Bork A. Berghoff

Photooxidative stress-induced and abundant small RNAs in Rhodobacter sphaeroides.

Exposure to oxygen and light generates photooxidative stress by the bacteriochlorophyll a mediated formation of singlet oxygen (1O2) in Rhodobacter sphaeroides. Our study reports the genome‐wide search for small RNAs (sRNAs) involved in the regulatory response to 1O2. By using 454 pyrosequencing and Northern blot analysis, we identified 20 sRNAs from R. sphaeroides aerobic cultures or following treatment with 1O2 or superoxide (O–2). One sRNA was specifically induced by 1O2 and its expression depends on the extracytoplasmic function sigma factor RpoE. Two sRNAs induced by 1O2 and O–2 were cotranscribed with upstream genes preceded by promoters with target sequences for the alternative sigma factors RpoHI and RpoHII. The most abundant sRNA was processed in the presence of 1O2 but not by O–2. From this and a second sRNA a conserved 3′‐segment accumulated from a larger precursor. Absence of the RNA chaperone Hfq changed the half‐lives, abundance and processing of 1O2‐affected sRNAs. Orthologues of three sRNA genes are present in different alpha‐proteobacteria, but the majority was unique to R. sphaeroides or Rhodobacterales species. Our discovery that abundant sRNAs are affected by 1O2 exposure extends the knowledge on the role of sRNAs and Hfq in the regulatory response to oxidative stress.

Singlet Oxygen Stress in Microorganisms

Singlet oxygen is the primary agent of photooxidative stress in microorganisms. In photosynthetic microorganisms, sensitized generation by pigments of the photosystems is the main source of singlet oxygen and, in nonphotosynthetic microorganisms, cellular cofactors such as flavins, rhodopsins, quinones, and porphyrins serve as photosensitizer. Singlet oxygen rapidly reacts with a wide range of cellular macromolecules including proteins, lipids, DNA, and RNA, and thereby further reactive substances including organic peroxides and sulfoxides are formed. Microorganisms that face high light intensities or exhibit potent photosensitizers have evolved specific mechanisms to prevent photooxidative stress. These mechanisms include the use of quenchers, such as carotenoids, which interact either with excited photosensitizer molecules or singlet oxygen itself to prevent damage of cellular molecules. Scavengers like glutathione react with singlet oxygen. Despite those protection mechanisms, damage by reactions with singlet oxygen on cellular macromolecules disturbs cellular functions. Microorganisms that regularly face photooxidative stress have evolved specific systems to sense singlet oxygen and tightly control the removal of singlet oxygen reaction products. Responses to photooxidative stress have been investigated in a range of photosynthetic and nonphotosynthetic microorganisms. However, detailed knowledge on the regulation of this response has only been obtained for the phototrophic alpha-proteobacterium Rhodobacter sphaeroides. In this organism and in related proteobacteria, the extracytoplasmic function (ECF) sigma factor RpoE is released from the cognate antisigma factor ChrR in the presence of singlet oxygen and triggers the expression of genes providing protection against photooxidative stress. Recent experiments show that singlet oxygen acts as a signal, which is sensed by yet unknown components and leads to proteolysis of ChrR. RpoE induces expression of a second alternative sigma factor, RpoH(II), which controls a large set of genes that partially overlaps with the heat-shock response controlled by RpoH(I). In addition to the transcriptional control of gene regulation by alternative sigma factors, a set of noncoding small RNAs (sRNAs) appear to affect the synthesis of several proteins involved in the response to photooxidative stress. The interaction of mRNA targets with those sRNAs is usually mediated by the RNA chaperone Hfq. Deletion of the gene encoding Hfq leads to a singlet oxygen-sensitive phenotype, which underlines the control of gene regulation on the posttranscriptional level by sRNAs in R. sphaeroides. Hence, a complex network of different regulatory components controls the defense against photooxidative stress in anoxygenic photosynthetic bacteria.

Contribution of Hfq to photooxidative stress resistance and global regulation in Rhodobacter sphaeroides

The photosynthetic alphaproteobacterium Rhodobacter sphaeroides has to cope with photooxidative stress that is caused by the bacteriochlorophyll a‐mediated formation of singlet oxygen (1O2). Exposure to 1O2 induces the alternative sigma factors RpoE and RpoHII which then promote transcription of photooxidative stress‐related genes, including small RNAs (sRNAs). The ubiquitous RNA chaperone Hfq is well established to interact with and facilitate the base‐pairing of sRNAs and target mRNAs to influence mRNA stability and/or translation. Here we report on the pleiotropic phenotype of a Δhfq mutant of R. sphaeroides, which is less pigmented, produces minicells and is more sensitive to 1O2. The higher 1O2 sensitivity of the Δhfq mutant is paralleled by a reduced RpoE activity and a disordered induction of RpoHII‐dependent genes. We used co‐immunoprecipitation of FLAG‐tagged Hfq combined with RNA‐seq to identify association of at least 25 sRNAs and of mRNAs encoding cell division proteins and ribosomal proteins with Hfq. Remarkably, > 70% of the Hfq‐bound sRNAs are 1O2‐affected. Proteomics analysis of the Hfq‐deficient strain revealed an impact of Hfq on amino acid transport and metabolic functions. Our data demonstrate for the first time an involvement of Hfq in regulation of photosynthesis genes and in the photooxidative stress response.

Integrative ''Omics''-Approach Discovers Dynamic and Regulatory Features of Bacterial Stress Responses

Bacteria constantly face stress conditions and therefore mount specific responses to ensure adaptation and survival. Stress responses were believed to be predominantly regulated at the transcriptional level. In the phototrophic bacterium Rhodobacter sphaeroides the response to singlet oxygen is initiated by alternative sigma factors. Further adaptive mechanisms include post-transcriptional and post-translational events, which have to be considered to gain a deeper understanding of how sophisticated regulation networks operate. To address this issue, we integrated three layers of regulation: (1) total mRNA levels at different time-points revealed dynamics of the transcriptome, (2) mRNAs in polysome fractions reported on translational regulation (translatome), and (3) SILAC-based mass spectrometry was used to quantify protein abundances (proteome). The singlet oxygen stress response exhibited highly dynamic features regarding short-term effects and late adaptation, which could in part be assigned to the sigma factors RpoE and RpoH2 generating distinct expression kinetics of corresponding regulons. The occurrence of polar expression patterns of genes within stress-inducible operons pointed to an alternative of dynamic fine-tuning upon stress. In addition to transcriptional activation, we observed significant induction of genes at the post-transcriptional level (translatome), which identified new putative regulators and assigned genes of quorum sensing to the singlet oxygen stress response. Intriguingly, the SILAC approach explored the stress-dependent decline of photosynthetic proteins, but also identified 19 new open reading frames, which were partly validated by RNA-seq. We propose that comparative approaches as presented here will help to create multi-layered expression maps on the system level (“expressome”). Finally, intense mass spectrometry combined with RNA-seq might be the future tool of choice to re-annotate genomes in various organisms and will help to understand how they adapt to alternating conditions.

Regulation of bacterial photosynthesis genes by the small noncoding RNA PcrZ

The small RNA PcrZ (photosynthesis control RNA Z) of the facultative phototrophic bacterium Rhodobacter sphaeroides is induced upon a drop of oxygen tension with similar kinetics to those of genes for components of photosynthetic complexes. High expression of PcrZ depends on PrrA, the response regulator of the PrrB/PrrA two-component system with a central role in redox regulation in R. sphaeroides. In addition the FnrL protein, an activator of some photosynthesis genes at low oxygen tension, is involved in redox-dependent expression of this small (s)RNA. Overexpression of full-length PcrZ in R. sphaeroides affects expression of a small subset of genes, most of them with a function in photosynthesis. Some mRNAs from the photosynthetic gene cluster were predicted to be putative PcrZ targets and results from an in vivo reporter system support these predictions. Our data reveal a negative effect of PcrZ on expression of its target mRNAs. Thus, PcrZ counteracts the redox-dependent induction of photosynthesis genes, which is mediated by protein regulators. Because PrrA directly activates photosynthesis genes and at the same time PcrZ, which negatively affects photosynthesis gene expression, this is one of the rare cases of an incoherent feed-forward loop including an sRNA. Our data identified PcrZ as a trans acting sRNA with a direct regulatory function in formation of photosynthetic complexes and provide a model for the control of photosynthesis gene expression by a regulatory network consisting of proteins and a small noncoding RNA.

Anoxygenic photosynthesis and photooxidative stress: a particular challenge for Roseobacter

Roseobacter clade aerobic anoxygenic phototrophic bacteria (AAnP) are abundant in photic zone environments of marine ecosystems. These bacteria form a photosynthetic apparatus at oxygen saturation, a situation expected to generate high levels of singlet oxygen (¹O₂) when light is present. Rhodobacter sphaeroides, an anaerobic anoxygenic phototroph, represses photosynthesis genes at high oxygen tension. Here we report that Roseobacter denitrificans showed higher sensitivity to ¹O₂ compared with Rhb. sphaeroides. While photosynthetic membranes of Rsb. denitrificans generated more ¹O₂ during light exposure, key regulator genes rpoE and rpoH(II) were more strongly induced in response to ¹O₂ stress compared with Rhb. sphaeroides. The regulon controlled by RpoE was different in Rsb. denitrificans and Rhb. sphaeroides. Patterns of synthesized soluble proteins strongly changed upon high light exposure in Rsb. denitrificans but not in Rhb. sphaeroides, and most changes were not further promoted by artificial ¹O₂ generation. The strong increase of small RNA RDs2461 levels by photooxidative stress implies a role for sRNAs in post-transcriptional regulation of the response to ¹O₂ in AAnPs. Our data reveal similarities but also significant differences in the response of Rsb. denitrificans and Rhb. sphaeroides to ¹O₂, most likely a consequence of their different lifestyles.

Riboregulators and the role of Hfq in photosynthetic bacteria

Anoxygenic and oxygenic bacteria directly convert solar energy into biomass using photosynthesis. The formation and composition of photosynthetic complexes has to be tightly controlled in response to environmental conditions, as exposure to sunlight can be harmful due to the generation of reactive oxygen species and the damaging effects of UV irradiation. Therefore, photosynthetic bacteria are exposed to a particular set of regulatory challenges in addition to those that also affect other bacteria, requiring sophisticated regulatory systems. Indeed, hundreds of potential regulatory RNAs have been identified in photosynthetic model bacteria as well as antisense RNAs (asRNAs) of up to several kb in length that protect certain mRNAs from degradation. The trans-acting small non-coding RNAs (sRNAs), PcrZ and PsrR1, control pigment and photosystem biogenesis in Rhodobacter sphaeroides and cyanobacteria, respectively. The asRNAs IsrR and As1_flv4 act as negative regulators and the asRNAs PsbA2R and PsbA3R as positive effectors of photosynthesis gene expression in Synechocystis 6803.

Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides

BackgroundHigh intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the α-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen.ResultsOne third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation.ConclusionSome R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a “core iron response” that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.

A Cluster of Four Homologous Small RNAs Modulates C1 Metabolism and the Pyruvate Dehydrogenase Complex in Rhodobacter sphaeroides under Various Stress Conditions

UNLABELLED In bacteria, regulatory RNAs play an important role in the regulation and balancing of many cellular processes and stress responses. Among these regulatory RNAs, trans-encoded small RNAs (sRNAs) are of particular interest since one sRNA can lead to the regulation of multiple target mRNAs. In the purple bacterium Rhodobacter sphaeroides, several sRNAs are induced by oxidative stress. In this study, we focused on the functional characterization of four homologous sRNAs that are cotranscribed with the gene for the conserved hypothetical protein RSP_6037, a genetic arrangement described for only a few sRNAs until now. Each of the four sRNAs is characterized by two stem-loops that carry CCUCCUCCC motifs in their loops. They are induced under oxidative stress, as well as by various other stress conditions, and were therefore renamed here sRNAs CcsR1 to CcsR4 (CcsR1-4) for conserved CCUCCUCCC motif stress-induced RNAs 1 to 4. Increased CcsR1-4 expression decreases the expression of genes involved in C1 metabolism or encoding components of the pyruvate dehydrogenase complex either directly by binding to their target mRNAs or indirectly. One of the CcsR1-4 target mRNAs encodes the transcriptional regulator FlhR, an activator of glutathione-dependent methanol/formaldehyde metabolism. Downregulation of this glutathione-dependent pathway increases the pool of glutathione, which helps to counteract oxidative stress. The FlhR-dependent downregulation of the pyruvate dehydrogenase complex reduces a primary target of reactive oxygen species and reduces aerobic electron transport, a main source of reactive oxygen species. Our findings reveal a previously unknown strategy used by bacteria to counteract oxidative stress. IMPORTANCE Phototrophic organisms have to cope with photo-oxidative stress due to the function of chlorophylls as photosensitizers for the formation of singlet oxygen. Our study assigns an important role in photo-oxidative stress resistance to a cluster of four homologous sRNAs in the anoxygenic phototrophic bacterium Rhodobacter sphaeroides. We reveal a function of these regulatory RNAs in the fine-tuning of C1 metabolism. A model that relates oxidative stress defense to C1 metabolism is presented.

Two regulatory RNA elements affect TisB-dependent depolarization and persister formation

Bacterial survival strategies involve phenotypic diversity which is generated by regulatory factors and noisy expression of effector proteins. The question of how bacteria exploit regulatory RNAs to make decisions between phenotypes is central to a general understanding of these universal regulators. We investigated the TisB/IstR‐1 toxin‐antitoxin system of Escherichia coli to appreciate the role of the RNA antitoxin IstR‐1 in TisB‐dependent depolarization of the inner membrane and persister formation. Persisters are phenotypic variants that have become transiently drug‐tolerant by arresting growth. The RNA antitoxin IstR‐1 sets a threshold for TisB‐dependent depolarization under DNA‐damaging conditions, resulting in two sub‐populations: polarized and depolarized cells. Furthermore, our data indicate that an inhibitory 5′ UTR structure in the tisB mRNA serves as a regulatory RNA element that delays TisB translation to avoid inappropriate depolarization when DNA damage is low. Investigation of the persister sub‐population further revealed that both regulatory RNA elements affect persister levels as well as persistence time. This work provides an intriguing example of how bacteria exploit regulatory RNAs to control phenotypic heterogeneity.