Liquid-Liquid Phase Separation of the DEAD-Box Cyanobacterial RNA Helicase Redox (CrhR) into Dynamic Membraneless Organelles in Synechocystis sp. Strain PCC 6803.

Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.

Multiple Light-Dark Signals Regulate Expression of the DEAD-Box RNA Helicase CrhR in Synechocystis PCC 6803.

Since oxygenic photosynthesis evolved in the common ancestor of cyanobacteria during the Archean, a range of sensing and response strategies evolved to allow efficient acclimation to the fluctuating light conditions experienced in the diverse environments they inhabit. However, how these regulatory mechanisms are assimilated at the molecular level to coordinate individual gene expression is still being elucidated. Here, we demonstrate that integration of a series of three distinct light signals generate an unexpectedly complex network regulating expression of the sole DEAD-box RNA helicase, CrhR, encoded in Synechocystis sp. PCC 6803. The mechanisms function at the transcriptional, translational and post-translation levels, fine-tuning CrhR abundance to permit rapid acclimation to fluctuating light and temperature regimes. CrhR abundance is enhanced 15-fold by low temperature stress. We initially confirmed that the primary mechanism controlling crhR transcript accumulation at 20 °C requires a light quantity-driven reduction of the redox poise in the vicinity of the plastoquinone pool. Once transcribed, a specific light quality cue, a red light signal, was required for crhR translation, far-red reversal of which indicates a phytochrome-mediated mechanism. Examination of CrhR repression at 30 °C revealed that a redox- and light quality-independent light signal was required to initiate CrhR degradation. The crucial role of light was further revealed by the observation that dark conditions superseded the light signals required to initiate each of these regulatory processes. The findings reveal an unexpected complexity of light-dark sensing and signaling that regulate expression of an individual gene in cyanobacteria, an integrated mechanism of environmental perception not previously reported.

Degron-mediated proteolysis of CrhR-like DEAD-box RNA helicases in cyanobacteria.

Conditional proteolytic degradation is an irreversible and highly regulated process that fulfills crucial regulatory functions in all organisms. As proteolytic targets tend to be critical metabolic or regulatory proteins, substrates are targeted for degradation only under appropriate conditions through the recognition of an amino acid sequence referred to as a "degron". DEAD-box RNA helicases mediate all aspects of RNA metabolism, contributing to cellular fitness. However, the mechanism by which abiotic-stress modulation of protein stability regulates bacterial helicase abundance has not been extensively characterized. Here, we provide in vivo evidence that proteolytic degradation of the cyanobacterial DEAD-box RNA helicase CrhR is conditional, being initiated by a temperature upshift from 20 to 30 °C in the model cyanobacterium, Synechocystis sp. PCC 6803. We show degradation requires a unique, highly conserved, inherently bipartite degron located in the C-terminal extension found only in CrhR-related RNA helicases in the phylum Cyanobacteria. However, although necessary, the degron is not sufficient for proteolysis, as disruption of RNA helicase activity and/or translation inhibits degradation. These results suggest a positive feedback mechanism involving a role for CrhR in expression of a crucial factor required for degradation. Furthermore, AlphaFold structural prediction indicated the C-terminal extension is a homodimerization domain with homology to other bacterial RNA helicases, and mass photometry data confirmed that CrhR exists as a dimer in solution at 22 °C. These structural data suggest a model wherein the CrhR degron is occluded at the dimerization interface but could be exposed if dimerization was disrupted by nonpermissive conditions.

The temperature-regulated DEAD-box RNA helicase CrhR interactome: Autoregulation and photosynthesis-related transcripts.

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the ΔcrhR mutant exhibits a severe cold-sensitive phenotype, the physiological function(s) performed by CrhR have not been described. To identify transcripts interacting with CrhR, we performed RNA co-immunoprecipitation with extracts from a Synechocystis crhR deletion mutant expressing the FLAG-tagged native CrhR or a K57A mutated version with an anticipated enhanced RNA binding. The composition of the interactome was strikingly biased towards photosynthesis-associated and redox-controlled transcripts. A transcript highly enriched in all experiments was the crhR mRNA, suggesting an auto-regulatory molecular mechanism. The identified interactome explains the described physiological role of CrhR in response to the redox poise of the photosynthetic electron transport chain and characterizes CrhR as an enzyme with a diverse range of transcripts as molecular targets.

Genera specific distribution of DEAD-box RNA helicases in cyanobacteria.

Although RNA helicases are essentially ubiquitous and perform roles in all stages of RNA metabolism, phylogenetic analysis of the DEAD (Asp-Glu-Ala-Asp)-box RNA helicase family in a single phylum has not been performed. Here, we performed a phylogenetic analysis on DEAD-box helicases from all currently available cyanobacterial genomes, comprising a total of 362 helicase protein sequences from 280 strains. DEAD-box helicases belonging to three distinct clades were observed. Two clades, the CsdA (cold shock DEAD-box A)-like and RhlE (RNA helicase E)-like helicases, cluster with the homologous proteins from Escherichia coli. The third clade, the CrhR (cyanobacterial RNA helicase Redox)-like helicases, is unique to cyanobacteria and characterized by a conserved sequence motif in the C-terminal extension. Restricted distribution is observed across cyanobacterial diversity with respect to both helicase type and strain. CrhR-like and CsdA-like helicases essentially never occur together, while RhlE always occurs with either a CrhR-like or CsdA-like helicase. CrhR-like and RhlE-like proteins occurred in filamentous cyanobacteria of the orders Nostocales, Oscillatoriales and Synechococcales. Similarly, CsdA- and RhlE-like proteins are restricted to unicellular cyanobacteria of the genera Cyanobium and Synechococcus. In addition, the unexpected occurrence of RhlE in two Synechococcus strains suggests recent acquisition and evolutionary divergence. This study, therefore, raises physiological and evolutionary questions as to why DEAD-box RNA helicases encoded in cyanobacterial lineages display restricted distributions, suggesting niches that require either CrhR or CsdA RNA helicase activity but not both. Extensive conservation of gene synteny surrounding the previously described rimO-crhR operon is also observed, indicating a role in the maintenance of photosynthesis. The analysis provides insights into the evolution, origin and dissemination of sequences within a single gene family to yield divergent functional roles.

RNA helicase-regulated processing of the Synechocystis rimO-crhR operon results in differential cistron expression and accumulation of two sRNAs.

The arrangement of functionally-related genes in operons is a fundamental element of how genetic information is organized in prokaryotes. This organization ensures coordinated gene expression by co-transcription. Often, however, alternative genetic responses to specific stress conditions demand the discoordination of operon expression. During cold temperature stress, accumulation of the gene encoding the sole Asp-Glu-Ala-Asp (DEAD)-box RNA helicase in Synechocystis sp. PCC 6803, crhR (slr0083), increases 15-fold. Here, we show that crhR is expressed from a dicistronic operon with the methylthiotransferase rimO/miaB (slr0082) gene, followed by rapid processing of the operon transcript into two monocistronic mRNAs. This cleavage event is required for and results in destabilization of the rimO transcript. Results from secondary structure modeling and analysis of RNase E cleavage of the rimO-crhR transcript in vitro suggested that CrhR plays a role in enhancing the rate of the processing in an auto-regulatory manner. Moreover, two putative small RNAs are generated from additional processing, degradation, or both of the rimO transcript. These results suggest a role for the bacterial RNA helicase CrhR in RNase E-dependent mRNA processing in Synechocystis and expand the known range of organisms possessing small RNAs derived from processing of mRNA transcripts.

Evidence for convergent sensing of multiple abiotic stresses in cyanobacteria.

BACKGROUND: Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons. METHODS: Electron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR. RESULTS: Alterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between QA and cytochrome b6f, with reduction favoring CrhR accumulation. CONCLUSIONS: The results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of QB/PQH2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression. GENERAL SIGNIFICANCE: The potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated.

Inactivation of the RNA helicase CrhR impacts a specific subset of the transcriptome in the cyanobacterium Synechocystis sp. PCC 6803.

DEAD-box RNA-helicases catalyze the reorganization of structured RNAs and the formation of RNP complexes. The cyanobacterium Synechocystis sp. PCC 6803 encodes a single DEAD-box RNA helicase, CrhR (Slr0083), whose expression is regulated by abiotic stresses that alter the redox potential of the photosynthetic electron transport chain, including temperature downshift. Despite its proposed effect on RNA metabolism and its known relevance in cold-stress adaptation, the reported impact of a CrhR knockout on the cold adaption of the transcriptome only identified eight affected genes. Here, we utilized a custom designed microarray to assess the impact of the absence of CrhR RNA helicase activity on the transcriptome, independent of cold stress. CrhR truncation impacts an RNA subset comprising ~10% of the ncRNA and also ~10% of the mRNA transcripts. While equal numbers of mRNAs showed increased as well as decreased abundance, more than 90% of the ncRNAs showed enhanced expression in the absence of CrhR, indicative of a negative effect on ncRNA transcription or stability. We further tested the effect of CrhR on the stability of strongly responding RNAs that identify examples of post-transcriptional and transcriptional regulation. The data suggest that CrhR impacts multiple aspects of RNA metabolism in Synechocystis.

UV radiation limited the expansion of cyanobacteria in early marine photic environments.

Prior to atmospheric oxygenation, ecosystems were exposed to higher UV radiation fluxes relative to modern surface environments. Iron-silica mineral coatings have been evoked as effective UV radiation shields in early terrestrial settings. Here we test whether similar protection applied to planktonic cyanobacteria within the Archean water column. Based on experiments done under Archean seawater conditions, we report that Fe(III)-Si-rich precipitates absorb up to 70% of incoming UV-C radiation, with a reduction of <20% in photosynthetically active radiation flux. However, we demonstrate that even short periods of UV-C irradiation in the presence of Fe(III)-Si precipitates resulted in high mortality rates, and suggest that these effects would have persisted throughout much of the photic zone. Our findings imply that despite the shielding properties of Fe(III)-Si-rich precipitates in the early water column, UV radiation would continue to limit cyanobacterial expansion and likely had a greater effect on Archean ecosystem structure before the formation of an ozone layer.

Determination of the Settling Rate of Clay/Cyanobacterial Floccules.

The mechanisms underpinning the deposition of fine-grained, organic-rich sediments are still largely debated. Specifically, the impact of the interaction of clay particles with reactive, planktonic cyanobacterial cells to the sedimentary record is under studied. This interaction is a potentially major contributor to shale depositional models. Within a lab setting, the flocculation and sedimentation rates of these materials can be examined and measured in a controlled environment. Here, we detail a protocol for measuring the sedimentation rate of cyanobacterial/clay mixtures. This methodology is demonstrated through the description of two sample experiments: the first uses kaolin (a dehydrated form of kaolinite) and Synechococcus sp. PCC 7002 (a marine coccoid cyanobacteria), and the second uses kaolin and Synechocystis sp. PCC 6803 (a freshwater coccoid cyanobacteria). Cyanobacterial cultures are mixed with varying amounts of clay within a specially designed tank apparatus optimized to allow continuous, real-time video and photographic recording. The sampling procedures are detailed as well as a post-collection protocol for precise measurement of chlorophyll a from which the concentration of cyanobacterial cells remaining in suspension can be determined. Through experimental replication, a profile is constructed that displays sedimentation rate.

Cyanobacterial RNA Helicase CrhR Localizes to the Thylakoid Membrane Region and Cosediments with Degradosome and Polysome Complexes in Synechocystis sp. Strain PCC 6803.

UNLABELLED: The cyanobacterium Synechocystis sp. strain PCC 6803 encodes a single DEAD box RNA helicase, CrhR, whose expression is tightly autoregulated in response to cold stress. Subcellular localization and proteomic analysis results indicate that CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with polysome and RNA degradosome components. Evidence is presented that either functional RNA helicase activity or a C-terminal localization signal was required for polysome but not thylakoid membrane localization. Polysome fractionation and runoff translation analysis results indicate that CrhR associates with actively translating polysomes. The data implicate a role for CrhR in translation or RNA degradation in the thylakoid region related to thylakoid biogenesis or stability, a role that is enhanced at low temperature. Furthermore, CrhR cosedimentation with polysome and RNA degradosome complexes links alteration of RNA secondary structure with a potential translation-RNA degradation complex in Synechocystis IMPORTANCE: The interaction between mRNA translation and degradation is a major determinant controlling gene expression. Regulation of RNA function by alteration of secondary structure by RNA helicases performs crucial roles, not only in both of these processes but also in all aspects of RNA metabolism. Here, we provide evidence that the cyanobacterial RNA helicase CrhR localizes to both the cytoplasmic and thylakoid membrane regions and cosediments with actively translating polysomes and RNA degradosome components. These findings link RNA helicase alteration of RNA secondary structure with translation and RNA degradation in prokaryotic systems and contribute to the data supporting the idea of the existence of a macromolecular machine catalyzing these reactions in prokaryotic systems, an association hitherto recognized only in archaea and eukarya.

Conditional, temperature-induced proteolytic regulation of cyanobacterial RNA helicase expression.

Conditional proteolysis is a crucial process regulating the abundance of key regulatory proteins associated with the cell cycle, differentiation pathways, or cellular response to abiotic stress in eukaryotic and prokaryotic organisms. We provide evidence that conditional proteolysis is involved in the rapid and dramatic reduction in abundance of the cyanobacterial RNA helicase, CrhR, in response to a temperature upshift from 20 to 30°C. The proteolytic activity is not a general protein degradation response, since proteolysis is only present and/or functional in cells grown at 30°C and is only transiently active at 30°C. Degradation is also autoregulatory, since the CrhR proteolytic target is required for activation of the degradation machinery. This suggests that an autoregulatory feedback loop exists in which the target of the proteolytic machinery, CrhR, is required for activation of the system. Inhibition of translation revealed that only elongation is required for induction of the temperature-regulated proteolysis, suggesting that translation of an activating factor was already initiated at 20°C. The results indicate that Synechocystis responds to a temperature shift via two independent pathways: a CrhR-independent sensing and signal transduction pathway that regulates induction of crhR expression at low temperature and a CrhR-dependent conditional proteolytic pathway at elevated temperature. The data link the potential for CrhR RNA helicase alteration of RNA secondary structure with the autoregulatory induction of conditional proteolysis in the response of Synechocystis to temperature upshift.

RNA helicases: diverse roles in prokaryotic response to abiotic stress.

Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes.

Autoregulation of RNA helicase expression in response to temperature stress in Synechocystis sp. PCC 6803.

RNA helicases are ubiquitous enzymes whose modification of RNA secondary structure is known to regulate RNA function. The pathways controlling RNA helicase expression, however, have not been well characterized. Expression of the cyanobacterial RNA helicase, crhR, is regulated in response to environmental signals that alter the redox poise of the electron transport chain, including light and temperature. Here we analyze crhR expression in response to alteration of abiotic conditions in wild type and a crhR mutant, providing evidence that CrhR autoregulates its own expression through a combination of transcriptional and post-transcriptional mechanisms. Temperature regulates crhR expression through alteration of both transcript and protein half-life which are significantly extended at low temperature (20°C). CrhR-dependent mechanisms regulate both the transient accumulation of crhR transcript at 20°C and stability of the CrhR protein at all temperatures. CrhR-independent mechanisms regulate temperature sensing and induction of crhR transcript accumulation at 20°C and the temperature regulation of crhR transcript stability, suggesting CrhR is not directly associated with crhR mRNA turnover. Many of the processes are CrhR- and temperature-dependent and occur in the absence of a correlation between crhR transcript and protein abundance. The data provide important insights into not only how RNA helicase gene expression is regulated but also the role that rearrangement of RNA secondary structure performs in the molecular response to temperature stress. We propose that the crhR-regulatory pathway exhibits characteristics similar to the heat shock response rather than a cold stress-specific mechanism.

RNA helicases in cyanobacteria: biochemical and molecular approaches.

RNA helicases are associated with every aspect of RNA metabolism and function. A diverse range of RNA helicases are encoded by essentially every organism. While RNA helicases alter gene expression, RNA helicase expression is itself regulated, frequently in response to abiotic stress. Photosynthetic cyanobacteria present a unique model system to investigate RNA helicase expression and function. This chapter describes methodology to study the expression and cellular localization of RNA helicases, providing insights into the metabolic pathway(s) in which these enzymes function in cyanobacteria. The approaches are applicable to other systems as well.

Inactivation of a low temperature-induced RNA helicase in Synechocystis sp. PCC 6803: physiological and morphological consequences.

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

Plant RNA helicases: linking aberrant and silencing RNA.

RNA helicases are ATPases that are capable of rearranging RNA and ribonucleoprotein (RNP) structure, and they can potentially function in any aspect of RNA metabolism. The RNA helicase gene family of plant genomes is larger and more diverse than genome families observed in other systems and provides an ideal model for investigation of the physiological importance of RNA secondary structure rearrangement in plant development. Numerous plant RNA helicases are associated with a variety of physiological functions, but this review will focus on the thirteen RNA helicases associated with the metabolism of aberrant and silencing RNAs. The results emphasize the crucial role RNA helicase activity has in the regulation of mRNA quality control and gene expression in plant development.

A Synechocystis LexA-orthologue binds direct repeats in target genes.

Although evidence for LexA-orthologues, which do not regulate DNA damage repair, is accumulating, identification of binding sites and regulon members remains poorly characterized. In the cyanobacterium, Synechocystis sp. strain PCC 6803, we have recently identified a LexA-related protein that regulates expression of the crhR RNA helicase gene. Here we show that the Synechocystis LexA-orthologue binds as a dimer to 12 bp direct repeats containing a CTA-N9-CTA sequence conserved in two target genes, lexA and crhR. Characterization of this site provides the basis for identification of additional LexA targets and further evidence for LexA's divergence during evolution.

Cold-stress-altered phosphorylation of EF-Tu in the cyanobacterium Anabaena sp. strain PCC 7120.

Growth of prokaryotes at reduced temperature results in the formation of a cold-adapted ribosome through association with de novo synthesized polypeptides. In vitro and in vivo phosphorylation studies combined with affinity purification and mass spectrometry identified that the phosphorylation status of translation elongation factor EF-Tu was altered in response to cold stress in the photosynthetic, Gram-negative cyanobacterium Anabaena sp. strain PCC 7120. In response to a temperature downshift from 30 to 20 degrees C, EF-Tu was rapidly and transiently hyperphosphorylated during the acclimation phase followed by a reduction in phosphorylation below background levels in response to prolonged exposure. EF-Tu was identified as a phosphothreonine protein. Unexpectedly, ribosomal protein S2 was also observed to be a phosphoprotein continuously phosphorylated during cold stress. The phosphorylation status of EF-Tu has previously been associated with translational regulation in other systems, with a reduction in translation elongation occurring in response to phosphorylation. These results provide evidence for a novel mechanism by which translation is initially downregulated in response to cold stress in Anabaena.

A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR.

Expression of the cyanobacterial DEAD-box RNA helicase, crhR, is regulated in response to conditions, which elicit reduction of the photosynthetic electron transport chain. A combination of electrophoretic mobility shift assay (EMSA), DNA affinity chromatography and mass spectrometry identified that a LexA-related protein binds specifically to the crhR gene. Transcript analysis indicates that lexA and crhR are divergently expressed, with lexA and crhR transcripts accumulating differentially under conditions, which respectively oxidize and reduce the electron transport chain. In addition, expression of the Synechocystis lexA gene is not DNA damage inducible and its amino acid sequence lacks two of three residues required for activity of prototypical LexA proteins, which repress expression of DNA repair genes in a range of prokaryotes. A direct effect of recombinant LexA protein on crhR expression was confirmed from the observation that LexA reduces crhR expression in a linear manner in an in vitro transcription/translation assay. The results indicate that the Synechocystis LexA-related protein functions as a regulator of redox-responsive crhR gene expression, and not DNA damage repair genes.