaviour. General mechanisms are known to facilitate this adaptation by sensing the environmental change and coordinating gene expression. However, the existence of such mechanisms among the Alphaproteobacteria remains unclear. This study focusses on global changes in transcript levels during growth under carbon-limiting conditions in a model Alphaproteobacterium, Rhodobacter sphaeroides, a metabolically diverse organism capable of multiple modes of growth including aerobic and anaerobic respiration, anaerobic anoxygenic photosynthesis and fermentation. We identified genes that showed changed transcript levels independently of oxygen levels during the adaptation to stationary phase. We selected a subset of these genes and subjected them to mutational analysis, including genes predicted to be involved in manganese uptake, polyhydroxybutyrate production and quorum sensing and an alternative sigma factor. Although these genes have not been previously associated with the adaptation to stationary phase, we found that all were important to varying degrees. We conclude that while R. sphaeroides appears to lack a rpoS-like master regulator of stationary phase adaptation, this adaptation is nonetheless enabled through the impact of multiple genes, each responding to environmental conditions and contributing to the adaptation to stationary phase.

The PhyR homolog RSP_1274 of Rhodobacter sphaeroides is involved in defense of membrane stress and has a moderate effect on RpoE (RSP_1092) activity.

BACKGROUND: A major role of the PhyR-NepR-σ(EcfG) cascade in the general stress response was demonstrated for some bacterial species and considered as conserved in Alphaproteobacteria. The σ(EcfG) factor activates its target genes in response to diverse stresses and NepR represents its anti-sigma factor. PhyR comprises a response regulator domain and a sigma factor domain and acts as anti-sigma factor antagonist. The facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides harbours a PhyR homolog in the same genomic context as found in other members of this class. RESULTS: Our study reveals increased expression of the phyR gene in response to superoxide, singlet oxygen, and diamide and also an effect of PhyR on rpoE expression. RpoE has a central role in mounting the response to singlet oxygen in R. sphaeroides. Despite these findings a mutant lacking PhyR was not significantly impeded in resistance to oxidative stress, heat stress or osmotic stress. However a role of PhyR in membrane stress is demonstrated. CONCLUSION: These results support the view that the effect of the PhyR-NepR-σ(EcfG) cascade on diverse stress responses varies among members of the Alphaproteobacteria. In the facultative phototroph Rhodobacter sphaeroides PhyR plays no major role in the general stress or the oxidative stress response but rather has a more specialized role in defense of membrane stress.

6S RNA in Rhodobacter sphaeroides: 6S RNA and pRNA transcript levels peak in late exponential phase and gene deletion causes a high salt stress phenotype.

The function of 6S RNA, a global regulator of transcription, was studied in the photosynthetic α-proteobacterium Rhodobacter sphaeroides. The cellular levels of R. sphaeroides 6S RNA peak toward the transition to stationary phase and strongly decrease during extended stationary phase. The synthesis of so-called product RNA transcripts (mainly 12-16-mers) on 6S RNA as template by RNA polymerase was found to be highest in late exponential phase. Product RNA ≥ 13-mers are expected to trigger the dissociation of 6S RNA:RNA polymerase complexes. A 6S RNA deletion in R. sphaeroides had no impact on growth under various metabolic and oxidative stress conditions (with the possible exception of tert-butyl hydroperoxide stress). However, the 6S RNA knockout resulted in a robust growth defect under high salt stress (0.25 M NaCl). Remarkably, the sspA gene encoding the putative salt stress-induced membrane protein SspA and located immediately downstream of the 6S RNA (ssrS) gene on the antisense strand was expressed at elevated levels in the ΔssrS strain when grown in the presence of 250 mM NaCl.

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

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.