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Featured researches published by Stephan Klähn.


Environmental Microbiology | 2011

Compatible solute biosynthesis in cyanobacteria

Stephan Klähn; Martin Hagemann

Compatible solutes are a functional group of small, highly soluble organic molecules that demonstrate compatibility in high amounts with cellular metabolism. The accumulation of compatible solutes is often observed during the acclimation of organisms to adverse environmental conditions, particularly to salt and drought stress. Among cyanobacteria, sucrose, trehalose, glucosylglycerol and glycine betaine are used as major compatible solutes. Interestingly, a close correlation has been discovered between the final salt tolerance limit and the primary compatible solute in these organisms. In addition to the dominant compatible solutes, many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline as secondary or tertiary solutes. In particular, the accumulation of sucrose and trehalose results in an increase in tolerance to general stresses such as desiccation and high temperatures. During recent years, the biochemical and molecular basis of compatible solute accumulation has been characterized using cyanobacterial model strains that comprise different salt tolerance groups. Based on these data, the distribution of genes involved in compatible solute synthesis among sequenced cyanobacterial genomes is reviewed, and thereby, the major compatible solutes and potential salt tolerance of these strains can be predicted. Knowledge regarding cyanobacterial salt tolerance is not only useful to characterize strain-specific adaptations to ecological niches, but it can also be used to generate cells with increased tolerance to adverse environmental conditions for biotechnological purposes.


DNA Research | 2014

Comparative Analysis of the Primary Transcriptome of Synechocystis sp. PCC 6803

Matthias Kopf; Stephan Klähn; Ingeborg Scholz; Jasper K.F. Matthiessen; Wolfgang R. Hess; Björn Voß

RNA-seq and especially differential RNA-seq-type transcriptomic analyses (dRNA-seq) are powerful analytical tools, as they not only provide insights into gene expression changes but also provide detailed information about all promoters active at a given moment, effectively giving a deep insight into the transcriptional landscape. Synechocystis sp. PCC 6803 (Synechocystis 6803) is a unicellular model cyanobacterium that is widely used in research fields from ecology, photophysiology to systems biology, modelling and biotechnology. Here, we analysed the response of the Synechocystis 6803 primary transcriptome to different, environmentally relevant stimuli. We established genome-wide maps of the transcriptional start sites active under 10 different conditions relevant for photosynthetic growth and identified 4,091 transcriptional units, which provide information about operons, 5′ and 3′ untranslated regions (UTRs). Based on a unique expression factor, we describe regulons and relevant promoter sequences at single-nucleotide resolution. Finally, we report several sRNAs with an intriguing expression pattern and therefore likely function, specific for carbon depletion (CsiR1), nitrogen depletion (NsiR4), phosphate depletion (PsiR1), iron stress (IsaR1) or photosynthesis (PsrR1). This dataset is accompanied by comprehensive information providing extensive visualization and data access to allow an easy-to-use approach for the design of experiments, the incorporation into modelling studies of the regulatory system and for comparative analyses.


Environmental Microbiology | 2010

Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments

Stephan Klähn; Claudia Steglich; Wolfgang R. Hess; Martin Hagemann

The synthesis and accumulation of compatible solutes represent an essential part of the salt acclimation strategy of microorganisms. Glucosylglycerol is considered to be the typical compatible solute among marine cyanobacteria. However, genes that encode enzymes for the synthesis of glucosylglycerol were not detected in the genome sequences of marine picoplanktonic Prochlorococcus strains. Instead, we noticed the presence of genes that putatively encode for glucosylglycerate (GGA) synthesis among Prochlorococcus and most other closely related marine picocyanobacteria. Recombinant proteins from Prochlorococcus marinus SS120 and Synechococcus sp. PCC 7002 exhibited glucosyl-phosphoglycerate synthase (GpgS) activity, and GpgS is a key enzyme of GGA synthesis. GGA accumulation was found to be salt- as well as nitrogen-regulated in the coastal strain Synechococcus sp. PCC 7002. Moreover, GGA was also detected in all picoplanktonic Prochlorococcus and Synechococcus strains harbouring gpgS genes, especially under N-limiting conditions. These results suggest that marine picocyanobacteria acquired the capacity to synthesize the negatively charged compound GGA during their evolution. Our results establish GGA as the fifth most widespread compatible solute among cyanobacteria. Additionally, GGA appears to replace glutamate as an anion to counter monovalent cations in marine picocyanobacteria from N-poor environments.


Journal of Biological Chemistry | 2012

The antisense RNA As1_flv4 in the cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply

Marion Eisenhut; Jens Georg; Stephan Klähn; Isamu Sakurai; Henna Mustila; Pengpeng Zhang; Wolfgang R. Hess; Eva-Mari Aro

Background: Flavodiiron proteins encoded by the flv4-2 operon are photoprotective for photosystem II, but their regulation of expression has remained enigmatic. Results: Expression of flv4-2 is controlled jointly by NdhR and the antisense RNA As1_flv4, whereas As1_flv4 is controlled by an AbrB-like factor. Conclusion: As1_flv4 provides a safety threshold preventing premature expression. Significance: Regulatory networks controlling photosynthetic photoprotection are highly complex. The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.


The Plant Cell | 2014

The Small Regulatory RNA SyR1/PsrR1 Controls Photosynthetic Functions in Cyanobacteria

Jens Georg; Dennis Dienst; Nils Schürgers; Thomas Wallner; Dominik Kopp; Damir Stazic; Ekaterina Kuchmina; Stephan Klähn; Heiko Lokstein; Wolfgang R. Hess; Annegret Wilde

This work describes the role of a small noncoding RNA in the regulation of photosynthetic gene expression in cyanobacteria. Advanced computational target prediction combined with extensive experimental data reveal the photosynthesis regulatory RNA 1 as a key modulator of photosynthetic functions upon high-light exposure. Little is known so far about RNA regulators of photosynthesis in plants, algae, or cyanobacteria. The small RNA PsrR1 (formerly SyR1) has been discovered in Synechocystis sp PCC 6803 and appears to be widely conserved within the cyanobacterial phylum. Expression of PsrR1 is induced shortly after a shift from moderate to high-light conditions. Artificial overexpression of PsrR1 led to a bleaching phenotype under moderate light growth conditions. Advanced computational target prediction suggested that several photosynthesis-related mRNAs could be controlled by PsrR1, a finding supported by the results of transcriptome profiling experiments upon pulsed overexpression of this small RNA in Synechocystis sp PCC 6803. We confirmed the interaction between PsrR1 and the ribosome binding regions of the psaL, psaJ, chlN, and cpcA mRNAs by mutational analysis in a heterologous reporter system. Focusing on psaL as a specific target, we show that the psaL mRNA is processed by RNase E only in the presence of PsrR1. Furthermore, we provide evidence for a posttranscriptional regulation of psaL by PsrR1 in the wild type at various environmental conditions and analyzed the consequences of PsrR1-based regulation on photosystem I. In summary, computational and experimental data consistently establish the small RNA PsrR1 as a regulatory factor controlling photosynthetic functions.


Journal of Bacteriology | 2008

The Plant-Associated Bacterium Stenotrophomonas rhizophila Expresses a New Enzyme for the Synthesis of the Compatible Solute Glucosylglycerol

Martin Hagemann; Kathrin Ribbeck-Busch; Stephan Klähn; Dirk Hasse; Robert Steinbruch; Gabriele Berg

The rhizobacterium Stenotrophomonas rhizophila accumulates the compatible solutes glucosylglycerol (GG) and trehalose under salt stress conditions. The complete gene for the GG synthesis enzyme was cloned and sequenced. This enzyme from S. rhizophila represented a novel fusion protein composed of a putative C-terminal GG-phosphate synthase domain and an N-terminal putative GG-phosphate phosphatase domain, which was named GgpPS. A similar gene was cloned from Pseudomonas sp. strain OA146. The ggpPS gene was induced after a salt shock in S. rhizophila cells. After the salt-loaded cells reached stationary phase, the ggpPS mRNA content returned to the low level characteristic of the control cells, and GG was released into the medium. The complete ggpPS gene and a truncated version devoid of the phosphatase part were obtained as recombinant proteins. Enzyme activity tests revealed the expected abilities of the full-length protein to synthesize GG and the truncated GgpPS to synthesize GG-phosphate. However, dephosphorylation of GG-phosphate was detected only with the complete GgpPS protein. These enzyme activities were confirmed by complementation experiments using defined GG-defective mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Genes coding for proteins very similar to the newly identified fusion protein GgpPS for GG synthesis in S. rhizophila were found in genome sequences of related bacteria, where these genes are often linked to a gene coding for a transporter of the Mfs superfamily.


Proceedings of the National Academy of Sciences of the United States of America | 2015

The sRNA NsiR4 is involved in nitrogen assimilation control in cyanobacteria by targeting glutamine synthetase inactivating factor IF7

Stephan Klähn; Christoph Schaal; Jens Georg; Desirée Baumgartner; Gernot Knippen; Martin Hagemann; Alicia M. Muro-Pastor; Wolfgang R. Hess

Significance A key enzyme in inorganic nitrogen assimilation, glutamine synthetase, is the target of multiple regulatory mechanisms. Here we describe the nitrogen stress-induced RNA 4 (NsiR4), a small regulatory RNA that reduces the expression of inactivating factor 7 (IF7), an inhibitory factor of glutamine synthetase in cyanobacteria. The expression of NsiR4 is under positive control through the nitrogen control transcription factor (NtcA). NtcA also induces the transcription of the glutamine synthetase gene and represses the gene encoding IF7. Therefore, NsiR4 is a new player in the NtcA-mediated regulation of nitrogen assimilation, which is important for adaptations to rapid changes in available nitrogen sources and concentrations. Glutamine synthetase (GS), a key enzyme in biological nitrogen assimilation, is regulated in multiple ways in response to varying nitrogen sources and levels. Here we show a small regulatory RNA, NsiR4 (nitrogen stress-induced RNA 4), which plays an important role in the regulation of GS in cyanobacteria. NsiR4 expression in the unicellular Synechocystis sp. PCC 6803 and in the filamentous, nitrogen-fixing Anabaena sp. PCC 7120 is stimulated through nitrogen limitation via NtcA, the global transcriptional regulator of genes involved in nitrogen metabolism. NsiR4 is widely conserved throughout the cyanobacterial phylum, suggesting a conserved function. In silico target prediction, transcriptome profiling on pulse overexpression, and site-directed mutagenesis experiments using a heterologous reporter system showed that NsiR4 interacts with the 5′UTR of gifA mRNA, which encodes glutamine synthetase inactivating factor (IF)7. In Synechocystis, we observed an inverse relationship between the levels of NsiR4 and the accumulation of IF7 in vivo. This NsiR4-dependent modulation of gifA (IF7) mRNA accumulation influenced the glutamine pool and thus NH4+ assimilation via GS. As a second target, we identified ssr1528, a hitherto uncharacterized nitrogen-regulated gene. Competition experiments between WT and an ΔnsiR4 KO mutant showed that the lack of NsiR4 led to decreased acclimation capabilities of Synechocystis toward oscillating nitrogen levels. These results suggest a role for NsiR4 in the regulation of nitrogen metabolism in cyanobacteria, especially for the adaptation to rapid changes in available nitrogen sources and concentrations. NsiR4 is, to our knowledge, the first identified bacterial sRNA regulating the primary assimilation of a macronutrient.


Frontiers in Bioengineering and Biotechnology | 2014

Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

Stephan Klähn; Desirée Baumgartner; Ulrike Pfreundt; Karsten Voigt; Verena Schön; Claudia Steglich; Wolfgang R. Hess

In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria.


Journal of Experimental Botany | 2009

Expression of the ggpPS gene for glucosylglycerol biosynthesis from Azotobacter vinelandii improves the salt tolerance of Arabidopsis thaliana.

Stephan Klähn; Daniel M. Marquardt; Inga Rollwitz; Martin Hagemann

Many organisms accumulate compatible solutes in response to salt or desiccation stress. Moderate halotolerant cyanobacteria and some heterotrophic bacteria synthesize the compatible solute glucosylglycerol (GG) as their main protective compound. In order to analyse the potential of GG to improve salt tolerance of higher plants, the model plant Arabidopsis thaliana was transformed with the ggpPS gene from the γ-proteobacterium Azotobacter vinelandii coding for a combined GG-phosphate synthase/phosphatase. The heterologous expression of the ggpPS gene led to the accumulation of high amounts of GG. Three independent Arabidopsis lines showing different GG contents were characterized in growth experiments. Plants containing a low (1–2 μmol g−1 FM) GG content in leaves showed no altered growth performance under control conditions but an increased salt tolerance, whereas plants accumulating a moderate (2–8 μmol g−1 FM) or a high GG content (around 17 μmol g−1 FM) showed growth retardation and no improvement of salt resistance. These results indicate that the synthesis of the compatible solute GG has a beneficial effect on plant stress tolerance as long as it is accumulated to an extent that does not negatively interfere with plant metabolism.


Scientific Reports | 2015

Variations in the non-coding transcriptome as a driver of inter-strain divergence and physiological adaptation in bacteria

Matthias Kopf; Stephan Klähn; Ingeborg Scholz; Wolfgang R. Hess; Björn Voß

In all studied organisms, a substantial portion of the transcriptome consists of non-coding RNAs that frequently execute regulatory functions. Here, we have compared the primary transcriptomes of the cyanobacteria Synechocystis sp. PCC 6714 and PCC 6803 under 10 different conditions. These strains share 2854 protein-coding genes and a 16S rRNA identity of 99.4%, indicating their close relatedness. Conserved major transcriptional start sites (TSSs) give rise to non-coding transcripts within the sigB gene, from the 5′UTRs of cmpA and isiA, and 168 loci in antisense orientation. Distinct differences include single nucleotide polymorphisms rendering promoters inactive in one of the strains, e.g., for cmpR and for the asRNA PsbA2R. Based on the genome-wide mapped location, regulation and classification of TSSs, non-coding transcripts were identified as the most dynamic component of the transcriptome. We identified a class of mRNAs that originate by read-through from an sRNA that accumulates as a discrete and abundant transcript while also serving as the 5′UTR. Such an sRNA/mRNA structure, which we name ‘actuaton’, represents another way for bacteria to remodel their transcriptional network. Our findings support the hypothesis that variations in the non-coding transcriptome constitute a major evolutionary element of inter-strain divergence and capability for physiological adaptation.

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Jens Georg

University of Freiburg

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Björn Voß

University of Freiburg

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