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 The crucial role of light was further revealed by the observation that dark conditions superseded the light signals required to initiate each of these regulatory processes.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.

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.

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.