TY - JOUR A1 - Babski, Julia A1 - Haas, Karina A. A1 - Näther-Schindler, Daniela A1 - Pfeiffer, Friedhelm A1 - Förstner, Konrad U. A1 - Hammelmann, Matthias A1 - Hilker, Rolf A1 - Becker, Anke A1 - Sharma, Cynthia M. A1 - Marchfelder, Anita A1 - Soppa, Jörg T1 - Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq) JF - BMC Genomics N2 - Background Differential RNA-Seq (dRNA-Seq) is a recently developed method of performing primary transcriptome analyses that allows for the genome-wide mapping of transcriptional start sites (TSSs) and the identification of novel transcripts. Although the transcriptomes of diverse bacterial species have been characterized by dRNA-Seq, the transcriptome analysis of archaeal species is still rather limited. Therefore, we used dRNA-Seq to characterize the primary transcriptome of the model archaeon Haloferax volcanii. Results Three independent cultures of Hfx. volcanii grown under optimal conditions to the mid-exponential growth phase were used to determine the primary transcriptome and map the 5′-ends of the transcripts. In total, 4749 potential TSSs were detected. A position weight matrix (PWM) was derived for the promoter predictions, and the results showed that 64 % of the TSSs were preceded by stringent or relaxed basal promoters. Of the identified TSSs, 1851 belonged to protein-coding genes. Thus, fewer than half (46 %) of the 4040 protein-coding genes were expressed under optimal growth conditions. Seventy-two percent of all protein-coding transcripts were leaderless, which emphasized that this pathway is the major pathway for translation initiation in haloarchaea. A total of 2898 of the TSSs belonged to potential non-coding RNAs, which accounted for an unexpectedly high fraction (61 %) of all transcripts. Most of the non-coding TSSs had not been previously described (2792) and represented novel sequences (59 % of all TSSs). A large fraction of the potential novel non-coding transcripts were cis-antisense RNAs (1244 aTSSs). A strong negative correlation between the levels of antisense transcripts and cognate sense mRNAs was found, which suggested that the negative regulation of gene expression via antisense RNAs may play an important role in haloarchaea. The other types of novel non-coding transcripts corresponded to internal transcripts overlapping with mRNAs (1153 iTSSs) and intergenic small RNA (sRNA) candidates (395 TSSs). Conclusion This study provides a comprehensive map of the primary transcriptome of Hfx. volcanii grown under optimal conditions. Fewer than half of all protein-coding genes have been transcribed under these conditions. Unexpectedly, more than half of the detected TSSs belonged to several classes of non-coding RNAs. Thus, RNA-based regulation appears to play a more important role in haloarchaea than previously anticipated. KW - Archaea KW - dRNA-Seq KW - Promoter KW - Non-coding RNAs KW - sRNA KW - Haloferax volcanii KW - Transcriptome KW - Leaderless transcript KW - Antisense RNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164553 VL - 17 IS - 629 ER - TY - JOUR A1 - Čuklina, Jelena A1 - Hahn, Julia A1 - Imakaev, Maxim A1 - Omasits, Ulrich A1 - Förstner, Konrad U. A1 - Ljubimov, Nikolay A1 - Goebel, Melanie A1 - Pessi, Gabriella A1 - Fischer, Hans-Martin A1 - Ahrens, Christian H. A1 - Gelfand, Mikhail S. A1 - Evguenieva-Hackenberg, Elena T1 - Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation JF - BMC Genomics N2 - Background Differential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters. Results A specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file. Conclusions The genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes. KW - Bradyrhizobium KW - RNA-seq KW - Promoter prediction KW - Genome re-annotation KW - Internal transcription start site KW - Nodule KW - Transcription start site KW - Proteogenomics KW - Antisense RNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-164565 VL - 17 ER - TY - JOUR A1 - Krohn-Molt, Ines A1 - Alawi, Malik A1 - Förstner, Konrad U. A1 - Wiegandt, Alena A1 - Burkhardt, Lia A1 - Indenbirken, Daniela A1 - Thieß, Melanie A1 - Grundhoff, Adam A1 - Kehr, Julia A1 - Tholey, Andreas A1 - Streit, Wolfgang R. T1 - Insights into microalga and bacteria interactions of selected phycosphere biofilms using metagenomic, transcriptomic, and proteomic approaches JF - Frontiers in Microbiology N2 - Microalga are of high relevance for the global carbon cycling and it is well-known that they are associated with a microbiota. However, it remains unclear, if the associated microbiota, often found in phycosphere biofilms, is specific for the microalga strains and which role individual bacterial taxa play. Here we provide experimental evidence that \(Chlorella\) \(saccharophila\), \(Scenedesmus\) \(quadricauda\), and \(Micrasterias\) \(crux-melitensis\), maintained in strain collections, are associated with unique and specific microbial populations. Deep metagenome sequencing, binning approaches, secretome analyses in combination with RNA-Seq data implied fundamental differences in the gene expression profiles of the microbiota associated with the different microalga. Our metatranscriptome analyses indicates that the transcriptionally most active bacteria with respect to key genes commonly involved in plant–microbe interactions in the Chlorella (Trebouxiophyceae) and Scenedesmus (Chlorophyceae) strains belong to the phylum of the α-Proteobacteria. In contrast, in the Micrasterias (Zygnematophyceae) phycosphere biofilm bacteria affiliated with the phylum of the Bacteroidetes showed the highest gene expression rates. We furthermore show that effector molecules known from plant-microbe interactions as inducers for the innate immunity are already of relevance at this evolutionary early plant-microbiome level. KW - microbiology KW - microalga-bacteria interaction KW - phycosphere biofilm KW - metagenomics KW - metatranscriptomics KW - metaproteomics Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173701 VL - 2017 IS - 8 ER - TY - JOUR A1 - Gomes, Sara F. Martins A1 - Westermann, Alexander J. A1 - Sauerwein, Till A1 - Hertlein, Tobias A1 - Förstner, Konrad U. A1 - Ohlsen, Knut A1 - Metzger, Marco A1 - Shusta, Eric V. A1 - Kim, Brandon J. A1 - Appelt-Menzel, Antje A1 - Schubert-Unkmeir, Alexandra T1 - Induced pluripotent stem cell-derived brain endothelial cells as a cellular model to study Neisseria meningitidis infection JF - Frontiers in Microbiology N2 - Meningococcal meningitis is a severe central nervous system infection that occurs when Neisseria meningitidis (Nm) penetrates brain endothelial cells (BECs) of the meningeal blood-cerebrospinal fluid barrier. As a human-specific pathogen, in vivo models are greatly limited and pose a significant challenge. In vitro cell models have been developed, however, most lack critical BEC phenotypes limiting their usefulness. Human BECs generated from induced pluripotent stem cells (iPSCs) retain BEC properties and offer the prospect of modeling the human-specific Nm interaction with BECs. Here, we exploit iPSC-BECs as a novel cellular model to study Nm host-pathogen interactions, and provide an overview of host responses to Nm infection. Using iPSC-BECs, we first confirmed that multiple Nm strains and mutants follow similar phenotypes to previously described models. The recruitment of the recently published pilus adhesin receptor CD147 underneath meningococcal microcolonies could be verified in iPSC-BECs. Nm was also observed to significantly increase the expression of pro-inflammatory and neutrophil-specific chemokines IL6, CXCL1, CXCL2, CXCL8, and CCL20, and the secretion of IFN-γ and RANTES. For the first time, we directly observe that Nm disrupts the three tight junction proteins ZO-1, Occludin, and Claudin-5, which become frayed and/or discontinuous in BECs upon Nm challenge. In accordance with tight junction loss, a sharp loss in trans-endothelial electrical resistance, and an increase in sodium fluorescein permeability and in bacterial transmigration, was observed. Finally, we established RNA-Seq of sorted, infected iPSC-BECs, providing expression data of Nm-responsive host genes. Altogether, this model provides novel insights into Nm pathogenesis, including an impact of Nm on barrier properties and tight junction complexes, and suggests that the paracellular route may contribute to Nm traversal of BECs. KW - Neisseria meningitidis KW - meningococcus KW - bacteria KW - stem cells KW - blood-cerebrospinal fluid barrier KW - blood-brain barrier KW - brain endothelial cells Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201562 VL - 10 IS - 1181 ER - TY - JOUR A1 - Yu, Sung-Huan A1 - Vogel, Jörg A1 - Förstner, Konrad U. T1 - ANNOgesic: a Swiss army knife for the RNA-seq based annotation of bacterial/archaeal genomes JF - GigaScience N2 - To understand the gene regulation of an organism of interest, a comprehensive genome annotation is essential. While some features, such as coding sequences, can be computationally predicted with high accuracy based purely on the genomic sequence, others, such as promoter elements or noncoding RNAs, are harder to detect. RNA sequencing (RNA-seq) has proven to be an efficient method to identify these genomic features and to improve genome annotations. However, processing and integrating RNA-seq data in order to generate high-resolution annotations is challenging, time consuming, and requires numerous steps. We have constructed a powerful and modular tool called ANNOgesic that provides the required analyses and simplifies RNA-seq-based bacterial and archaeal genome annotation. It can integrate data from conventional RNA-seq and differential RNA-seq and predicts and annotates numerous features, including small noncoding RNAs, with high precision. The software is available under an open source license (ISCL) at https://pypi.org/project/ANNOgesic/. KW - genome annotation KW - RNA-seq KW - transcriptomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-178942 VL - 7 ER - TY - JOUR A1 - Weidner, Magdalena T. A1 - Lardenoije, Roy A1 - Eijssen, Lars A1 - Mogavero, Floriana A1 - De Groodt, Lilian P. M. T. A1 - Popp, Sandy A1 - Palme, Rupert A1 - Förstner, Konrad U. A1 - Strekalova, Tatyana A1 - Steinbusch, Harry W. M. A1 - Schmitt-Böhrer, Angelika G. A1 - Glennon, Jeffrey C. A1 - Waider, Jonas A1 - van den Hove, Daniel L. A. A1 - Lesch, Klaus-Peter T1 - Identification of cholecystokinin by genome-wide profiling as potential mediator of serotonin-dependent behavioral effects of maternal separation in the amygdala JF - Frontiers in Neuroscience N2 - Converging evidence suggests a role of serotonin (5-hydroxytryptamine, 5-HT) and tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme of 5-HT synthesis in the brain, in modulating long-term, neurobiological effects of early-life adversity. Here, we aimed at further elucidating the molecular mechanisms underlying this interaction, and its consequences for socio-emotional behaviors, with a focus on anxiety and social interaction. In this study, adult, male Tph2 null mutant (Tph2\(^{-/-}\)) and heterozygous (Tph2\(^{+/-}\)) mice, and their wildtype littermates (Tph2\(^{+/+}\)) were exposed to neonatal, maternal separation (MS) and screened for behavioral changes, followed by genome-wide RNA expression and DNA methylation profiling. In Tph2\(^{-/-}\) mice, brain 5-HT deficiency profoundly affected socio-emotional behaviors, i.e., decreased avoidance of the aversive open arms in the elevated plus-maze (EPM) as well as decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Tph2\(^{+/-}\) mice showed an ambiguous profile with context-dependent, behavioral responses. In the EPM they showed similar avoidance of the open arm but decreased prosocial and increased rule breaking behavior in the resident-intruder test when compared to their wildtype littermates. Notably, MS effects on behavior were subtle and depended on the Tph2 genotype, in particular increasing the observed avoidance of EPM open arms in wildtype and Tph2\(^{+/-}\) mice when compared to their Tph2\(^{-/-}\) littermates. On the genomic level, the interaction of Tph2 genotype with MS differentially affected the expression of numerous genes, of which a subset showed an overlap with DNA methylation profiles at corresponding loci. Remarkably, changes in methylation nearby and expression of the gene encoding cholecystokinin, which were inversely correlated to each other, were associated with variations in anxiety-related phenotypes. In conclusion, next to various behavioral alterations, we identified gene expression and DNA methylation profiles to be associated with TPH2 inactivation and its interaction with MS, suggesting a gene-by-environment interaction-dependent, modulatory function of brain 5-HT availability. KW - serotonin KW - maternal separation KW - mouse KW - emotional behavior KW - DNA methylation KW - RNA expression Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201340 VL - 13 ER - TY - JOUR A1 - Westermann, Alexander J. A1 - Venturini, Elisa A1 - Sellin, Mikael E. A1 - Förstner, Konrad U. A1 - Hardt, Wolf-Dietrich A1 - Vogel, Jörg T1 - The major RNA-binding protein ProQ impacts virulence gene expression in Salmonella enterica serovar Typhimurium JF - mBio N2 - FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs. IMPORTANCE The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria. KW - Hfq KW - noncoding RNA KW - ProQ KW - RNA-seq KW - bacterial pathogen KW - posttranscriptional control Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177722 VL - 10 IS - 1 ER - TY - JOUR A1 - Esken, Jens A1 - Goris, Tobias A1 - Gadkari, Jennifer A1 - Bischler, Thorsten A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Diekert, Gabriele A1 - Schubert, Torsten T1 - Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two‐component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies JF - MicrobiologyOpen N2 - Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE‐respiring representatives of the genus, uncovered the genetic inactivation of a two‐component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low‐level expression of the TCS operon in fumarate‐adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR‐family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter‐binding assays. The RR bound a cis‐regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the −35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine‐tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide‐respiring bacteria. KW - genomics KW - organohalide respiration KW - RNA sequencing KW - tetrachloroethene KW - transcriptomics KW - two‐component system Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225754 VL - 9 IS - 12 ER - TY - JOUR A1 - Sharan, Malvika A1 - Förstner, Konrad U. A1 - Eulalio, Ana A1 - Vogel, Jörg T1 - APRICOT: an integrated computational pipeline for the sequence-based identification and characterization of RNA-binding proteins JF - Nucleic Acids Research N2 - RNA-binding proteins (RBPs) have been established as core components of several post-transcriptional gene regulation mechanisms. Experimental techniques such as cross-linking and co-immunoprecipitation have enabled the identification of RBPs, RNA-binding domains (RBDs) and their regulatory roles in the eukaryotic species such as human and yeast in large-scale. In contrast, our knowledge of the number and potential diversity of RBPs in bacteria is poorer due to the technical challenges associated with the existing global screening approaches. We introduce APRICOT, a computational pipeline for the sequence-based identification and characterization of proteins using RBDs known from experimental studies. The pipeline identifies functional motifs in protein sequences using position-specific scoring matrices and Hidden Markov Models of the functional domains and statistically scores them based on a series of sequence-based features. Subsequently, APRICOT identifies putative RBPs and characterizes them by several biological properties. Here we demonstrate the application and adaptability of the pipeline on large-scale protein sets, including the bacterial proteome of Escherichia coli. APRICOT showed better performance on various datasets compared to other existing tools for the sequence-based prediction of RBPs by achieving an average sensitivity and specificity of 0.90 and 0.91 respectively. The command-line tool and its documentation are available at https://pypi.python.org/pypi/bio-apricot. KW - RNA-binding proteins KW - identification KW - characterization Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-157963 VL - 45 IS - 11 ER - TY - JOUR A1 - Meier, Doreen A1 - Kruse, Janis A1 - Buttlar, Jann A1 - Friedrich, Michael A1 - Zenk, Fides A1 - Boesler, Benjamin A1 - Forstner, Konrad U. A1 - Hammann, Christian A1 - Nellen, Wolfgang T1 - Analysis of the Microprocessor in Dictyostelium: The Role of RbdB, a dsRNA Binding Protein JF - PLoS Genetics N2 - We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB- strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class. KW - microprocessor KW - Dictyostelium discoideum KW - dsRNA binding protein KW - RbdB Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-166687 VL - 12 IS - 6 ER -