@article{NguyenMuellerParketal.2014, author = {Nguyen, Tu N. and M{\"u}ller, Laura S. M. and Park, Sung Hee and Siegel, T. Nicolai and G{\"u}nzl, Arthur}, title = {Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei}, series = {Nucleic Acid Research}, volume = {42}, journal = {Nucleic Acid Research}, number = {5}, issn = {1362-4962}, doi = {10.1093/nar/gkt1301}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117232}, pages = {3164-3176}, year = {2014}, abstract = {Monoallelic expression within a gene family is found in pathogens exhibiting antigenic variation and in mammalian olfactory neurons. Trypanosoma brucei, a lethal parasite living in the human bloodstream, expresses variant surface glycoprotein (VSG) from 1 of 15 bloodstream expression sites (BESs) by virtue of a multifunctional RNA polymerase I. The active BES is transcribed in an extranucleolar compartment termed the expression site body (ESB), whereas silent BESs, located elsewhere within the nucleus, are repressed epigenetically. The regulatory mechanisms, however, are poorly understood. Here we show that two essential subunits of the basal class I transcription factor A (CITFA) predominantly occupied the promoter of the active BES relative to that of a silent BES, a phenotype that was maintained after switching BESs in situ. In these experiments, high promoter occupancy of CITFA was coupled to high levels of both promoter-proximal RNA abundance and RNA polymerase I occupancy. Accordingly, fluorescently tagged CITFA-7 was concentrated in the nucleolus and the ESB. Because a ChIP-seq analysis found that along the entire BES, CITFA-7 is specifically enriched only at the promoter, our data strongly indicate that monoallelic BES transcription is activated by a mechanism that functions at the level of transcription initiation.}, language = {en} } @article{BischlerKopfVoss2014, author = {Bischler, Thorsten and Kopf, Matthias and Voss, Bjoern}, title = {Transcript mapping based on dRNA-seq data}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {122}, issn = {1471-2105}, doi = {10.1186/1471-2105-15-122}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116663}, year = {2014}, abstract = {Background: RNA-seq and its variant differential RNA-seq (dRNA-seq) are today routine methods for transcriptome analysis in bacteria. While expression profiling and transcriptional start site prediction are standard tasks today, the problem of identifying transcriptional units in a genome-wide fashion is still not solved for prokaryotic systems. Results: We present RNASEG, an algorithm for the prediction of transcriptional units based on dRNA-seq data. A key feature of the algorithm is that, based on the data, it distinguishes between transcribed and un-transcribed genomic segments. Furthermore, the program provides many different predictions in a single run, which can be used to infer the significance of transcriptional units in a consensus procedure. We show the performance of our method based on a well-studied dRNA-seq data set for Helicobacter pylori. Conclusions: With our algorithm it is possible to identify operons and 5'- and 3'-UTRs in an automated fashion. This alleviates the need for labour intensive manual inspection and enables large-scale studies in the area of comparative transcriptomics.}, language = {en} } @article{WagnerVolkmerSharanetal.2014, author = {Wagner, Ines and Volkmer, Michael and Sharan, Malvika and Villaveces, Jose M. and Oswald, Felix and Surendranath, Vineeth and Habermann, Bianca H.}, title = {morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring}, series = {BMC Bioinformatics}, volume = {15}, journal = {BMC Bioinformatics}, number = {263}, doi = {10.1186/1471-2105-15-263}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115590}, year = {2014}, abstract = {Background: Searching the orthologs of a given protein or DNA sequence is one of the most important and most commonly used Bioinformatics methods in Biology. Programs like BLAST or the orthology search engine Inparanoid can be used to find orthologs when the similarity between two sequences is sufficiently high. They however fail when the level of conservation is low. The detection of remotely conserved proteins oftentimes involves sophisticated manual intervention that is difficult to automate. Results: Here, we introduce morFeus, a search program to find remotely conserved orthologs. Based on relaxed sequence similarity searches, morFeus selects sequences based on the similarity of their alignments to the query, tests for orthology by iterative reciprocal BLAST searches and calculates a network score for the resulting network of orthologs that is a measure of orthology independent of the E-value. Detecting remotely conserved orthologs of a protein using morFeus thus requires no manual intervention. We demonstrate the performance of morFeus by comparing it to state-of-the-art orthology resources and methods. We provide an example of remotely conserved orthologs, which were experimentally shown to be functionally equivalent in the respective organisms and therefore meet the criteria of the orthology-function conjecture. Conclusions: Based on our results, we conclude that morFeus is a powerful and specific search method for detecting remotely conserved orthologs.}, language = {en} } @article{TalmanPrietoMarquesetal.2014, author = {Talman, Arthur M. and Prieto, Judith H. and Marques, Sara and Ubaida-Mohien, Ceereena and Lawniczak, Mara and Wass, Mark N. and Xu, Tao and Frank, Roland and Ecker, Andrea and Stanway, Rebecca S. and Krishna, Sanjeev and Sternberg, Michael J. E. and Christophides, Georges K. and Graham, David R. and Dinglasan, Rhoel R. and Yates, John R., III and Sinden, Robert E.}, title = {Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility}, series = {Malaria Journal}, volume = {13}, journal = {Malaria Journal}, number = {315}, doi = {10.1186/1475-2875-13-315}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115572}, year = {2014}, abstract = {Background: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. Methods: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. Results: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. Conclusions: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization.}, language = {en} } @article{RemesBerghoffFoerstneretal.2014, author = {Remes, Bernhard and Berghoff, Bork A. and F{\"o}rstner, Konrad U. and Klug, Gabriele}, title = {Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides}, series = {BMC Genomics}, volume = {15}, journal = {BMC Genomics}, number = {794}, issn = {1471-2164}, doi = {10.1186/1471-2164-15-794}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115357}, year = {2014}, abstract = {Background: High intracellular levels of unbound iron can contribute to the production of reactive oxygen species (ROS) via the Fenton reaction, while depletion of iron limits the availability of iron-containing proteins, some of which have important functions in defence against oxidative stress. Vice versa increased ROS levels lead to the damage of proteins with iron sulphur centres. Thus, organisms have to coordinate and balance their responses to oxidative stress and iron availability. Our knowledge of the molecular mechanisms underlying the co-regulation of these responses remains limited. To discriminate between a direct cellular response to iron limitation and indirect responses, which are the consequence of increased levels of ROS, we compared the response of the alpha-proteobacterium Rhodobacter sphaeroides to iron limitation in the presence or absence of oxygen. Results: One third of all genes with altered expression under iron limitation showed a response that was independent of oxygen availability. The other iron-regulated genes showed different responses in oxic or anoxic conditions and were grouped into six clusters based on the different expression profiles. For two of these clusters, induction in response to iron limitation under oxic conditions was dependent on the OxyR regulatory protein. An OxyR mutant showed increased ROS production and impaired growth under iron limitation. Conclusion: Some R. sphaeroides genes respond to iron limitation irrespective of oxygen availability. These genes therefore reflect a "core iron response" that is independent of potential ROS production under oxic, iron-limiting conditions. However, the regulation of most of the iron-responsive genes was biased by oxygen availability. Most strikingly, the OxyR-dependent activation of a subset of genes upon iron limitation under oxic conditions, including many genes with a role in iron metabolism, revealed that elevated ROS levels were an important trigger for this response. OxyR thus provides a regulatory link between the responses to oxidative stress and to iron limitation in R. sphaeroides.}, language = {en} } @article{LindgreenUmuLaietal.2014, author = {Lindgreen, Stinus and Umu, Sinan Uğur and Lai, Alicia Sook-Wei and Eldai, Hisham and Liu, Wenting and McGimpsey, Stephanie and Wheeler, Nicole E. and Biggs, Patrick J. and Thomson, Nick R. and Barquist, Lars and Poole, Anthony M. and Gardner, Paul P.}, title = {Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling}, series = {PLOS Computational Biology}, volume = {10}, journal = {PLOS Computational Biology}, number = {10}, doi = {10.1371/journal.pcbi.1003907}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115259}, pages = {e1003907}, year = {2014}, abstract = {Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.}, language = {en} } @article{RicoYepesRodriguezetal.2014, author = {Rico, Sergio and Yepes, Ana and Rodriguez, Hector and Santamaria, Jorge and Antoraz, Sergio and Krause, Eva M. and Diaz, Margarita and Santamaria, Ramon I.}, title = {Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {10}, issn = {1932-6203}, doi = {10.1371/journal.pone.0109844}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115151}, pages = {e109844}, year = {2014}, abstract = {The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant DabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the DabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.}, language = {en} } @article{MoellerOverloeperFoerstneretal.2014, author = {M{\"o}ller, Philip and Overl{\"o}per, Aaron and F{\"o}rstner, Konrad U. and Wen, Tuan-Nan and Sharma, Cynthia M. and Lai, Erh-Min and Narberhaus, Franz}, title = {Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens}, series = {PLOS ONE}, volume = {9}, journal = {PLOS ONE}, number = {10}, doi = {10.1371/journal.pone.0110427}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114874}, pages = {e110427}, year = {2014}, abstract = {As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq 3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55\% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens.}, language = {en} } @article{ZukherNovikovaTikhonovetal.2014, author = {Zukher, Inna and Novikova, Maria and Tikhonov, Anton and Nesterchuk, Mikhail V. and Osterman, Ilya A. and Djordjevic, Marko and Sergiev, Petr V. and Sharma, Cynthia M. and Severinov, Konstantin}, title = {Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C}, series = {Nucleic Acids Research}, volume = {42}, journal = {Nucleic Acids Research}, number = {19}, issn = {0305-1048}, doi = {10.1093/nar/gku880}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114839}, pages = {11891-11902}, year = {2014}, abstract = {Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori.}, 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} }