Biology subjects

Bilger, R.

Publications and source records attributed to Bilger, R..

5 recordsLinked to original sources

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↗

bioRxiv2025

Involvement of RNase J in CRISPR RNA maturation and degradosome formation in cyanobacteria

Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5'-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3 exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.

microbiology↗

bioRxiv2023

Control of a type III-Dv CRISPR-Cas system by the transcription factor RpaB and interaction of its transcribed leader with the DEAD-box RNA helicase CrhR

CRISPR-Cas systems in bacteria and archaea provide powerful defense against phages and other foreign genetic elements. The principles of CRISPR-Cas activity are well understood, but less is known about how their expression is regulated. The cyanobacterium Synechocystis sp. PCC 6803 encodes three different CRISPR-Cas systems. The expression of one of these, a type III-Dv system, responds to changes in environmental conditions, such as nitrogen starvation or varying light intensities. Here, we found that the promoter of the six-gene cas operon for the type III-Dv system is controlled by the light-and redox-responsive transcription factor RpaB. RpaB binds to an HLR1 motif located 53 to 70 nt upstream of the transcription start site, 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, we found that the 125 nt leader transcript is bound by the redox-sensitive RNA helicase CrhR. Crosslinking coupled to mass spectrometry analysis revealed six residues involved in the CrhR-RNA interaction. Of these, L103, F104, H225, and C371 were predicted to be on the surface of a dimeric CrhR model, while C184 was not on the surface, and P443 could not be assigned to a structural element. These results showed that the expression of the CRISPR-Cas system is linked to the redox status of the photosynthetic cyanobacterial cell at two different levels. While RpaB affects transcription, CrhR interacts with the leader transcript posttranscription. These results highlight the complex interplay between a CRISPR-Cas system and its host cell.

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↗

bioRxiv2020

Grad-seq analysis of a photosynthetic bacterium rich in internal membrane systems

Regulatory sRNAs in photosynthetic cyanobacteria have been reported, but the lack of plausible RNA chaperones involved in this regulation appears enigmatic. Here, we analyzed the full ensemble 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 a high quality of the approach. Clustering of the in-gradient distribution profiles followed by several additional criteria yielded a short list of potential RNA chaperones that include a 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. One-sentence summaryWe provide the first global analysis of a cyanobacterium using Grad-seq, providing a comprehensive resource for the in-depth analysis of the complexome in a photosynthetic organism.

microbiology↗