Biology subjects

Reimann, V.

Publications and source records attributed to Reimann, V..

10 recordsLinked to original sources

bioRxiv2026

The relevance of GGCC methylation for cyanobacterial gene expression regulation

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In the Synechocystis 6803 genome, the DNA-specific methyltransferase M.Ssp6803II modifies the first cytosine in the 38,512 GGCC motifs, forming N4-methylcytosine (GGm4CC). Previous analyses showed that deletion of the gene encoding M.Ssp6803II ({triangleup}sll0729), caused a bluish phenotype due to reduced expression of the hemJ gene. This effect was caused by the lacking GGCC methylation in the discriminator sequence of the hemJ promoter in {triangleup}sll0729. In the present study, we analyzed the impact of GGm4CC methylation on the overall gene expression. Bioinformatic analyses of multiple, independently obtained DNA microarray datasets revealed reproducible changes in the expression of more than 300 genes. We selected 26 genes that had the GGCC methylation motif in different locations of their promoter sequences and verified their differential expression through qPCR experiments. Classification of the promoters according to the relative position of GGCC motifs suggested context-specific effects on gene expression. These findings imply that GGm4CC methylation via DNA methyltransferase M.Ssp6803II has an important impact on proper gene expression and thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely many other bacteria.

microbiology↗

bioRxiv2026

Deletion of the gene for a cyanobacterial ribosome-associated protein affects the carbon/nitrogen metabolism

In contrast to their important structural and regulatory functions, such as in the metabolism of cyanobacteria, genes encoding small proteins are often not well characterized. Cyanobacteria use redox equivalents and energy from oxygenic photosynthesis to produce organic carbon compounds from inorganic carbon (Ci) and organic nitrogen compounds from inorganic nitrogen sources. Therefore, the assimilation and metabolism of carbon and nitrogen are coordinated at multiple levels in cyanobacteria. Here, we analyzed the Synechocystis sp. PCC 6803 gene ssr3189 encoding a 55 amino acids protein. Orthologs were detected in 665 cyanobacterial genomes defining COG5794 in the Database of Clusters of Orthologous Genes. Homologs in several eukaryotic algae suggest that Ssr3189 is an important protein that originated in cyanobacteria, was retained in algae after endosymbiosis, but was lost in plants. Polynucleotide kinase assays validated Ssr3189 as an RNA-binding protein. Deletion of ssr3189 resulted in lower pigmentation, delayed growth, and alterations in the expression of genes encoding transporters for nitrogen and Ci, and metabolic enzymes. Metabolomic analysis revealed a substantial overaccumulation of glutamine and tricarboxylic acid cycle intermediates in the deletion mutant, and further differences in the amino acid and organic acid pools compared to the wild type. Co-immunoprecipitation analysis yielded ribosomal protein S21, enolase and the Cas6-1 endoribonuclease as the most strongly co-enriched proteins, together with all other ribosomal proteins and a small set of metabolic enzymes. These findings are consistent with observations that ssr3189 encodes the ribosome-associated protein cS24 and suggest that it connects translation with metabolic control, and, potentially, RNA decay. IMPACT STATEMENTDespite considerable progress in analyzing microbial genomes, there are still substantial numbers of uncharacterized gene functions. Here, we analyzed a mutant lacking gene ssr3189 that is widely conserved, but phenotypically uncharacterized in cyanobacteria. This gene is important for growth at the optimum temperature and essential at lower temperatures. In its absence, important metabolites were overaccumulated, while genes involved in nitrogen and Ci uptake were dysregulated. The encoded protein binds RNA and interacts with proteins involved in translation and metabolism. The findings are consistent with a function as a ribosomal protein bridging protein synthesis and the regulation of metabolism.

microbiology↗

bioRxiv2026

Genomic Isomerism and Environmental Adaptation in Cyanophages Infecting Freshwater Cyanobacteria

Cyanophages infecting multicellular, heterocyst-forming cyanobacteria play diverse pivotal roles in freshwater ecosystems, yet their infection strategies and genomic adaptability remain poorly understood. Here, we isolated and characterized three Caudoviricetes cyanophages (A-Lf14, A-Alj1 and A-Hlh1) specifically infecting the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120. Super-resolution microscopy revealed heterogeneous infection outcomes even among adjacent cells within the same filament, highlighting host-level defense variability. Comparative genomics placed these phages within a cluster that includes the previously isolated phages A-1(L) and N1, and revealed a conserved [~]30 kb invertible genomic region flanked by inverted repeats (IR). This region exists as two stable isomers simultaneously within phage populations, a previously unreported genomic plasticity trait in cyanophages. Consistent with such plasticity, infection kinetics were modulated by light and nitrogen availability, indicating a resource-responsive infection strategy. The novel phages encode an alkaline phosphatase of non-cyanobacterial origin, which was highly upregulated during infection indicating a role in phosphate acquisition in phosphate-limited waters. In contrast, they lack a tnpB gene present in A-1(L), which is identical in sequence to five genes in the host genome. We detected protein-coding potential for both strands of tnpB and upregulated transcription during infection, consistent with a role in this process. The 347 residues protein encoded on the tnpB reverse strand exhibited only limited similarity to other proteins or folding potential, underlining its novelty. Our work illustrates how genomic isomerism, accessory genes, and environmental sensing collectively drive functional diversification in cyanophages, providing insights into phage-host coevolution and the impact of phages on cyanobacterial blooms in dynamic freshwater ecosystems.

microbiology↗

bioRxiv2025

A revised genome annotation of the model cyanobacterium Synechocystis based on start and stop codon-enriched ribosome profiling and proteogenomics

Cyanobacteria are important primary producers and are used as microbial cell factories due to their ability to use solar light for oxygenic photosynthesis. Synechocystis sp. PCC 6803 is a popular model cyanobacterium, yet there are ambiguities in the precise coding regions of many genes, and numerous genes encoding small proteins have remained undetected. Here we present the results of a riboproteogenomic analysis, combining ribosome profiling (Ribo-seq) analysis involving inhibitors that stall ribosomes at translation initiation and termination sites (TIS- and TTS-Ribo-seq), with a proteogenomic reevaluation and reannotation of its entire genome. We report evidence for the translation of 3,055 annotated genes based on proteogenomics (83%), of 3,492 based on Ribo-seq (95.2%), and of 3,018 supported by both methods (82%). The data suggested unannotated protein-coding genes and corrections for annotated ones. We validated 15 small proteins translated from antisense RNAs, from intergenic and intragenic regions and provide proteogenomic support for up to 69 further, mostly small proteins. With slr0489, slr1079 and slr1082 we identified three genes with intragenic out-of-frame translons and show that both the internal and host reading frames are translated and that the resulting proteins interact with each other. Our data can be accessed via an intuitive and interactive genome browser platform at https://www.bioinf.uni-freiburg.de/[~]ribobase/. They illustrate the enormous value of consolidating genome annotations in the context of integrated experimental data.

microbiology↗

bioRxiv2024

RAPDOR: Using Jensen-Shannon Distance for the computational analysis of complex proteomics datasets

The computational analysis of large proteomics datasets, such as those from gradient profiling or spatially resolved proteomics, is often as crucial as the experimental design. We present RAPDOR, a tool for intuitive analyzing and visualizing such datasets, based on the Jensen-Shannon distance and subsequent analysis of similarities between replicates, applied to three datasets. First, we examined the in-gradient distribution profiles of protein complexes with or without RNase treatment (GradR) to identify the set of RNA-binding proteins (RBPs) in the cyanobacterium Synechocystis sp. PCC 6803. RBPs play pivotal regulatory and structural roles; although numerous RBPs have been identified, the complete set is unknown for any species. RAPDOR identified 80 potential RBPs, including ribosomal proteins, likely RNA-modifying enzymes, and several proteins not previously associated with RNA binding. High-ranking putative RBPs, such as the universal stress protein Sll1388, or the translation inhibitor LrtA/RaiA, were predicted by RAPDOR but not the TriPepSVM algorithm, indicating uncharacterized RBP domains. These data are available online at https://synecho-rapdor.biologie.uni-freiburg.de, providing a comprehensive resource for RNase-sensitive protein complexes in cyanobacteria. We then show by reanalyzing existing datasets, that RAPDOR is effective in examining the intracellular redistribution of proteins under stress conditions. RAPDOR is a generic, non-parametric tool for the intuitive and versatile analysis of highly complex data sets such as the study of protein distributions using fractionation protocols.

bioinformatics↗

bioRxiv2024

The RRM domain-containing protein Rbp3 interacts with ribosomes and the 3' ends of mRNAs encoding photosynthesis proteins

RNA recognition motif (RRM) domain proteins are crucial RNA-binding proteins (RBPs) across all domains of life. In cyanobacteria, single RRM domain proteins are involved in mRNA targeting to the thylakoid membrane and acclimation to certain stress conditions, but many details of their physiological functions and molecular targets have remained unknown. The model cyanobacterium Synechocystis sp. PCC 6803 has a family of three genes encoding the RRM domain-containing proteins Rbp1, Rbp2 and Rbp3. Here, we verified the RNA-binding activity of Rbp3 in vivo and show that cells of a {Delta}rbp3 deletion strain had a lower PSI:PSII ratio and decreased pigment content and were significantly smaller than wild-type cells. To identify the set of interacting molecules, co-immunoprecipitation experiments were performed with a strain expressing a C-terminally FLAG-tagged Rbp3. Mass spectrometry of the elution fraction suggested physical proximity between Rbp3, ribosomes, and a very small number of other proteins. The most highly enriched transcript in the co-eluting RNA fraction was the psaAB mRNA. This was corroborated by fluorescent in situ hybridization (FISH) analyses showing decreased psaA mRNA signals in {Delta}rbp3, and colocalization with Rbp3-GFP in the wild type. Other mRNAs co-enriched with Rbp3 encode thylakoid, plasma membrane and carboxysome proteins. Binding assays using Bio-layer Interferometry validated the Rbp3-psaAB mRNA interaction, indicating a preference for folded RNA segments near or overlapping the respective stop codons. Significance statementThe mechanisms by which proteins are produced at specific sites and inserted into the intricate membrane systems of photosynthetic cyanobacteria are only partially understood. While RRM domain proteins are well-studied RNA-binding proteins in eukaryotes, their functions in bacteria remain underexplored. This study reveals that the RRM domain protein Rbp3 in the cyanobacterium Synechocystis sp. PCC 6803 binds mRNAs encoding photosynthetic proteins, plasma membrane proteins and carboxysome proteins and localizes near ribosomes. The bound RNA segments are typically near the ends of coding regions, or in 5' untranslated regions. These findings suggest that Rbp3 is involved in targeting mRNAs to various intracellular locations by interacting with structural elements within these mRNA molecules.

microbiology↗

bioRxiv2023

The role of the 5' sensing function of ribonuclease E in cyanobacteria

RNA degradation is crucial for many processes in pro- and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y towards 5-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5 sensing. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologs. Here, we constructed a Synechocystis strain lacking the 5 sensing function of RNase E and mapped on a transcriptome-wide level 292 5-sensing-dependent cleavage sites. These included so far unknown targets such as the 5 untranslated region of the response regulator gene lsiR; trxA, apcE and atpI mRNAs, encoding proteins related to energy metabolism; as well as sbtB and rbcLXS encoding proteins relevant for carbon fixation. Cyanobacterial 5 sensing is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and most prokaryotes. We found that increased RNase activities leads to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. The results provide a first step towards understanding the relative importance of different target mechanisms of RNase E outside Escherichia coli.

microbiology↗

bioRxiv2022

Regulation of pSYSA defense plasmid copy number in Synechocystis through RNase E and a highly transcribed asRNA

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-located 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.

microbiology↗

bioRxiv2022

CvkR, a novel MerR-type transcriptional regulator, is a repressor of class 2 type V-K CRISPR-associated transposase systems

CRISPR-associated transposons (CASTs) exist in different groups of bacteria, including certain cyanobacteria, which contain type V-K CAST systems. These systems contain genes encoding Tn7-like transposase subunits and a divergent number of cargo genes. How the activity of these systems is controlled in situ has remained largely unknown but possibly regulatory genes within these elements are prime candidates. Deletion of the respective regulator gene alr3614 in the cyanobacterium Anabaena (Nostoc) sp. PCC 7120 led to the overexpression of CRISPR tracrRNA, precursor crRNAs and mRNAs encoding the Cas12k effector protein (all3613) and Tn7-like transposase subunits. Upon complementation, these same genes were repressed again. DNase I footprinting and electrophoretic mobility shift assays verified the direct interaction between Alr3614 and the promoter of cas12k and identified a widely conserved binding motif. Structural analysis of Alr3614 at 1.5 [A] resolution revealed that it belongs to the MerR-type transcription factor family but with distinct dimerization and effector-binding domains. This protein assembles into a homodimer interacting with DNA through its N-terminal winged helix-turn-helix (wHTH) domain and binds an effector molecule through a C-terminal -helical domain lacking a conserved cysteine. These results identify Alr3614 as a transcriptional repressor of the CAST system in Anabaena sp. PCC 7120. We suggest naming this family of repressors CvkR for Cas V-K repressors, which are at the core of a widely conserved regulatory mechanism that controls type V-K CAST systems.

microbiology↗

bioRxiv2020

Specificities of and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups

The majority of bacteria and archaea possess an RNA-guided adaptive and inheritable immune system against viruses and other foreign genetic elements 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. In some bacteria, multiple instances of cas gene-free (orphan) repeat-spacer arrays exist, while additional instances of arrays that are linked to cas gene cassettes are present elsewhere in the genome. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR-Cas repeat-spacer arrays are present, but only two cas gene cassettes plus a Tn7-associated eleventh array are observed. 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 a strong tendency for array fragmentation in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our data demonstrate the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.

microbiology↗