Biology subjects

Steglich, C.

Publications and source records attributed to Steglich, C..

10 recordsLinked to original sources

bioRxiv2025

Diel rhythms shape viral community structure and activity across the host domains of life

Circadian rhythms, driven by endogenous clocks and synchronized with environmental cues, are fundamental to life on Earth. While extensively studied in diverse organisms, their influence on viral ecology remains largely unexplored. This study takes the temporal dynamics of viruses in Daya Bay as an example to uncover the rhythmic control of viral replication and activity. Using a high-resolution time-series dataset collected every 2 hours over a 3-day period, we identified a total of 22,151 viral operational taxonomic units (vOTUs) and 414 Nucleocytoplasmic Large DNA Virus (NCLDV) genomes. Our analysis revealed significant diel fluctuations, with 14.48% of vOTUs exhibiting diel patterns in metagenomic abundance and 1.97% showing diel transcriptional activity. We found that these abundant diel viruses infect hosts across the domains of life, including cyanobacteria, pelagibacteria, Marine Group II (MGII) archaea and protists, all known for their diel metabolism. The expanded spectrum of host diversity with diel viral interactions significantly broadens our understanding of virus-host rhythmic dynamics in natural environmental settings. A strong positive correlation was detected between the transcriptional activities of these diel viruses and their respective hosts. Contrary to bacteriophages, which mostly peaked during the day, we demonstrated that NCLDVs showed nocturnal diel abundance with a co-fluctuating diel transcriptomic activity pattern tightly hitched to their hosts, peaking at night and declining during the day. Furthermore, we identified a rich compendium of viral genes with significant diel expression patterns, including those related to structural protein production, DNA replication, and stress response. Notably, several essential viral genes involved in stress response and repair were found to be diel transcribed for the first time, including UV-endonuclease (UvdE), peroxidase, chaperones, and early light-induced protein (ELIP). Our findings suggest that viruses across host domains actively synchronize with environmental cues to optimize their replication and transmission, despite their dependence on host metabolism. This study provides novel insights into the rhythmic control of viral communities and their intricate interactions with hosts, with profound implications for microbial community succession and biogeochemical cycles.

ecology↗

bioRxiv2025

The infection cycle of the haloarchaeal virus HFTV1 is tightly regulated and strongly inhibits motility of its host

Although viruses have been shown to infect all domains of life, our understanding of the genetic program behind the exploitation of host resources to produce progeny virions is thus far limited to several bacterial viruses. Therefore, to elucidate the transcriptome of euryarchaeal viruses and their hosts, we employed RNAseq analysis of samples taken at different time points from Haloferax gibbonsii LR2-5 cultures infected with the lytic model virus Haloferax Tailed Virus 1 (HFTV1). While following the transcription of viral genes throughout the infective life cycle, we observed a tight temporal regulation of viral transcripts as well as differential expression from within viral gene clusters. Furthermore, anti-sense RNAs (asRNAs) appear to play an important role in support of the timing of late-expressed viral genes. Therefore, with many differentially expressed transcripts, including intragenic transcripts and asRNAs, the regulatory machinery employed by HFTV1 contrasts with viral model systems (based on phages), in which antitermination and/or alternative polymerases (seemingly lacking in HFTV1) are more widespread. When looking into differentially expressed host genes, we observed a strong downregulation of genes involved in motility, such as the archaellum and chemotaxis machinery, which was confirmed with swimming assays of HFTV1 infected cells. This might be a strategy of the virus to redirect energy flowing into movement towards the production of virions. In conclusion, this work thus provides a stepping stone for further exploration of the intriguing strategies of viral transcriptional regulation of their infection cycle across the domains of life. IMPORTANCEViruses infect members of all three domains of life, including Archaea. Euryarchaea are widespread microorganisms found in various environments such as the human gut and solar salterns. Due to the exceptional availability of cell biology and genetic tools of some salt-loving archaea, they are a model system to extrapolate from. Insights into the regulation of viral infections are of particular importance, especially since HFTV1, has been adopted as a model virus by the archaeal viral community. We found that, while harboring parallels with bacterial viruses, such as tight temporal regulation, HFTV1 harbors an impressive number of differentially expressed transcriptional units. Furthermore, anti-sense RNAs and intragenic regulatory elements seem to play a much more prominent role in HFTV1 gene expression. Thus, this work challenges current models and provides valuable new insights into the gene regulation of viral infection of archaea, which mark similarities and differences with viruses from other domains of life.

microbiology↗

bioRxiv2024

Emergence of metabolic coupling to the heterotroph Alteromonas promotes dark survival in Prochlorococcus

Prochlorococcus is found throughout the euphotic zone in the oligotrophic open ocean. Deep mixing and sinking while attached to particles can, however, transport Prochlorococcus cells below this sunlit zone, depriving them of light for extended periods of time. Previous work has shown that Prochlorococcus by itself cannot survive extended periods of darkness. However, when co-cultured with a heterotrophic microbe and subjected to repeated periods of extended darkness, Prochlorococcus cells develop an epigenetically inherited dark-tolerant phenotype that can survive longer periods of darkness. Here we examine the metabolic and physiological changes underlying this adaptation using co-cultures of dark-tolerant and parental strains of Prochlorococcus, each grown with the heterotroph Alteromonas under diel light:dark conditions. The relative abundance of Alteromonas was higher in dark-tolerant than parental co-cultures, while dark-tolerant Prochlorococcus cells were larger, contained less chlorophyll, and were less synchronized to the light:dark cycle. Meta-transcriptome analysis revealed that dark-tolerant co-cultures undergo a joint change, in which Prochlorococcus undergoes a relative shift from photosynthesis to respiration, while Alteromonas shifts towards using more organic acids instead of sugars. Furthermore, the transcriptome data suggested enhanced biosynthesis of amino acids and purines in dark-tolerant Prochlorococcus and enhanced degradation of these compounds in Alteromonas. Collectively, our results demonstrate that dark adaptation involves a strengthening of the metabolic coupling between Prochlorococcus and Alteromonas, presumably mediated by an enhanced, and compositionally modified, carbon exchange between the two species.

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↗

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

Feedback regulation of RNase E during UV-stress response in the cyanobacterium Synechocystis sp. PCC 6803

Endoribonucleases govern the maturation and degradation of RNA and are indispensable in the posttranscriptional regulation of gene expression. A key endoribonuclease in many bacteria is RNase E. To ensure an appropriate supply of RNase E, some bacteria, such as E. coli, have evolved tightly functioning feedback regulation of RNase E that is mediated in cis by the rne 5'-untranslated region (5' UTR); 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. Here, we investigated the global gene expression response in the cyanobacterium Synechocystis sp. PCC 6803 after exposure to UV light and discovered a unique response of RNase E: a rapidly increasing enzymatic activity, although the stability of the protein was decreased. In parallel, we observed an increased accumulation of full-length rne mRNA that was 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 via its own 5' UTR. These observations imply that RNase E in cyanobacteria contributes substantially to reshaping the transcriptome during the UV stress response and that its required activity level is maintained despite enhanced turnover of the protein by posttranscriptional feedback regulation.

microbiology↗

bioRxiv2021

Transcriptome-wide in vivo mapping of cleavage sites for the compact cyanobacterial ribonuclease E reveals insights into its function and substrate recognition

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 spontaneously occurring V94A. This enabled us to perform RNA-seq after the transient inactivation of RNase E by a temperature shift (TIER-seq) and to map 1,472 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 putative RNase-E-dependent instances of operon discoordination, mRNAs likely regulated jointly by RNase E and an sRNA, potential 3 end-derived sRNAs and a dual-acting mechanism for the glutamine riboswitch. 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.

microbiology↗

bioRxiv2021

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

RNA helicases play crucial functions in RNA biology. In plants, RNA helicases are encoded by large gene families, performing roles in abiotic stress responses, development, the post-transcriptional regulation of gene expression as well as house-keeping functions. Several of these RNA helicases are targeted to the organelles, mitochondria and chloroplasts. Cyanobacteria are the direct evolutionary ancestors of plant chloroplasts. The cyanobacterium Synechocystis 6803 encodes a single DEAD-box RNA helicase, CrhR, that is induced by a range of abiotic stresses, including low temperature. Though the {Delta}crhR 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. HighlightThe cyanobacterial DEAD-box RNA helicase CrhR binds mainly photosynthesis-associated and redox-controlled transcripts connecting its regulation, localization and phenotypes of mutants for the first time with a set of potential RNA targets.

plant biology↗

bioRxiv2020

Discovery of a novel small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria

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. This degradation is tightly coordinated, follows a genetic program and involves small proteins serving as proteolysis adaptors. Here, we describe the role of NblD, a novel factor in this process in cyanobacteria. NblD is a cysteine-rich, 66-amino acid small protein that becomes rapidly induced upon nitrogen starvation. Deletion of the nblD gene in the cyanobacterium Synechocystis prevents the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype. Competition experiments provided direct evidence for the physiological importance of NblD. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type. Overexpression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. Transcriptome analysis revealed that nitrogen starvation correctly induces nblA1/2 transcription in the {Delta}nblD strain implying that NblD does not act as a transcriptional (co-)regulator. However, fractionation and coimmunoprecipitation experiments indicated the presence of NblD in the phycobilisome fraction and identified the {beta}-phycocyanin subunit as its target. These data add NblD as a new factor to the genetically programmed response to nitrogen starvation and demonstrate that it plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. Significance StatementDuring genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the small protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells run low in nitrogen, a condition that frequently occurs in nature. Similar to the NblA proteins that label phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration.

microbiology↗