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Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deleting the sll0729 gene (∆sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of seven suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoter's discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in ∆sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, hemJ promoter variants with varying discriminator sequences were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC discriminator motif, resulting in wild-type-like pigmentation, whereas freshly prepared ∆sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression. Thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely other cyanobacteria., (© The Author(s) 2024. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

How CRISPR-Cas systems defend bacteria and archaea against invading genetic elements is well understood, but less is known about their regulation. In the cyanobacterium Synechocystis sp. PCC 6803, the expression of one of the three different CRISPR-Cas systems responds to changes in environmental conditions. The cas operon promoter of this system is controlled by the light- and redox-responsive transcription factor RpaB binding to an HLR1 motif, resulting in transcriptional activation at low light intensities. However, the strong promoter that drives transcription of the cognate repeat-spacer array is not controlled by RpaB. Instead, the leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled with mass spectrometry analysis and site-directed mutagenesis revealed six residues involved in the CrhR-RNA interaction, with C371 being critically important. Thus, the expression of a type III-Dv CRISPR-Cas system is linked to the redox status of the photosynthetic cell at the transcriptional and post-transcriptional levels., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. It has been hypothesized that the excreted nitrite represents excess nitrogen that cannot be further assimilated due to the missing carbon, but the underlying molecular mechanisms are unclear. Here, we identified a protein that interacts with nitrite reductase, regulates nitrogen metabolism and promotes nitrite excretion. The protein, which we named NirP1, is encoded by an unannotated gene that is upregulated under low carbon conditions and controlled by transcription factor NtcA, a central regulator of nitrogen homeostasis. Ectopic overexpression of nirP1 in Synechocystis sp. PCC 6803 resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments indicated that NirP1 interacts with nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite. Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting nitrite reductase., (© 2024. The Author(s).)

Numerous cyanobacteria capable of oxygenic photosynthesis possess multiple large plasmids exceeding 100 kbp in size. These plasmids are believed to have distinct replication and distribution mechanisms, as they coexist within cells without causing incompatibilities between plasmids. However, information on plasmid replication proteins (Rep) in cyanobacteria is limited. Synechocystis sp. PCC 6803 hosts four large plasmids, pSYSM, pSYSX, pSYSA, and pSYSG, but Rep proteins for these plasmids, except for CyRepA1 on pSYSA, are unknown. Using Autonomous Replication sequencing (AR-seq), we identified two potential Rep genes in Synechocystis 6803, slr6031 and slr6090 , both located on pSYSX. The corresponding Rep candidates, Slr6031 and Slr6090, share structural similarities with Rep-associated proteins of other bacteria and homologs were also identified in various cyanobacteria. We observed autonomous replication activity for Slr6031 and Slr6090 in Synechococcus elongatus PCC 7942 by fusing their genes with a construct expressing GFP and introducing them via transformation. The slr6031/slr6090 -containing plasmids exhibited lower copy numbers and instability in Synechococcus 7942 cells compared to the expression vector pYS. While recombination occurred in the case of slr6090 , the engineered plasmid with slr6031 coexisted with plasmids encoding CyRepA1 or Slr6090 in Synechococcus 7942 cells, indicating the compatibility of Slr6031 and Slr6090 with CyRepA1. Based on these results, we designated Slr6031 and Slr6090 as CyRepX1 (Cyanobacterial Rep-related protein encoded on pSYSX) and CyRepX2, respectively, demonstrating that pSYSX is a plasmid with "two Reps in one plasmid." Furthermore, we determined the copy number and stability of plasmids with cyanobacterial Reps in Synechococcus 7942 and Synechocystis 6803 to elucidate their potential applications. The novel properties of CyRepX1 and 2, as revealed by this study, hold promise for the development of innovative genetic engineering tools in cyanobacteria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ohdate, Sakata, Maeda, Sakamaki, Nimura-Matsune, Ohbayashi, Hess and Watanabe.)

Throughout the tree of life RNA-binding proteins play important roles, but they are poorly characterized in cyanobacteria. Overexpression of the predicted RNA-binding protein Ssr1238 in the cyanobacterium Synechocystis 6803 for 24 h led to higher levels of RNase P RNA, tRNAs, and stress-related mRNAs. Co-immunoprecipitation of proteins followed by MS analysis and sequencing of UV crosslinked, co-immunoprecipitated RNA samples identified potential interaction partners of Ssr1238. The most enriched transcript was RNase P RNA, and RnpA, the protein component of RNase P, was among the most highly enriched proteins. A second highly enriched transcript is derived from gene ssl3177 , which encodes a central enzyme in cell wall remodelling during cell division. The data also showed a strong connection to the RNA maturation and modification system indicated by co-precipitation of RNA modifying enzymes, riboendonuclease E and enolase. Surprisingly, cyanophycin synthetase and urease were highly enriched as well. In conclusion, Ssr1238 specifically binds to two different transcripts and could be involved in the coordination of RNA maturation, translation, cell division, and aspects of nitrogen metabolism. Our results are consistent with recent findings that the B. subtilis YlxR protein functions as an RNase P modulator (RnpM), extending its proposed role to the phylum cyanobacteria, and suggesting additional functionalities.

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 ( Synechocystis ), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNA Glu UUC . This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli .

Summary: RNA molecules play crucial roles in various biological processes. They mediate their function mainly by interacting with other RNAs or proteins. At present, information about these interactions is distributed over different resources, often providing the data in simple tab-delimited formats that differ between the databases. There is no standardized data format that can capture the nature of all these different interactions in detail., Availability and Implementation: Here, we propose the RNA interaction format (RIF) for the detailed representation of RNA-RNA and RNA-Protein interactions and provide reference implementations in C/C++, Python, and JavaScript. RIF is released under licence GNU General Public License version 3 (GNU GPLv3) and is available on https://github.com/RNABioInfo/rna-interaction-format., (© The Author(s) 2023. Published by Oxford University Press.)

An increasing number of small proteins has been identified in the genomes of well-annotated organisms, including the model cyanobacterium Synechocystis sp. PCC 6803. We describe a newly assigned protein comprising 37 amino acids that is encoded upstream of the superoxide dismutase SodB encoding gene. To clarify the role of SliP4, we analyzed a Synechocystis sliP4 mutant and a strain containing a fully active, Flag-tagged variant of SliP4 (SliP4.f). The initial hypothesis that this small protein might be functionally related to SodB could not be supported. Instead, we provide evidence that it fulfills important functions related to the organization of photosynthetic complexes. Therefore, we named it a small light-induced protein of 4 kDa, SliP4. This protein is strongly induced under high-light conditions. The lack of SliP4 causes a light-sensitive phenotype due to impaired cyclic electron flow and state transitions. Interestingly, SliP4.f was co-isolated with NDH1 complex and both photosystems. The interaction between SliP4.f and all three types of complexes was further confirmed by additional pulldowns and 2D-electrophoreses. We propose that the dimeric SliP4 serves as a molecular glue promoting the aggregation of thylakoid complexes, which contributes to different electron transfer modes and energy dissipation under stress conditions., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Certain CRISPR-Cas elements integrate into Tn7-like transposons, forming CRISPR-associated transposon (CAST) systems. How the activity of these systems is controlled in situ has remained largely unknown. Here we characterize the MerR-type transcriptional regulator Alr3614 that is encoded by one of the CAST (AnCAST) system genes in the genome of cyanobacterium Anabaena sp. PCC 7120. We identify a number of Alr3614 homologs across cyanobacteria and suggest naming these regulators CvkR for Cas V-K repressors. Alr3614/CvkR is translated from leaderless mRNA and represses the AnCAST core modules cas12k and tnsB directly, and indirectly the abundance of the tracr-CRISPR RNA. We identify a widely conserved CvkR binding motif 5'-AnnACATnATGTnnT-3'. Crystal structure of CvkR at 1.6 Å resolution reveals that it comprises distinct dimerization and potential effector-binding domains and that it assembles into a homodimer, representing a discrete structural subfamily of MerR regulators. CvkR repressors are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems., (© 2023. The Author(s).)

Synthetic biology approaches toward the development of cyanobacterial producer strains require the availability of appropriate sets of plasmid vectors. A factor for the industrial usefulness of such strains is their robustness against pathogens, such as bacteriophages infecting cyanobacteria. Therefore, it is of great interest to understand the native plasmid replication systems and the CRISPR-Cas based defense mechanisms already present in cyanobacteria. In the model cyanobacterium Synechocystis sp. PCC 6803, four large and three smaller plasmids exist. The ~100 kb plasmid pSYSA is specialized in defense functions by encoding all three CRISPR-Cas systems and several toxin-antitoxin systems. The expression of genes located on pSYSA depends on the plasmid copy number in the cell. The pSYSA copy number is positively correlated with the expression level of the endoribonuclease E. As molecular basis for this correlation we identified the RNase E-mediated cleavage within the pSYSA-encoded ssr7036 transcript. Together with a cis-encoded abundant antisense RNA (asRNA1), this mechanism resembles the control of ColE1-type plasmid replication by two overlapping RNAs, RNA I and II. In the ColE1 mechanism, two non-coding RNAs interact, supported by the small protein Rop, which is encoded separately. In contrast, in pSYSA the similar-sized protein Ssr7036 is encoded within one of the interacting RNAs and it is this mRNA that likely primes pSYSA replication. Essential for plasmid replication is furthermore the downstream encoded protein Slr7037 featuring primase and helicase domains. Deletion of slr7037 led to the integration of pSYSA into the chromosome or the other large plasmid pSYSX. Moreover, the presence of slr7037 was required for successful replication of a pSYSA-derived vector in another model cyanobacterium, Synechococcus elongatus PCC 7942. Therefore, we annotated the protein encoded by slr7037 as Cyanobacterial Rep protein A1 (CyRepA1). Our findings open new perspectives on the development of shuttle vectors for genetic engineering of cyanobacteria and of modulating the activity of the entire CRISPR-Cas apparatus in Synechocystis sp. PCC 6803., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kaltenbrunner, Reimann, Hoffmann, Aoyagi, Sakata, Nimura-Matsune, Watanabe, Steglich, Wilde and Hess.)

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in Gram-negative bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as Escherichia coli , feedback-regulate RNase E expression via the rne 5'-untranslated region (5' UTR) in cis . However, the mechanisms involved in the control of RNase E in other bacteria largely remain unknown. Cyanobacteria rely on solar light as an energy source for photosynthesis, despite the inherent ultraviolet (UV) irradiation. In this study, we first investigated globally the changes in gene expression in the cyanobacterium Synechocystis sp. PCC 6803 after a brief exposure to UV. Among the 407 responding genes 2 h after UV exposure was a prominent upregulation of rne  mRNA level. Moreover, the enzymatic activity of RNase E rapidly increased as well, although the protein stability decreased. This unique response was underpinned by the increased accumulation of full-length rne mRNA caused by the stabilization of its 5' UTR and suppression of premature transcriptional termination, but not by an increased transcription rate. Mapping of RNA 3' ends and in vitro cleavage assays revealed that RNase E cleaves within a stretch of six consecutive uridine residues within the rne 5' UTR, indicating autoregulation. These observations suggest that RNase E in cyanobacteria contributes to reshaping the transcriptome during the UV stress response and that its required activity level is secured at the RNA level despite the enhanced turnover of the protein., Competing Interests: The authors declare no conflict of interests., (© 2023 The Authors. mLife published by John Wiley & Sons Australia, Ltd. on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

Motivation: The CRISPR-Cas9 system is a Type II CRISPR system that has rapidly become the most versatile and widespread tool for genome engineering. It consists of two components, the Cas9 effector protein, and a single guide RNA that combines the spacer (for identifying the target) with the tracrRNA, a trans-activating small RNA required for both crRNA maturation and interference. While there are well-established methods for screening Cas effector proteins and CRISPR arrays, the detection of tracrRNA remains the bottleneck in detecting Class 2 CRISPR systems., Results: We introduce a new pipeline CRISPRtracrRNA for screening and evaluation of tracrRNA candidates in genomes. This pipeline combines evidence from different components of the Cas9-sgRNA complex. The core is a newly developed structural model via covariance models from a sequence-structure alignment of experimentally validated tracrRNAs. As additional evidence, we determine the terminator signal (required for the tracrRNA transcription) and the RNA-RNA interaction between the CRISPR array repeat and the 5'-part of the tracrRNA. Repeats are detected via an ML-based approach (CRISPRidenify). Providing further evidence, we detect the cassette containing the Cas9 (Type II CRISPR systems) and Cas12 (Type V CRISPR systems) effector protein. Our tool is the first for detecting tracrRNA for Type V systems., Availability and Implementation: The implementation of the CRISPRtracrRNA is available on GitHub upon requesting the access permission, (https://github.com/BackofenLab/CRISPRtracrRNA). Data generated in this study can be obtained upon request to the corresponding person: Rolf Backofen (backofen@informatik.uni-freiburg.de)., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

The FoF1 ATP synthase (ATPase) is one of the most important protein complexes in energy metabolism. The isolation of functional ATPase complexes is fundamental to address questions about its assembly, regulation, and functions. This protocol describes the purification of intact and active ATPase from the model cyanobacterium Synechocystis sp. PCC 6803. Basis for purification is a 3×FLAG tag fused to the beta subunit. The ATPase is enzymatically active and its purity is demonstrated using mass spectrometry, denaturing, and blue-native PAGE. For complete details on the use and execution of this protocol, please refer to Song et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Oxygenic photosynthesis evolved in cyanobacteria, primary producers of striking ecological importance. Like plants, cyanobacteria use the Calvin-Benson-Bassham cycle for CO 2 fixation, fuelled by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). In a competitive reaction this enzyme also fixes O 2 which makes it rather ineffective. To mitigate this problem, cyanobacteria evolved a CO 2 concentrating mechanism (CCM) to pool CO 2 in the vicinity of RuBisCO. However, the regulation of these carbon (C) assimilatory systems is understood only partially. Using the model Synechocystis sp. PCC 6803 we characterized an essential LysR-type transcriptional regulator encoded by gene sll0998. Transcript profiling of a knockdown mutant revealed diminished expression of several genes involved in C acquisition, including rbcLXS, sbtA and ccmKL encoding RuBisCO and parts of the CCM, respectively. We demonstrate that the Sll0998 protein binds the rbcL promoter and acts as a RuBisCO regulator (RbcR). We propose ATTA(G/A)-N 5 -(C/T)TAAT as the binding motif consensus. Our data validate RbcR as a regulator of inorganic C assimilation and define the regulon controlled by it. Biological CO 2 fixation can sustain efforts to reduce its atmospheric concentrations and is fundamental for the light-driven production of chemicals directly from CO 2 . Information about the involved regulatory and physiological processes is crucial to engineer cyanobacterial cell factories., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

F o F 1 ATP synthases produce ATP, the universal biological energy source. ATP synthase complexes on cyanobacterial thylakoid membranes use proton gradients generated either by photosynthesis or respiration. AtpΘ is an ATP synthase regulator in cyanobacteria which is encoded by the gene atpT . AtpΘ prevents the hydrolysis of ATP (reverse reaction) that otherwise would occur under unfavorable conditions. In the cyanobacterium Synechocystis sp. PCC 6803, AtpΘ is expressed maximum in darkness but at very low levels under optimum phototrophic growth conditions or in the presence of glucose. DNA coimmunoprecipitation experiments followed by mass spectrometry identified the binding of the two transcriptional regulators cyAbrB1 and cyAbrB2 to the promoter and the histone-like protein HU to the 5'UTR of atpT. Analyses of nucleotide substitutions in the promoter and GFP reporter assays identified a functionally relevant sequence motif resembling the HLR1 element bound by the RpaB transcription factor. Electrophoretic mobility shift assays confirmed interaction of cyAbrB1, cyAbrB2, and RpaB with the promoter DNA. However, overall the effect of transcriptional regulation was comparatively low. In contrast, atpT transcript stabilities differed dramatically, half-lives were 1.6 min in the light, 33 min in the dark and substantial changes were observed if glucose or DCMU were added. These findings show that transcriptional control of atpT involves nucleoid-associated DNA-binding proteins, positive regulation through RpaB, while the major effect on the condition-dependent regulation of atpT expression is mediated by controlling mRNA stability, which is related to the cellular redox and energy status. IMPORTANCE F o F 1 ATP synthases produce ATP, the universal biological energy source. Under unfavorable conditions, ATP synthases can operate in a futile reverse reaction, pumping protons while ATP is used up. Cyanobacteria perform plant-like photosynthesis, but they cannot use the same mechanism as plant chloroplasts to inhibit ATP synthases during the night because respiratory and photosynthetic complexes are both located in the same membrane system. AtpΘ is a small protein encoded by the gene atpT in cyanobacteria that can prevent the ATP synthase reverse reaction (ATPase activity). Here we found that three transcription factors contribute to the regulation of atpT expression. However, the control of mRNA stability was identified as the major regulatory process governing atpT expression. Thus, it is the interplay between transcriptional and posttranscriptional regulation that position the AtpΘ-based regulatory mechanism within the context of the cellular redox and energy balance.

The amount of inorganic carbon (C i ) fluctuates in aquatic environments. Cyanobacteria evolved a C i -concentrating mechanism (CCM) that is regulated at different levels. The regulator SbtB binds to the second messengers cAMP or c-di-AMP and is involved in acclimation to low C i (LC) in Synechocystis sp. PCC 6803. Here, we investigated the role of SbtB and of associated second messengers at different C i conditions. The transcriptome of wild-type (WT) Synechocystis and the ΔsbtB mutant were compared with Δcya1, a mutant defective in cAMP production, and ΔdacA, a mutant defective in generating c-di-AMP. A defined subset of LC-regulated genes in the WT was already changed in ΔsbtB under high C i (HC) conditions. This response of ΔsbtB correlated with a diminished induction of many CCM-associated genes after LC shift in this mutant. The Δcya1 mutant showed less deviation from WT, whereas ΔdacA induced CCM-associated genes under HC. Metabolome analysis also revealed differences between the strains, whereby ΔsbtB showed slower accumulation of 2-phosphoglycolate and ΔdacA differences among amino acids compared to WT. Collectively, these results indicate that SbtB regulates a subset of LC acclimation genes while c-di-AMP and especially cAMP appear to have a lesser impact on gene expression under different C i availabilities., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

RNA turnover plays critical roles in the regulation of gene expression and allows cells to respond rapidly to environmental changes. In bacteria, the mechanisms of RNA turnover have been extensively studied in the models Escherichia coli and Bacillus subtilis , but not much is known in other bacteria. Cyanobacteria are a diverse group of photosynthetic organisms that have great potential for the sustainable production of valuable products using CO 2 and solar energy. A better understanding of the regulation of RNA decay is important for both basic and applied studies of cyanobacteria. Genomic analysis shows that cyanobacteria have more than 10 ribonucleases and related proteins in common with E. coli and B. subtilis, and only a limited number of them have been experimentally investigated. In this review, we summarize the current knowledge about these RNA-turnover-related proteins in cyanobacteria. Although many of them are biochemically similar to their counterparts in E. coli and B. subtilis, they appear to have distinct cellular functions, suggesting a different mechanism of RNA turnover regulation in cyanobacteria. The identification of new players involved in the regulation of RNA turnover and the elucidation of their biological functions are among the future challenges in this field., (© 2022 The Authors. mLife published by John Wiley & Sons Australia, Ltd. on behalf of Institute of Microbiology, Chinese Academy of Sciences.)

Biological processes in all living cells are powered by ATP, a nearly universal molecule of energy transfer. ATP synthases produce ATP utilizing proton gradients that are usually generated by either respiration or photosynthesis. However, cyanobacteria are unique in combining photosynthetic and respiratory electron transport chains in the same membrane system, the thylakoids. How cyanobacteria prevent the futile reverse operation of ATP synthase under unfavorable conditions pumping protons while hydrolyzing ATP is mostly unclear. Here, we provide evidence that the small protein AtpΘ, which is widely conserved in cyanobacteria, is mainly fulfilling this task. The expression of AtpΘ becomes induced under conditions such as darkness or heat shock, which can lead to a weakening of the proton gradient. Translational fusions of AtpΘ to the green fluorescent protein revealed targeting to the thylakoid membrane. Immunoprecipitation assays followed by mass spectrometry and far western blots identified subunits of ATP synthase as interacting partners of AtpΘ. ATP hydrolysis assays with isolated membrane fractions, as well as purified ATP synthase complexes, demonstrated that AtpΘ inhibits ATPase activity in a dose-dependent manner similar to the F 0 F 1 -ATP synthase inhibitor N,N-dicyclohexylcarbodimide. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration and indicate that ATP synthase activity can be controlled in surprisingly different ways., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

As the only oxygenic phototrophs among prokaryotes, cyanobacteria employ intricate mechanisms to regulate common metabolic pathways. These mechanisms include small protein inhibitors exerting their function by protein-protein interaction with key metabolic enzymes and regulatory small RNAs (sRNAs). Here we show that the sRNA NsiR4, which is highly expressed under nitrogen limiting conditions, interacts with the mRNA of the recently described small protein PirA in the model strain Synechocystis sp. PCC 6803. In particular, NsiR4 targets the pirA 5'UTR close to the ribosome binding site. Heterologous reporter assays confirmed that this interaction interferes with pirA translation. PirA negatively impacts arginine synthesis under ammonium excess by competing with the central carbon/nitrogen regulator P II that binds to and thereby activates the key enzyme of arginine synthesis, N-acetyl-L-glutamate-kinase (NAGK). Consistently, ectopic nsiR4 expression in Synechocystis resulted in lowered PirA accumulation in response to ammonium upshifts, which also affected intracellular arginine pools. As NsiR4 and PirA are inversely regulated by the global nitrogen transcriptional regulator NtcA, this regulatory axis enables fine tuning of arginine synthesis and conveys additional metabolic flexibility under highly fluctuating nitrogen regimes. Pairs of small protein inhibitors and of sRNAs that control the abundance of these enzyme effectors at the post-transcriptional level appear as fundamental building blocks in the regulation of primary metabolism in cyanobacteria.

Ribonucleases are crucial enzymes in RNA metabolism and post-transcriptional regulatory processes in bacteria. Cyanobacteria encode the two essential ribonucleases RNase E and RNase J. Cyanobacterial RNase E is shorter than homologues in other groups of bacteria and lacks both the chloroplast-specific N-terminal extension as well as the C-terminal domain typical for RNase E of enterobacteria. In order to investigate the function of RNase E in the model cyanobacterium Synechocystis sp. PCC 6803, we engineered a temperature-sensitive RNase E mutant by introducing two site-specific mutations, I65F and the spontaneously occurred V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1472 RNase-E-dependent cleavage sites. We inferred a dominating cleavage signature consisting of an adenine at the -3 and a uridine at the +2 position within a single-stranded segment of the RNA. The data identified mRNAs likely regulated jointly by RNase E and an sRNA and potential 3' end-derived sRNAs. Our findings substantiate the pivotal role of RNase E in post-transcriptional regulation and suggest the redundant or concerted action of RNase E and RNase J in cyanobacteria., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation., (© 2021. The Author(s).)

The Ferric uptake regulator (Fur) is crucial to both pathogenic and non-pathogenic bacteria for the maintenance of iron homeostasis as well as the defence against reactive oxygen species. Based on datasets from the genome-wide mapping of transcriptional start sites and transcriptome data, we identified a high confidence regulon controlled by Fur for the model cyanobacterium Synechocystis sp. PCC 6803 and its close relative, strain 6714, based on the conserved strong iron starvation response and Fur-binding site occurrence. This regulon comprises 33 protein-coding genes and the sRNA IsaR1 that are under the control of 16 or 14 individual promoters in strains 6803 and 6714, respectively. The associated gene functions are mostly restricted to transporters and enzymes involved in the uptake and storage of iron ions, with few exceptions or unknown functional relevance. Within the isiABC operon, we identified a previously neglected gene encoding a small cysteine-rich protein, which we suggest calling, IsiE. The regulation of iron uptake, storage, and utilization ultimately results from the interplay between the Fur regulon, several other transcription factors, the FtsH3 protease, and the sRNA IsaR1., (© The Author(s) 2021. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Although regulatory small RNAs have been reported in photosynthetic cyanobacteria, the lack of clear RNA chaperones involved in their regulation poses a conundrum. Here, we analyzed the full complement of cellular RNAs and proteins using gradient profiling by sequencing (Grad-seq) in Synechocystis 6803. Complexes with overlapping subunits such as the CpcG1-type versus the CpcL-type phycobilisomes or the PsaK1 versus PsaK2 photosystem I pre(complexes) could be distinguished, supporting the high quality of this approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include an YlxR homolog and a cyanobacterial homolog of the KhpA/B complex. The data suggest previously undetected complexes between accessory proteins and CRISPR-Cas systems, such as a Csx1-Csm6 ribonucleolytic defense complex. Moreover, the exclusive association of either RpoZ or 6S RNA with the core RNA polymerase complex and the existence of a reservoir of inactive sigma-antisigma complexes is suggested. The Synechocystis Grad-seq resource is available online at https://sunshine.biologie.uni-freiburg.de/GradSeqExplorer/ providing a comprehensive resource for the functional assignment of RNA-protein complexes and multisubunit protein complexes in a photosynthetic organism., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Phycobilisomes (PBSs), the principal cyanobacterial antenna, are among the most efficient macromolecular structures in nature, and are used for both light harvesting and directed energy transfer to the photosynthetic reaction center. However, under unfavorable conditions, excess excitation energy needs to be rapidly dissipated to avoid photodamage. The orange carotenoid protein (OCP) senses light intensity and induces thermal energy dissipation under stress conditions. Hence, its expression must be tightly controlled; however, the molecular mechanism of this regulation remains to be elucidated. Here, we describe the discovery of a posttranscriptional regulatory mechanism in Synechocystis sp. PCC 6803 in which the expression of the operon encoding the allophycocyanin subunits of the PBS is directly and in an inverse fashion linked to the expression of OCP. This regulation is mediated by ApcZ, a small regulatory RNA that is derived from the 3'-end of the tetracistronic apcABC-apcZ operon. ApcZ inhibits ocp translation under stress-free conditions. Under most stress conditions, apc operon transcription decreases and ocp translation increases. Thus, a key operon involved in the collection of light energy is functionally connected to the expression of a protein involved in energy dissipation. Our findings support the view that regulatory RNA networks in bacteria evolve through the functionalization of mRNA 3'-UTRs., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

NsiR3 (nitrogen stress-inducible RNA 3) is a small noncoding RNA strongly conserved in heterocyst-forming cyanobacteria. In Nostoc sp. PCC 7120, transcription of NsiR3 is induced by nitrogen starvation and depends on the global nitrogen regulator NtcA. A conserved NtcA-binding site is centered around position -42.5 with respect to the transcription start site of NsiR3 homologs, and NtcA binds in vitro to a DNA fragment containing this sequence. In the absence of combined nitrogen, NsiR3 expression is induced in all cells along the Nostoc filament but much more strongly in heterocysts, differentiated cells devoted to nitrogen fixation. Co-expression analysis of transcriptomic data obtained from microarrays hybridized with RNA obtained from Nostoc wild-type or mutant strains grown in the presence of ammonium or in the absence of combined nitrogen revealed that the expression profile of gene putA (proline oxidase) correlates negatively with that of NsiR3. Using a heterologous system in Escherichia coli, we show that NsiR3 binds to the 5'-UTR of putA mRNA, resulting in reduced expression of a reporter gene. Overexpression of NsiR3 in Nostoc resulted in strong reduction of putA mRNA accumulation, further supporting the negative regulation of putA by NsiR3. The higher expression of NsiR3 in heterocysts versus vegetative cells of the N 2 -fixing filament could contribute to the previously described absence of putA mRNA and of the catabolic pathway to produce glutamate from arginine via proline specifically in heterocysts. Post-transcriptional regulation by NsiR3 represents an indirect NtcA-operated regulatory mechanism of putA expression. DATABASE: Microarray data are available in GEO database under accession numbers GSE120377 and GSE150191., (© 2020 Federation of European Biochemical Societies.)

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte, and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded following an intricate genetic program. Here, we describe the role of NblD, a cysteine-rich, small protein in this process in cyanobacteria. Deletion of the nblD gene in the cyanobacterium Synechocystis sp. PCC 6803 prevented the degradation of phycobilisomes, leading to a nonbleaching ( nbl ) phenotype, which could be complemented by a plasmid-localized gene copy. Competitive growth experiments between the Δ nblD and the wild-type strain provided direct evidence for the physiological importance of NblD under nitrogen-limited conditions. Ectopic expression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation. Transcriptome analysis indicated increased nblA1/2 transcript levels in the Δ nblD strain during nitrogen starvation, implying that NblD does not act as a transcriptional (co)regulator. However, immunoprecipitation and far-western experiments identified the chromophorylated (holo form) of the phycocyanin β-subunit (CpcB) as its target, while apo-CpcB was not bound. The addition of recombinant NblD to isolated phycobilisomes caused a reduction in phycocyanin absorbance and a broadening and shifting of the peak to lower wavelengths, indicating the occurrence of structural changes. These data demonstrate that NblD plays a crucial role in the coordinated dismantling of phycobilisomes and add it as a factor to the genetically programmed response to nitrogen starvation., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Small proteins are critically involved in the acclimation response of photosynthetic cyanobacteria to nitrogen starvation. NblD is the 66-amino-acid effector of nitrogen-limitation-induced phycobilisome breakdown, which is believed to replenish the cellular amino acid pools. To address the physiological functions of NblD, the concentrations of amino acids, intermediates of the arginine catabolism pathway and several organic acids were measured during the response to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 wild type and in an nblD deletion strain. A characteristic signature of metabolite pool composition was identified, which shows that NblD-mediated phycobilisome degradation is required to maintain the cellular amino acid and organic acid pools during nitrogen starvation. Specific deviations from the wild type suggest wider-reaching effects that also affect such processes as redox homeostasis via glutathione and tetrapyrrole biosynthesis, both of which are linked to the strongly decreased glutamate pool, and transcriptional reprogramming via an enhanced concentration of 2-oxoglutarate, the metabolite co-regulator of the NtcA transcription factor. The essential role played by NblD in metabolic homeostasis is consistent with the widespread occurrence of NblD throughout the cyanobacterial radiation and the previously observed strong positive selection for the nblD gene under fluctuating nitrogen supply. Importance Cyanobacteria play important roles in the global carbon and nitrogen cycles. In their natural environment, these organisms are exposed to fluctuating nutrient conditions. Nitrogen starvation induces a coordinated nitrogen-saving program that includes the breakdown of nitrogen-rich photosynthetic pigments, particularly phycobiliproteins. The small protein NblD was recently identified as an effector of phycobilisome breakdown in cyanobacteria. In this study, we demonstrate that the NblD-mediated degradation of phycobiliproteins is needed to sustain cellular pools of soluble amino acids and other crucial metabolites. The essential role played by NblD in metabolic homeostasis explains why genes encoding this small protein are conserved in almost all members of cyanobacterial radiation.

Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo . We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavour other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighbouring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

Motivation: The correct prediction of bacterial sRNA homologs is a prerequisite for many downstream analyses based on comparative genomics, but it is frequently challenging due to the short length and distinct heterogeneity of such homologs. GLobal Automatic Small RNA Search go (GLASSgo) is an efficient tool for the prediction of sRNA homologs from a single input query. To make the algorithm available to a broader community, we offer a Docker container along with a free-access web service. For non-computer scientists, the web service provides a user-friendly interface. However, capabilities were lacking so far for batch processing, version control and direct interaction with compatible software applications as a workflow management system can provide., Results: Here, we present GLASSgo 1.5.2, an updated version that is fully incorporated into the workflow management system Galaxy. The improved version contains a new feature for extracting the upstream regions, allowing the search for conserved promoter elements. Additionally, it supports the use of accession numbers instead of the outdated GI numbers, which widens the applicability of the tool., Availability and Implementation: GLASSgo is available at https://github.com/lotts/GLASSgo/ under the MIT license and is accompanied by instruction and application data. Furthermore, it can be installed into any Galaxy instance using the Galaxy ToolShed., (© The Author(s) 2020. Published by Oxford University Press.)

Small regulatory RNAs and antisense RNAs play important roles in the regulation of gene expression in bacteria but are underexplored, especially in natural populations. While environmentally relevant microbes often are not amenable to genetic manipulation or cannot be cultivated in the laboratory, extensive metagenomic and metatranscriptomic datasets for these organisms might be available. Hence, dedicated workflows for specific analyses are needed to fully benefit from this information. Here, we identified abundant sRNAs from oceanic environmental populations of the ecologically important primary producer Prochlorococcus starting from a metatranscriptomic differential RNA-Seq (mdRNA-Seq) dataset. We tracked their homologs in laboratory isolates, and we provide a framework for their further detailed characterization. Several of the experimentally validated sRNAs responded to ecologically relevant changes in cultivation conditions. The expression of the here newly discovered sRNA Yfr28 was highly stimulated in low-nitrogen conditions. Its predicted top targets include mRNAs encoding cell division proteins, a sigma factor, and several enzymes and transporters, suggesting a pivotal role of Yfr28 in the coordination of primary metabolism and cell division. A cis-encoded antisense RNA was identified as a possible positive regulator of atpF encoding subunit b' of the ATP synthase complex. The presented workflow will also be useful for other environmentally relevant microorganisms for which experimental validation abilities are frequently limiting although there is wealth of sequence information available.

The introduction of alternative CO 2 -fixing pathways in photoautotrophic organism may improve the efficiency of biological carbon fixation such as minimizing the carbon loss due to photorespiration. Here, we analyzed the effects of creating a formate entry point into the primary metabolism of the cyanobacterium Synechocystis sp. PCC 6803. The formate-tetrahydrofolate ligase (FTL) from Methylobacterium extorquens AM1 was expressed in Synechocystis to enable formate assimilation and reducing the loss of fixed carbon in the photorespiratory pathway. Transgenic strains accumulated serine and 3-phosphoglycerate, and consumed more 2-phosphoglycolate and glycine, which seemed to reflect an efficient utilization of formate. However, labeling experiments showed that the serine accumulation was not due to the expected incorporation of formate. Subsequent DNA-microarray analysis revealed profound changes in transcript abundance due to ftl expression. Transcriptome changes were observed in relation to serine and glycine metabolism, C1-metabolism and particularly nitrogen assimilation. The data implied that ftl expression interfered with the signaling the carbon/nitrogen ratio in Synechocystis . Our results indicate that the expression of new enzymes could have a severe impact on the cellular regulatory network, which potentially hinders the establishment of newly designed pathways., (Copyright © 2020 Song, Timm, Lindner, Reimann, Hess, Hagemann and Brouwer.)

Synechocystis sp. PCC 6803 is a widely used model cyanobacterium whose genome has been well annotated. However, several additional small protein coding sequences (sORFs) have been recently identified, which might play important roles, for example in the regulation of cellular metabolism. Here, we analyzed the function of a sORF encoding a 44 amino acid peptide showing high similarity to the N-terminal part of aconitase (AcnB). The expression of the gene, which probably originated from a partial gene duplication of chromosomal acnB into the plasmid pSYSA, was verified and it was designated as acnSP . The protein-coding part of acnSP was inactivated by interposon mutagenesis. The obtained mutant displayed slower growth under photoautotrophic conditions with light exceeding 100 μmol photons m -2 s -1 and showed significant changes in the metabolome compared to wild type, including alterations in many metabolites associated to the tricarboxylic acid (TCA) cycle. To analyze a possible direct impact of AcnSP on aconitase, the recombinant Synechocystis enzyme was generated and biochemically characterized. Biochemical analysis revealed that addition of equimolar amounts of AcnSP resulted in an improved substrate affinity (lower K m ) and lowered V max of aconitase. These results imply that AcnSP can regulate aconitase activity, thereby impacting the carbon flow into the oxidative branch of the cyanobacterial TCA cycle, which is mainly responsible for the synthesis of carbon skeletons needed for ammonia assimilation., (Copyright © 2020 de Alvarenga, Hess and Hagemann.)

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., (© 2020 Rosana et al.)

Trans-acting small regulatory RNAs (sRNAs) are key players in the regulation of gene expression in bacteria. There are hundreds of different sRNAs in a typical bacterium, which in contrast to eukaryotic microRNAs are more heterogeneous in length, sequence composition, and secondary structure. The vast majority of sRNAs function post-transcriptionally by binding to other RNAs (mRNAs, sRNAs) through rather short regions of imperfect sequence complementarity. Besides, every single sRNA may interact with dozens of different target RNAs and impact gene expression either negatively or positively. These facts contributed to the view that the entirety of the regulatory targets of a given sRNA, its targetome, is challenging to identify. However, recent developments show that a more comprehensive sRNAs targetome can be achieved through the combination of experimental and computational approaches. Here, we give a short introduction into these methods followed by a description of two sRNAs, RyhB, and RsaA, to illustrate the particular strengths and weaknesses of these approaches in more details. RyhB is an sRNA involved in iron homeostasis in Enterobacteriaceae, while RsaA is a modulator of virulence in Staphylococcus aureus. Using such a combined strategy, a better appreciation of the sRNA-dependent regulatory networks is now attainable., (© 2019 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Marine cyanobacteria of the genus Prochlorococcus thrive in high cell numbers throughout the euphotic zones of the world's subtropical and tropical oligotrophic oceans, making them some of the most ecologically relevant photosynthetic microorganisms on Earth. The ecological success of these free-living phototrophs suggests that they are equipped with a regulatory system competent to address many different stress situations. However, Prochlorococcus genomes are compact and streamlined, with the majority encoding only five different sigma factors, five to six two-component systems and eight types of other transcriptional regulators. Here, we summarize the existing information about the functions of these protein regulators, about transcriptomic responses to defined stress conditions, and discuss the current knowledge about riboswitches, RNA-based regulation and the roles of certain metabolites as co-regulators. We focus on the best-studied isolate, Prochlorococcus MED4, but extend to other strains and ecotypes when appropriate, and we include some information gained from metagenomic and metatranscriptomic analyses., (© FEMS 2020.)

Cyanobacteria are photosynthetic bacteria that populate widely different habitats. Accordingly, cyanobacteria exhibit a wide spectrum of lifestyles, physiologies, and morphologies and possess genome sizes and gene numbers which may vary by up to a factor of ten within the phylum. Consequently, large differences exist between individual species in the size and complexity of their regulatory networks. Several non-coding RNAs have been identified that play crucial roles in the acclimation responses of cyanobacteria to changes in the environment. Some of these regulatory RNAs are conserved throughout the cyanobacterial phylum, while others exist only in a few taxa. Here we give an overview on characterized regulatory RNAs in cyanobacteria, with a focus on regulators of photosynthesis, carbon and nitrogen metabolism. However, chances are high that these regulators represent just the tip of the iceberg., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The membrane-embedded FtsH proteases found in bacteria, chloroplasts, and mitochondria are involved in diverse cellular processes including protein quality control and regulation. The genome of the model cyanobacterium Synechocystis sp PCC 6803 encodes four FtsH homologs designated FtsH1 to FtsH4. The FtsH3 homolog is present in two hetero-oligomeric complexes: FtsH2/3, which is responsible for photosystem II quality control, and the essential FtsH1/3 complex, which helps maintain Fe homeostasis by regulating the level of the transcription factor Fur. To gain a more comprehensive insight into the physiological roles of FtsH hetero-complexes, we performed genome-wide expression profiling and global proteomic analyses of Synechocystis mutants conditionally depleted of FtsH3 or FtsH1 grown under various nutrient conditions. We show that the lack of FtsH1/3 leads to a drastic reduction in the transcriptional response to nutrient stress of not only Fur but also the Pho, NdhR, and NtcA regulons. In addition, this effect is accompanied by the accumulation of the respective transcription factors. Thus, the FtsH1/3 complex is of critical importance for acclimation to iron, phosphate, carbon, and nitrogen starvation in Synechocystis. plantcell;31/12/2912/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

Cyanobacteria are a group of photosynthetic microorganisms with high commercial potential. They can utilize sunlight directly to convert carbon dioxide or even nitrogen into a variety of industrially relevant chemicals. However, commercial platforms for the renewable and sustainable production of chemicals have yet to be demonstrated for cyanobacteria. Diverse strategies have therefore been employed in recent years to improve the production yields and efficiency of target chemicals. These include the use of CRISPR/Cas systems for mutant selection, synthetic RNA elements for controlling transcription, metabolic network modelling for understanding pathway fluxes, enzyme engineering, improving growth rates, alleviating product toxicity and microbial consortia. More elaborate strategies for engineering cyanobacteria, however, are still very much required if we are to meet the grand challenge of employing cyanobacteria as photosynthetic workhorses for large-scale industrial applications., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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 .

Background: The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality., Results: The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated ( 5m CG 6m ATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5 CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5m C methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5 CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GG m4 CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system., Conclusions: The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5m C methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5m C methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5 CGATCG DNA-methylation marks in conjugation and DNA transformation.

DNA methylation plays a crucial role for gene regulation among eukaryotes, but its regulatory function is less documented in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803 five DNA methyltransferases have been identified. Among them, M.Ssp6803II is responsible for the specific methylation of the first cytosine in the frequently occurring motif GGCC, leading to N4-methylcytosine (GG m4 CC). The mutation of the corresponding gene sll0729 led to lowered chlorophyll/phycocyanin ratio and slower growth. Transcriptomics only showed altered expression of sll0470 and sll1526 , two genes encoding hypothetical proteins. Moreover, prolonged cultivation revealed instability of the initially obtained phenotype. Colonies with normal pigmentation and wild-type-like growth regularly appeared on agar plates. These colonies represent suppressor mutants, because the sll0729 gene was still completely inactivated and the GGCC sites remained unmethylated. The suppressor strains showed smaller cell size, lowered DNA content per cell, and decreased tolerance against UV compared to wild type. Promoter assays revealed that the transcription of the sll0470 gene was still stimulated in the suppressor clones. Proteomics identified decreased levels of DNA topoisomerase 4 subunit A in suppressor cells. Collectively, these results indicate that GG m4 CC methylation is involved in the regulation of gene expression, in the fine-tuning of DNA replication, and DNA repair mechanisms.