@article{MichauxHansenJennichesetal.2020, author = {Michaux, Charlotte and Hansen, Elisabeth E. and Jenniches, Laura and Gerovac, Milan and Barquist, Lars and Vogel, J{\"o}rg}, title = {Single-Nucleotide RNA Maps for the Two Major Nosocomial Pathogens Enterococcus faecalis and Enterococcus faecium}, series = {Frontiers in Cellular and Infection Microbiology}, volume = {10}, journal = {Frontiers in Cellular and Infection Microbiology}, issn = {2235-2988}, doi = {10.3389/fcimb.2020.600325}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217947}, year = {2020}, abstract = {Enterococcus faecalis and faecium are two major representative clinical strains of the Enterococcus genus and are sadly notorious to be part of the top agents responsible for nosocomial infections. Despite their critical implication in worldwide public healthcare, essential and available resources such as deep transcriptome annotations remain poor, which also limits our understanding of post-transcriptional control small regulatory RNA (sRNA) functions in these bacteria. Here, using the dRNA-seq technique in combination with ANNOgesic analysis, we successfully mapped and annotated transcription start sites (TSS) of both E. faecalis V583 and E. faecium AUS0004 at single nucleotide resolution. Analyzing bacteria in late exponential phase, we capture ~40\% (E. faecalis) and 43\% (E. faecium) of the annotated protein-coding genes, determine 5′ and 3′ UTR (untranslated region) length, and detect instances of leaderless mRNAs. The transcriptome maps revealed sRNA candidates in both bacteria, some found in previous studies and new ones. Expression of candidate sRNAs is being confirmed under biologically relevant environmental conditions. This comprehensive global TSS mapping atlas provides a valuable resource for RNA biology and gene expression analysis in the Enterococci. It can be accessed online at www.helmholtz-hiri.de/en/datasets/enterococcus through an instance of the genomic viewer JBrowse.}, language = {en} } @article{GorskiVogelSalibaetal.2014, author = {Gorski, Stanislaw A. and Vogel, J{\"o}rg and Saliba, Antoine-Emmanuel and Westermann, Alexander J.}, title = {Single-cell RNA-seq: advances and future challenges}, doi = {10.1093/nar/gku555}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-110993}, year = {2014}, abstract = {Phenotypically identical cells can dramatically vary with respect to behavior during their lifespan and this variation is reflected in their molecular composition such as the transcriptomic landscape. Singlecell transcriptomics using next-generation transcript sequencing (RNA-seq) is now emerging as a powerful tool to profile cell-to-cell variability on a genomic scale. Its application has already greatly impacted our conceptual understanding of diverse biological processes with broad implications for both basic and clinical research. Different single-cell RNAseq protocols have been introduced and are reviewed here - each one with its own strengths and current limitations. We further provide an overview of the biological questions single-cell RNA-seq has been used to address, the major findings obtained from such studies, and current challenges and expected future developments in this booming field.}, subject = {RNS}, language = {en} } @article{WestermannBarquistVogel2017, author = {Westermann, Alexander J. and Barquist, Lars and Vogel, J{\"o}rg}, title = {Resolving host-pathogen interactions by dual RNA-seq}, series = {PLoS Pathogens}, volume = {13}, journal = {PLoS Pathogens}, number = {2}, doi = {10.1371/journal.ppat.1006033}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171921}, year = {2017}, abstract = {The transcriptome is a powerful proxy for the physiological state of a cell, healthy or diseased. As a result, transcriptome analysis has become a key tool in understanding the molecular changes that accompany bacterial infections of eukaryotic cells. Until recently, such transcriptomic studies have been technically limited to analyzing mRNA expression changes in either the bacterial pathogen or the infected eukaryotic host cell. However, the increasing sensitivity of high-throughput RNA sequencing now enables "dual RNA-seq" studies, simultaneously capturing all classes of coding and noncoding transcripts in both the pathogen and the host. In the five years since the concept of dual RNA-seq was introduced, the technique has been applied to a range of infection models. This has not only led to a better understanding of the physiological changes in pathogen and host during the course of an infection but has also revealed hidden molecular phenotypes of virulence-associated small noncoding RNAs that were not visible in standard infection assays. Here, we use the knowledge gained from these recent studies to suggest experimental and computational guidelines for the design of future dual RNA-seq studies. We conclude this review by discussing prospective applications of the technique.}, language = {en} } @article{DimastrogiovanniFroehlichBandyraetal.2014, author = {Dimastrogiovanni, Daniela and Fr{\"o}hlich, Kathrin S. and Bandyra, Katarzyna J. and Bruce, Heather A. and Hohensee, Susann and Vogel, J{\"o}rg and Luisi, Ben F.}, title = {Recognition of the small regulatory RNA RydC by the bacterial Hfq protein}, series = {eLife}, volume = {3}, journal = {eLife}, number = {e05375}, issn = {2050-084X}, doi = {10.7554/eLife.05375}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114191}, year = {2014}, abstract = {Bacterial small RNAs (sRNAs) are key elements of regulatory networks that modulate gene expression. The sRNA RydC of Salmonella sp. and Escherichia coli is an example of this class of riboregulators. Like many other sRNAs, RydC bears a 'seed' region that recognises specific transcripts through base-pairing, and its activities are facilitated by the RNA chaperone Hfq. The crystal structure of RydC in complex with E. coli Hfq at 3.48 angstrom resolution illuminates how the protein interacts with and presents the sRNA for target recognition. Consolidating the protein-RNA complex is a host of distributed interactions mediated by the natively unstructured termini of Hfq. Based on the structure and other data, we propose a model for a dynamic effector complex comprising Hfq, small RNA, and the cognate mRNA target.}, language = {en} } @article{MuellerUlyshenSeiboldetal.2020, author = {M{\"u}ller, J{\"o}rg and Ulyshen, Mike and Seibold, Sebastian and Cadotte, Marc and Chao, Anne and B{\"a}ssler, Claus and Vogel, Sebastian and Hagge, Jonas and Weiß, Ingmar and Baldrian, Petr and Tl{\´a}skal, Vojtěch and Thorn, Simon}, title = {Primary determinants of communities in deadwood vary among taxa but are regionally consistent}, series = {Oikos}, volume = {129}, journal = {Oikos}, number = {10}, doi = {10.1111/oik.07335}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-228201}, pages = {1579 -- 1588}, year = {2020}, abstract = {The evolutionary split between gymnosperms and angiosperms has far-reaching implications for the current communities colonizing trees. The inherent characteristics of dead wood include its role as a spatially scattered habitat of plant tissue, transient in time. Thus, local assemblages in deadwood forming a food web in a necrobiome should be affected not only by dispersal ability but also by host tree identity, the decay stage and local abiotic conditions. However, experiments simultaneously manipulating these potential community drivers in deadwood are lacking. To disentangle the importance of spatial distance and microclimate, as well as host identity and decay stage as drivers of local assemblages, we conducted two consecutive experiments, a 2-tree species and 6-tree species experiment with 80 and 72 tree logs, respectively, located in canopy openings and under closed canopies of a montane and a lowland forest. We sampled saproxylic beetles, spiders, fungi and bacterial assemblages from logs. Variation partitioning for community metrics based on a unified framework of Hill numbers showed consistent results for both studies: host identity was most important for sporocarp-detected fungal assemblages, decay stage and host tree for DNA-detected fungal assemblages, microclimate and decay stage for beetles and spiders and decay stage for bacteria. Spatial distance was of minor importance for most taxa but showed the strongest effects for arthropods. The contrasting patterns among the taxa highlight the need for multi-taxon analyses in identifying the importance of abiotic and biotic drivers of community composition. Moreover, the consistent finding of microclimate as the primary driver for saproxylic beetles compared to host identity shows, for the first time that existing evolutionary host adaptions can be outcompeted by local climate conditions in deadwood.}, language = {en} } @article{VogelGossnerMergneretal.2020, author = {Vogel, Sebastian and Gossner, Martin M. and Mergner, Ulrich and M{\"u}ller, J{\"o}rg and Thorn, Simon}, title = {Optimizing enrichment of deadwood for biodiversity by varying sun exposure and tree species: An experimental approach}, series = {Journal of Applied Ecology}, volume = {57}, journal = {Journal of Applied Ecology}, number = {10}, doi = {10.1111/1365-2664.13648}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214614}, pages = {2075 -- 2085}, year = {2020}, abstract = {The enrichment of deadwood is essential for the conservation of saproxylic biodiversity in managed forests. However, existing strategies focus on a cost-intensive increase of deadwood amount, while largely neglecting increasing deadwood diversity. Deadwood objects, that is logs and branches, from six tree species were experimentally sun exposed, canopy shaded and artificially shaded for 4 years, after which the alpha-, beta- and gamma-diversity of saproxylic beetles, wood-inhabiting fungi and spiders were analysed. Analyses of beta-diversity included the spatial distance between exposed deadwood objects. A random-drawing procedure was used to identify the combination of tree species and sun exposure that yielded the highest gamma-diversity at a minimum of exposed deadwood amount. In sun-exposed plots, species numbers in logs were higher than in shaded plots for all taxa, while in branches we observed the opposite for saproxylic beetles. Tree species affected the species numbers only of saproxylic beetles and wood-inhabiting fungi. The beta-diversity of saproxylic beetles and wood-inhabiting fungi among logs was influenced by sun exposure and tree species, but beta-diversity of spiders by sun exposure only. For all saproxylic taxa recorded in logs, differences between communities increased with increasing spatial distance. A combination of canopy-shaded Carpinus logs and sun-exposed Populus logs resulted in the highest species numbers of all investigated saproxylic taxa among all possible combinations of tree species and sun-exposure treatments. Synthesis and applications. We recommend incorporating the enrichment of different tree species and particularly the variation in sun exposure into existing strategies of deadwood enrichment. Based on the results of our study, we suggest to combine the logs of softwood broadleaf tree species (e.g. Carpinus, Populus), hardwood broadleaf tree species (e.g. Quercus) and coniferous tree species (e.g. Pinus) under different conditions of sun exposure and distribute them spatially in a landscape to maximize the beneficial effects on overall diversity.}, language = {en} } @article{KellerLeidingerVogeletal.2014, author = {Keller, Andreas and Leidinger, Petra and Vogel, Britta and Backes, Christina and ElSharawy, Abdou and Galata, Valentina and Mueller, Sabine C. and Marquart, Sabine and Schrauder, Michael G. and Strick, Reiner and Bauer, Andrea and Wischhusen, J{\"o}rg and Beier, Markus and Kohlhaas, Jochen and Katus, Hugo A. and Hoheisel, J{\"o}rg and Franke, Andre and Meder, Benjamin and Meese, Eckart}, title = {miRNAs can be generally associated with human pathologies as exemplified for miR-144*}, series = {BMC MEDICINE}, volume = {12}, journal = {BMC MEDICINE}, issn = {1741-7015}, doi = {10.1186/s12916-014-0224-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114349}, pages = {224}, year = {2014}, abstract = {Background: miRNA profiles are promising biomarker candidates for a manifold of human pathologies, opening new avenues for diagnosis and prognosis. Beyond studies that describe miRNAs frequently as markers for specific traits, we asked whether a general pattern for miRNAs across many diseases exists. Methods: We evaluated genome-wide circulating profiles of 1,049 patients suffering from 19 different cancer and non-cancer diseases as well as unaffected controls. The results were validated on 319 individuals using qRT-PCR. Results: We discovered 34 miRNAs with strong disease association. Among those, we found substantially decreased levels of hsa-miR-144* and hsa-miR-20b with AUC of 0.751 ( 95\% CI: 0.703-0.799), respectively. We also discovered a set of miRNAs, including hsa-miR-155*, as rather stable markers, offering reasonable control miRNAs for future studies. The strong downregulation of hsa-miR-144* and the less variable pattern of hsa-miR-155* has been validated in a cohort of 319 samples in three different centers. Here, breast cancer as an additional disease phenotype not included in the screening phase has been included as the 20th trait. Conclusions: Our study on 1,368 patients including 1,049 genome-wide miRNA profiles and 319 qRT-PCR validations further underscores the high potential of specific blood-borne miRNA patterns as molecular biomarkers. Importantly, we highlight 34 miRNAs that are generally dysregulated in human pathologies. Although these markers are not specific to certain diseases they may add to the diagnosis in combination with other markers, building a specific signature. Besides these dysregulated miRNAs, we propose a set of constant miRNAs that may be used as control markers.}, language = {en} } @article{CorreiaSantosBischlerWestermannetal.2021, author = {Correia Santos, Sara and Bischler, Thorsten and Westermann, Alexander J. and Vogel, J{\"o}rg}, title = {MAPS integrates regulation of actin-targeting effector SteC into the virulence control network of Salmonella small RNA PinT}, series = {Cell Reports}, volume = {34}, journal = {Cell Reports}, number = {5}, doi = {10.1016/j.celrep.2021.108722}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259134}, pages = {108722}, year = {2021}, abstract = {A full understanding of the contribution of small RNAs (sRNAs) to bacterial virulence demands knowledge of their target suites under infection-relevant conditions. Here, we take an integrative approach to capturing targets of the Hfq-associated sRNA PinT, a known post-transcriptional timer of the two major virulence programs of Salmonella enterica. Using MS2 affinity purification and RNA sequencing (MAPS), we identify PinT ligands in bacteria under in vitro conditions mimicking specific stages of the infection cycle and in bacteria growing inside macrophages. This reveals PinT-mediated translational inhibition of the secreted effector kinase SteC, which had gone unnoticed in previous target searches. Using genetic, biochemical, and microscopic assays, we provide evidence for PinT-mediated repression of steC mRNA, eventually delaying actin rearrangements in infected host cells. Our findings support the role of PinT as a central post-transcriptional regulator in Salmonella virulence and illustrate the need for complementary methods to reveal the full target suites of sRNAs.}, language = {en} } @article{ElMoualiGerovacMineikaitėetal.2021, author = {El Mouali, Youssef and Gerovac, Milan and Mineikaitė, Raminta and Vogel, J{\"o}rg}, title = {In vivo targets of Salmonella FinO include a FinP-like small RNA controlling copy number of a cohabitating plasmid}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {9}, doi = {10.1093/nar/gkab281}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261072}, pages = {5319-5335}, year = {2021}, abstract = {FinO-domain proteins represent an emerging family of RNA-binding proteins (RBPs) with diverse roles in bacterial post-transcriptional control and physiology. They exhibit an intriguing targeting spectrum, ranging from an assumed single RNA pair (FinP/traJ) for the plasmid-encoded FinO protein, to transcriptome-wide activity as documented for chromosomally encoded ProQ proteins. Thus, the shared FinO domain might bear an unusual plasticity enabling it to act either selectively or promiscuously on the same cellular RNA pool. One caveat to this model is that the full suite of in vivo targets of the assumedly highly selective FinO protein is unknown. Here, we have extensively profiled cellular transcripts associated with the virulence plasmid-encoded FinO in Salmonella enterica. While our analysis confirms the FinP sRNA of plasmid pSLT as the primary FinO target, we identify a second major ligand: the RepX sRNA of the unrelated antibiotic resistance plasmid pRSF1010. FinP and RepX are strikingly similar in length and structure, but not in primary sequence, and so may provide clues to understanding the high selectivity of FinO-RNA interactions. Moreover, we observe that the FinO RBP encoded on the Salmonella virulence plasmid controls the replication of a cohabitating antibiotic resistance plasmid, suggesting cross-regulation of plasmids on the RNA level.}, language = {en} } @article{HombergerHaywardBarquistetal.2023, author = {Homberger, Christina and Hayward, Regan J. and Barquist, Lars and Vogel, J{\"o}rg}, title = {Improved bacterial single-cell RNA-seq through automated MATQ-seq and Cas9-based removal of rRNA reads}, series = {mBio}, volume = {14}, journal = {mBio}, number = {2}, doi = {10.1128/mbio.03557-22}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350059}, year = {2023}, abstract = {Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria. IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.}, language = {en} }