TY - JOUR A1 - Jäger, Dominik A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Santangelo, Thomas J. A1 - Reeve, John N. T1 - Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis JF - BMC Genomics N2 - Background Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions. Results Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile. Conclusion The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon. KW - riboswitch KW - hyperthermophile KW - hydrogen regulation KW - transcriptome KW - archaea KW - promoters KW - antisense RNAs KW - small non-coding RNAs Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120966 SN - 1471-2164 VL - 15 IS - 684 ER - TY - JOUR A1 - Glaser, Jan A1 - Schurigt, Uta A1 - Suzuki, Brian M. A1 - Caffrey, Connor R. A1 - Holzgrabe, Ulrike T1 - Anti-Schistosomal Activity of Cinnamic Acid Esters: Eugenyl JF - Molecules N2 - Bornyl caffeate (1) was previously isolated by us from Valeriana (V.) wallichii rhizomes and identified as an anti-leishmanial substance. Here, we screened a small compound library of synthesized derivatives 1–30 for activity against schistosomula of Schistosoma (S.) mansoni. Compound 1 did not show any anti-schistosomal activity. However, strong phenotypic changes, including the formation of vacuoles, degeneration and death were observed after in vitro treatment with compounds 23 (thymyl cinnamate) and 27 (eugenyl cinnamate). Electron microscopy analysis of the induced vacuoles in the dying parasites suggests that 23 and 27 interfere with autophagy. KW - thymyl cinnamate KW - vacuoles KW - autophagy KW - anti-schistosomal activity KW - schistosoma KW - schistosomula KW - parasite KW - eugenyl cinnamate Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125712 VL - 20 ER - TY - JOUR A1 - Schneider, Johannes A1 - Klein, Teresa A1 - Mielich-Süss, Benjamin A1 - Koch, Gudrun A1 - Franke, Christian A1 - Kuipers, Oskar P. A1 - Kovács, Ákos T. A1 - Sauer, Markus A1 - Lopez, Daniel T1 - Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium JF - PLoS Genetics N2 - Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. KW - membrane proteins KW - gene expression KW - bacillus subtilis KW - fluorescence microscopy KW - cell fusion KW - signal transduction KW - gene regulation KW - lipids Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125577 VL - 11 IS - 4 ER - TY - JOUR A1 - Masic, Anita A1 - Valencia Hernandez, Ana Maria A1 - Hazra, Sudipta A1 - Glaser, Jan A1 - Holzgrabe, Ulrike A1 - Hazra, Banasri A1 - Schurigt, Uta T1 - Cinnamic Acid Bornyl Ester Derivatives from Valeriana wallichii Exhibit Antileishmanial In Vivo Activity in Leishmania major-Infected BALB/c Mice JF - PLoS One N2 - Human leishmaniasis covers a broad spectrum of clinical manifestations ranging from self-healing cutaneous leishmaniasis to severe and lethal visceral leishmaniasis caused among other species by Leishmania major or Leishmania donovani, respectively. Some drug candidates are in clinical trials to substitute current therapies, which are facing emerging drug-resistance accompanied with serious side effects. Here, two cinnamic acid bornyl ester derivatives (1 and 2) were assessed for their antileishmanial activity. Good selectivity and antileishmanial activity of bornyl 3-phenylpropanoate (2) in vitro prompted the antileishmanial assessment in vivo. For this purpose, BALB/c mice were infected with Leishmania major promastigotes and treated with three doses of 50 mg/kg/day of compound 2. The treatment prevented the characteristic swelling at the site of infection and correlated with reduced parasite burden. Transmitted light microscopy and transmission electron microscopy of Leishmania major promastigotes revealed that compounds 1 and 2 induce mitochondrial swelling. Subsequent studies on Leishmania major promastigotes showed the loss of mitochondrial transmembrane potential (ΔΨm) as a putative mode of action. As the cinnamic acid bornyl ester derivatives 1 and 2 had exhibited antileishmanial activity in vitro, and compound 2 in Leishmania major-infected BALB/c mice in vivo, they can be regarded as possible lead structures for the development of new antileishmanial therapeutic approaches. KW - leishmania major KW - promastigotes KW - apoptosis KW - mitochondria KW - parasitic diseases KW - leishmania KW - leishmaniasis KW - mouse models Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125354 VL - 10 IS - 11 ER - TY - JOUR A1 - Frank, Benjamin A1 - Marcu, Ana A1 - de Oliveira Almeida Petersen, Antonio Luis A1 - Weber, Heike A1 - Stigloher, Christian A1 - Mottram, Jeremy C. A1 - Scholz, Claus Jürgen A1 - Schurigt, Uta T1 - Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210 JF - Parasites & Vectors N2 - Background Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed. Methods BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix® chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining. Results The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix® chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages. Conclusions Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients. KW - autophagy KW - BNIP3 KW - CTSE KW - electron tomography KW - leishmania major KW - macrophages KW - miRNAs KW - MTOR KW - siRNAs KW - transmission electron microscopy Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-124997 VL - 8 IS - 404 ER - TY - JOUR A1 - Amich, Jorge A1 - Krappmann, Sven T1 - Deciphering metabolic traits of the fungal pathogen Aspergillus fumigatus: redundancy vs. essentiality JF - Frontiers in Microbiology N2 - Incidence rates of infections caused by environmental opportunistic fungi have risen over recent decades. Aspergillus species have emerged as serious threat for the immunecompromised, and detailed knowledge about virulence-determining traits is crucial for drug target identification. As a prime saprobe, A. fumigatus has evolved to efficiently adapt to various stresses and to sustain nutritional supply by osmotrophy, which is characterized by extracellular substrate digestion followed by efficient uptake of breakdown products that are then fed into the fungal primary metabolism. These intrinsic metabolic features are believed to be related with its virulence ability. The plethora of genes that encode underlying effectors has hampered their in-depth analysis with respect to pathogenesis. Recent developments in Aspergillus molecular biology allow conditional gene expression or comprehensive targeting of gene families to cope with redundancy. Furthermore, identification of essential genes that are intrinsically connected to virulence opens accurate perspectives for novel targets in antifungal therapy. KW - Aspergillus fumigatus KW - aspergillosis KW - virulence KW - conditional promoter replacement KW - nutrients KW - gene family targeting Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-123669 VL - 3 ER - TY - THES A1 - Sturm, Volker Jörg Friedrich T1 - \(^{19}F\) Magnetresonanztomographie zur Bildgebung von Infektionen im Zeitverlauf T1 - \(^{19}F\) magnetic resonance imaging to monitor the timecourse of bacterial infections in vivo N2 - Im Rahmen dieser Arbeit sollten die Möglichkeiten der MR Tomographie erkundet werden bakterielle Infektionen im Zeitverlauf darzustellen. Genauer gesagt sollte das Potential der MR Tomographie anhand eines durch eine Infektion induzierten lokalisierten Abszesses unter Verwendung dreier unterschiedlicher MRT Methoden untersucht werden: Mittels nativem \(T_2\) Kontrast; der Verwendung von superparamagnetischen Eisenoxid Partieln (USPIO) als \(T_2^*\) Kontrastmittel; und dem Einsatz von Perfluorkarbonen (PFC) als \(^{19}F\) MRT Marker (siehe Kapitel 3). Wie erwartet führte die durch die Infektion hervorgerufene Entzündung zu veränderten \(T_2\)-Zeiten, welche auf \(T_2\)-gewichteten MR Bildern eine Lokalisierung des Abszessbereiches erlauben. Jedoch eigneten sich diese Daten aufgrund der graduellen Änderung der \(T_2\)-Zeiten nicht, um eine klare Grenze zwischen Abszess und umliegendem Gewebe zu ziehen. Superparamagnetische Eisenoxidpartikel andererseit haben als MRT Kontrastmittel bereits in den letzten Jahren ihre Fähigkeit unter Beweis gestellt Entzündungen [53, 58, 64] darzustellen. Die Anreicherung dieser Partikel am Rande des Abszesses [53], wie sie auch in unseren MR Daten zu beobachten war, erlaubte eine relativ scharfe Abgrenzung gegenüber dem umgebenden Gewebe in der chronischen Phase der Infektion (Tag 9 p.i.). Hingegen genügte die nur sehr spärlichen Anreicherung von USPIO Partikeln in der akuten Phase der Infektion (Tag 3 p.i.) nicht für eine entsprechende Abgrenzung [58]. Aufgrund der sehr geringen biologischen Häufigkeit und den sehr kurzen Relaxationszeiten von endogenem Fluor eignen sich Perfluorkarbone als Markersubstanz in der MR Tomographie von biologischen Systemen. Insbesondere da PFC Emulsionen durch phagozytierende Zellen aufgenommen werden und im Bereich von Entzündungen akkumulieren [30, 59]. In dieser Arbeit konnte anhand der erhaltenen MRT Daten eine Akkumulation von Perfluorkarbonen nicht nur in der chronischen Phase, sondern auch in der akuten Phase nachgewiesen werden. Diese Daten erlauben somit zu allen untersuchten Zeitpunkten eine Abgrenzung zwischen Infektion und umliegenden Gewebe. Aufgrund der besagten Vorteile wurden die Perfluorkarbone gewählt, um die Möglichkeiten der MR Tomographie zu testen, quantitative Informationen über die schwere der Infektion zu liefern. Als Referenz für die Bakterienbelastung wurden die Biolumineszenzbildgebung (BLI) [49, 50] und die Standardmethode zur Bestimmung der Bakterienbelastung cfu (koloniebildenden Einheiten) herangezogen. Eine Gegenüberstellung der zeitlichen Verläufe der durch die Biolumineszenzbildgebung und durch die cfu erhaltenen Daten liefert eine qualitative Übereinstimmung mit den durch die 19F MR Tomographie erhaltenen Daten. Dies trifft hierbei sowohl auf die über den gesamten Infektionsbereich hinweg summierten Signalamplituden, als auch auf das Volumen zu, in dem Fluor am Ort der Infektion akkumuliert wurde. Im Gegensatz zur Methode der cfu Bestimmung sind die MR Tomographie und die Biolumineszenzbildgebung nicht invasiv und erlauben die Verfolgung des Infektionsverlaufes an einem einzelnen Individuum. Hierzu benötigt, im Gegensatz zur MR Tomographie, die Methode der Biolumineszenzbildgebung jedoch einen speziellen Pathogenstamm. Darüber hinaus ist hervorzuheben, dass die MR Tomographie zudem die Möglichkeit bietet auch morphologische Informationen über den Infektionsbereich und seine Umgebung zu akquirieren. Gerade weil jede dieser Methoden die mit der Infektion einhergehenden Prozesse aus einer leicht anderen Blickrichtung betrachtet, erscheint es sinnvoll diese etablierte Untersuchungsplattform bestehend aus MRT, BLI und cfu über die in dieser Arbeit bearbeitete Fragestellung hinaus näher zu untersuchen. Insbesondere der Aspekt inwieweit die drei Methoden sich gegenseitig ergänzen, könnte einen tieferen Einblick in die Wechselwirkung zwischen Pathogen und Wirt erlauben. Auch wenn für die betrachtete Fragestellung bereits der hierdurchgeführte semiquanitative Ansatz zur Bestimmung der relativen Fluormengen am Ort der Infektion ausreichte, so ist doch im Allgemeinen wünschenswert probenbezogen die Sensitivität der Spule und damit die Güte der Spulenabstimmung zu bestimmen. Hierzu ist jedoch die Aufnahme von \(B_1\)-Karten unabdingbar und wird entsprechend im Kapitel 4 \(Bloch-Siegert B_1^+-Mapping\) näher addressiert. Der Schwerpunkt liegt hierbei, wie der Kapitelname bereits andeutet, auf der Bloch-Siegert Methode, die insbesondere in der präsentierten Implementierung in einer Turbo/ Multi Spin Echo Sequenz eine effiziente Nutzung der relativ langen \(T_\)2-Zeiten der Perfluorkarbone erlaubt. Da zudem die Bloch-Siegert-Methode eine rein phasenbasierte Methode ist, kann neben der aus den Daten erzeugten \(B_1\)-Karte zugleich ein unverfälschtes Magnitudenbild generiert werden, wodurch eine sehr effiziente Nutzung der vorhandenen Messzeit ermöglicht wird. Diese Eigenschaft ist insbesondere für \(^{19}F\) Bildgebung von besonderem Interesse, da hier für jede Messung, aufgrund der üblicherweise relativ geringen Konzentration an Fluoratomen, lange Messzeiten benötigt werden. Zusammenfassend konnte anhand des untersuchten Tiermodells sowohl die Fähigkeit der MR Tomographie nachgewiesen werden Infektionen im Zeitverlauf darzustellen, als auch die Fähigkeit der MR Tomographie quantitative Informationen über den Verlauf der Infektion zu liefern. Desweiteren konnte eine Möglichkeit aufgezeigt werden, welche das Potential hat in vertretbarem Zeitrahmen auch in vivo B1+-Karten auf dem Fluorkanal zu erstellen und so einen zentralen Unsicherheitsfaktor, für Relaxometry und absolute Quantifizierung von \(^{19}F\) Daten in vivo, zu beseitigen. N2 - The main focus of this work is to investigate the potential of magnetic resonance imaging (MRI) to monitor the timecourse of bacterial infections in vivo. More specifically, it focuses on the ability to localize and assess an infection-induced localized bulky abscess using three different MRI methods: the utilization of native \(T_2\) contrast; the usage of super paramagnetic iron oxide nanoparticles (USPIO) as MRI \(T_2^*\) contrast agents; and the application of perfluorcarbons (PFC) as \(^{19}F\) MRI marker (see chapter 3). Study results demonstrated that, as expected the altered \(T_2\) values present in the abscess area permit localization of the infection when using \(T_2\) weighted data. The precise boundary of the abscess, however, could not be determined due to the gradual change of the \(T_2\) values in the area of the infection. Conforming to other studies [53, 58], the MR-detected accumulation of USPIO particles along the abscess rim allowed definition of a fairly exact demarcation line between the abscess and surrounding tissue during the chronic phase of the infection (day 9 p.i.). During the acute phase of the infection (day 3 p.i.), however, the particle accumulation at the abscess rim was too sparse for precise boundary definition [58]. Because of their extremely low biological abundance and the very short relaxation times of endogenous fluorine, PFCs can be imaged background-free in a biological system. Moreover, as emulsified PFCs were taken up by phagocytosing cells and accumulated at the site of inflammation [30, 59], the acquired MRI data showed PFC accumulation during both the chronic and acute phases of infection. It was thus possible to differentiate between the abscess and surrounding tissue at each examined time point. Due to the described advantages, PFCs were chosen to evaluate with MRI the infection severity. As a bacterial burden reference, colony forming units (cfu) and bioluminescence imaging (BLI) [49, 50] were selected. Observation of BLI, cfu and \(^{19}F\) MRI data showed qualitative correlation during the investigated time course. This was true for the accumulated \(^{19}F\) MR signal in the area of infection and for the \(^{19}F\) MR signal volume. Additionally, unlike the cfu method MRI and BLI are non-invasive and thus data can be gathered at multiple time points. However, contrary to BLI, MRI does not require a special pathogen strain. Moreover, it can provide morphological data from an abscess and the surrounding tissue. Because the data delivered by each of these three methods (MRI, BLI and cfu), are based on alternative approaches, additional examinations of the established platform are suggested. For example, the extent to which the methods supplement each other may provide deeper insight into the interaction between pathogen and host. Even though the chosen semi quantitative approach was sufficient in the context of the evaluated issues to estimate the relative fluorine amount at the site of infection, it is in general desirable for each quantification to determine the sensitivity of the coil per sample. To address this issue the Bloch Siegert (BS) based \(B_1\) mapping method implemented in a turbo/ multi spin echo (TSE/MSE) sequence is presented in Chapter 4 Bloch-Siegert \(B_1^+\)-Mapping. Such a sequence allows effective use of the relatively long PFC \(T_2\) times and encodes BS information solely into the phase data. Thus, a \(B_1\) map can be created in addition to the unaltered TSE/MSE magnitude image. In the context of \(^{19}F\) imaging, this is of special interest due to the usually low amounts of fluorine resulting in long measurement times. In conclusion, it was shown that MRI not only enables visualization of the temporal behavior of infections on the investigated animal model, but it can also provide quantitative information about the progress of the infection. Additionally, a method potentially allowing in vivo B1+ mapping was introduced. This is an important step to improve the reliability of relaxometry and absolute quantification of in vivo \(^{19}F\) MRI. KW - Kernspintomografie KW - Bakterielle Infektion KW - 19F MR KW - Perfluorkarbon KW - Infektionsbildgebung KW - Bloch Siegert KW - B1 Mapping KW - Kontrastmittel Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-122851 ER - TY - JOUR A1 - Siegel, T. Nicolai A1 - Hon, Chung-Chau A1 - Zhang, Qinfeng A1 - Lopez-Rubio, Jose-Juan A1 - Scheidig-Benatar, Christine A1 - Martins, Rafeal M. A1 - Sismeiro, Odile A1 - Coppée, Jean-Yves T1 - Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum JF - BMC Genomics N2 - Background Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets. Results To gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3′-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes. Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3′ untranslated region length of 523 bp. Conclusions Our strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3′ UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum. KW - directional RNA-Seq KW - ncRNA KW - natural antisense transcripts KW - 3′ UTR KW - polyadenylation sites KW - genes KW - antisense RNA KW - plasmodium falciparum Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119892 VL - 15 ER - TY - JOUR A1 - Adelfinger, Marion A1 - Gentschev, Ivaylo A1 - de Guibert, Julio Grimm A1 - Weibel, Stephanie A1 - Langbein-Laugwitz, Johanna A1 - Härtl, Barbara A1 - Escobar, Hugo Murua A1 - Nolte, Ingo A1 - Chen, Nanhai G. A1 - Aguilar, Richard J. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Frentzen, Alexa A1 - Szalay, Aladar A. T1 - Evaluation of a New Recombinant Oncolytic Vaccinia Virus Strain GLV-5b451 for Feline Mammary Carcinoma Therapy JF - PLoS ONE N2 - Virotherapy on the basis of oncolytic vaccinia virus (VACV) infection is a promising approach for cancer therapy. In this study we describe the establishment of a new preclinical model of feline mammary carcinoma (FMC) using a recently established cancer cell line, DT09/06. In addition, we evaluated a recombinant vaccinia virus strain, GLV-5b451, expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as an oncolytic agent against FMC. Cell culture data demonstrate that GLV-5b451 virus efficiently infected, replicated in and destroyed DT09/06 cancer cells. In the selected xenografts of FMC, a single systemic administration of GLV-5b451 led to significant inhibition of tumor growth in comparison to untreated tumor-bearing mice. Furthermore, tumor-specific virus infection led to overproduction of functional scAb GLAF-2, which caused drastic reduction of intratumoral VEGF levels and inhibition of angiogenesis. In summary, here we have shown, for the first time, that the vaccinia virus strains and especially GLV-5b451 have great potential for effective treatment of FMC in animal model. KW - antibodies KW - cancer treatment KW - carcinomas KW - vaccinia virus KW - oncolytic viruses KW - viral replication KW - cell cultures KW - enzyme-linked immunoassays Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119387 VL - 9 IS - 8 ER - TY - JOUR A1 - Li, Lei A1 - Wong, Hin-chung A1 - Nong, Wenyan A1 - Cheung, Man Kit A1 - Law, Patrick Tik Wan A1 - Kam, Kai Man A1 - Kwan, Hoi Shan T1 - Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus JF - BMC Genomics N2 - Background: Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before. Results: Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp webcite) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium. Conclusions: We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium. " KW - comparative genomics KW - clinical KW - environment KW - vibrio parahaemolyticus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118080 SN - 1471-2164 VL - 15 IS - 1135 ER - TY - JOUR A1 - Reynolds, David A1 - Cliffe, Laura A1 - Förstner, Konrad U. A1 - Hon, Chung-Chau A1 - Siegel, T. Nicolai A1 - Sabatini, Robert T1 - Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei JF - Nucleic Acids Research N2 - Base J, beta-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase ( RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters. KW - RNA-polymerase-II KW - variant surface glycoprotein KW - SWI2/SNF2-like protein KW - messenger RNA KW - polycistronic transcription KW - DNA glycosation KW - hela cells KW - gene expression KW - genome KW - 5-bromodeoxyuridine Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117863 VL - 42 IS - 15 ER - TY - JOUR A1 - Jun, Kyong-Hwa A1 - Gholami, Spedideh A1 - Song, Tae-Jin A1 - Au, Joyce A1 - Haddad, Dana A1 - Carson, Joshua A1 - Chen, Chun-Hao A1 - Mojica, Kelly A1 - Zanzonico, Pat A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Szalay, Aladar A1 - Fong, Yuman T1 - A novel oncolytic viral therapy and imaging technique for gastric cancer using a genetically engineered vaccinia virus carrying the human sodium iodide symporter JF - Journal of Experimental & Clinical Cancer Research N2 - Background: Gastric cancers have poor overall survival despite recent advancements in early detection methods, endoscopic resection techniques, and chemotherapy treatments. Vaccinia viral therapy has had promising therapeutic potential for various cancers and has a great safety profile. We investigated the therapeutic efficacy of a novel genetically-engineered vaccinia virus carrying the human sodium iodide symporter (hNIS) gene, GLV-1 h153, on gastric cancers and its potential utility for imaging with Tc-99m pertechnetate scintigraphy and I-124 positron emission tomography (PET). Methods: GLV-1 h153 was tested against five human gastric cancer cell lines using cytotoxicity and standard viral plaque assays. In vivo, subcutaneous flank tumors were generated in nude mice with human gastric cancer cells, MKN-74. Tumors were subsequently injected with either GLV-1 h153 or PBS and followed for tumor growth. Tc-99m pertechnetate scintigraphy and I-124 microPET imaging were performed. Results: GFP expression, a surrogate for viral infectivity, confirmed viral infection by 24 hours. At a multiplicity of infection (MOI) of 1, GLV-1 h153 achieved > 90% cytotoxicity in MNK-74, OCUM-2MD3, and AGS over 9 days, and >70% cytotoxicity in MNK-45 and TMK-1. In vivo, GLV-1 h153 was effective in treating xenografts (p < 0.001) after 2 weeks of treatment. GLV-1 h153-infected tumors were readily imaged by Tc-99m pertechnetate scintigraphy and I-124 microPET imaging 2 days after treatment. Conclusions: GLV-1 h153 is an effective oncolytic virus expressing the hNIS protein that can efficiently regress gastric tumors and allow deep-tissue imaging. These data encourages its continued investigation in clinical settings. KW - oncolytic viral therapy KW - GLV-1 h153 KW - gastric cancer KW - human sodium iodide symporter (hNIS) KW - radioiodine therapy KW - gene therapy KW - expression KW - replication KW - stomach KW - tumors KW - surgery Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117716 SN - 1756-9966 VL - 33 IS - 2 ER - TY - JOUR A1 - Bielaszewska, Martina A1 - Schiller, Roswitha A1 - Lammers, Lydia A1 - Bauwens, Andreas A1 - Fruth, Angelika A1 - Middendorf, Barbara A1 - Schmidt, M. Alexander A1 - Tarr, Phillip I. A1 - Dobrindt, Ulrich A1 - Karch, Helge A1 - Mellmann, Alexander T1 - Heteropathogenic virulence and phylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6 JF - EMBO Molecular Medicine N2 - Extraintestinal pathogenic and intestinal pathogenic (diarrheagenic) Escherichia coli differ phylogenetically and by virulence profiles. Classic theory teaches simple linear descent in this species, where non-pathogens acquire virulence traits and emerge as pathogens. However, diarrheagenic Shiga toxin-producing E.coli (STEC) O2:H6 not only possess and express virulence factors associated with diarrheagenic and uropathogenic E.coli but also cause diarrhea and urinary tract infections. These organisms are phylogenetically positioned between members of an intestinal pathogenic group (STEC) and extraintestinal pathogenic E.coli. STEC O2:H6 is, therefore, a 'heteropathogen,' and the first such hybrid virulent E.coli identified. The phylogeny of these E.coli and the repertoire of virulence traits they possess compel consideration of an alternate view of pathogen emergence, whereby one pathogroup of E.coli undergoes phased metamorphosis into another. By understanding the evolutionary mechanisms of bacterial pathogens, rational strategies for counteracting their detrimental effects on humans can be developed. KW - phased metamorphosis KW - phylogeny KW - heteropathogenicity KW - Shiga toxin-producing Escherichia coli KW - hemolytic-uremic syndrome KW - urinary-tract-infection KW - cytolethal distending toxin KW - shiga toxin KW - Crohns-disease KW - outbreak KW - genes KW - island KW - strains KW - parallel evolution KW - uropathogenic Escherichia coli Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117254 SN - 1757-4684 VL - 6 IS - 3 ER - TY - JOUR A1 - Nguyen, Tu N. A1 - Müller, Laura S. M. A1 - Park, Sung Hee A1 - Siegel, T. Nicolai A1 - Günzl, Arthur T1 - Promoter occupancy of the basal class I transcription factor A differs strongly between active and silent VSG expression sites in Trypanosoma brucei JF - Nucleic Acid Research N2 - 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. KW - RNA-polymerase-I KW - blood-stream forms KW - acrican trypanosomes KW - gene expression KW - antigenic variation KW - ribosomal RNA KW - plasmodium falciparum KW - virulence genes KW - subunit KW - complex Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117232 SN - 1362-4962 VL - 42 IS - 5 ER - TY - JOUR A1 - Bischler, Thorsten A1 - Kopf, Matthias A1 - Voss, Bjoern T1 - Transcript mapping based on dRNA-seq data JF - BMC Bioinformatics N2 - 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. KW - transcriptional start site KW - dynamic programming KW - RNA-seq KW - differential KW - segmentation KW - transcriptional uni KW - transcriptome KW - reveals KW - model Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-116663 SN - 1471-2105 VL - 15 IS - 122 ER - TY - JOUR A1 - Wagner, Ines A1 - Volkmer, Michael A1 - Sharan, Malvika A1 - Villaveces, Jose M. A1 - Oswald, Felix A1 - Surendranath, Vineeth A1 - Habermann, Bianca H. T1 - morFeus: a web-based program to detect remotely conserved orthologs using symmetrical best hits and orthology network scoring JF - BMC Bioinformatics N2 - 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. KW - reciprocal best hit KW - finder using symmetrical best hits KW - sequences KW - annotation KW - identification KW - database KW - genomes KW - proteins KW - homologs KW - hidden markov-models KW - phylogenetic trees KW - PSI-blast KW - eigenvector centrality KW - meta-analysis based orthology KW - orthology KW - remote sequence conservation KW - alignment clustering KW - orthology network Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115590 VL - 15 IS - 263 ER - TY - JOUR A1 - Talman, Arthur M. A1 - Prieto, Judith H. A1 - Marques, Sara A1 - Ubaida-Mohien, Ceereena A1 - Lawniczak, Mara A1 - Wass, Mark N. A1 - Xu, Tao A1 - Frank, Roland A1 - Ecker, Andrea A1 - Stanway, Rebecca S. A1 - Krishna, Sanjeev A1 - Sternberg, Michael J. E. A1 - Christophides, Georges K. A1 - Graham, David R. A1 - Dinglasan, Rhoel R. A1 - Yates, John R., III A1 - Sinden, Robert E. T1 - Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility JF - Malaria Journal N2 - 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. KW - glycolysis KW - gamete KW - energy metabolism KW - tandem mass-spectra KW - YoelII-Nigeriensis KW - haemoproteus-columbae KW - chlamydomonas flagella KW - life cycle KW - microtubule motor KW - hexose transporter KW - membrane-protein topology KW - malaria parasite KW - subcellular localization KW - flagellum KW - plasmodium Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115572 N1 - Additional files are available here: http://www.malariajournal.com/content/13/1/315/additional VL - 13 IS - 315 ER - TY - JOUR A1 - Remes, Bernhard A1 - Berghoff, Bork A. A1 - Förstner, Konrad U. A1 - Klug, Gabriele T1 - Role of oxygen and the OxyR protein in the response to iron limitation in Rhodobacter sphaeroides JF - BMC Genomics N2 - 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. KW - oxidative stress KW - Rhodobacter sphaeroides KW - RNAseq KW - OxyR KW - iron limitation KW - transcriptomics KW - dependent gene-expression KW - hydrogen-peroxide KW - escherichia coli Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115357 SN - 1471-2164 VL - 15 IS - 794 ER - TY - JOUR A1 - Lindgreen, Stinus A1 - Umu, Sinan Uğur A1 - Lai, Alicia Sook-Wei A1 - Eldai, Hisham A1 - Liu, Wenting A1 - McGimpsey, Stephanie A1 - Wheeler, Nicole E. A1 - Biggs, Patrick J. A1 - Thomson, Nick R. A1 - Barquist, Lars A1 - Poole, Anthony M. A1 - Gardner, Paul P. T1 - Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling JF - PLOS Computational Biology N2 - 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. KW - protein families database KW - small nucleolar RNAs KW - bacterial genomes KW - comparative genomics KW - dark-matter KW - homology search KW - archaea KW - sequence KW - alignment KW - insights Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115259 VL - 10 IS - 10 ER - TY - JOUR A1 - Rico, Sergio A1 - Yepes, Ana A1 - Rodriguez, Hector A1 - Santamaria, Jorge A1 - Antoraz, Sergio A1 - Krause, Eva M. A1 - Diaz, Margarita A1 - Santamaria, Ramon I. T1 - 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 JF - PLOS ONE N2 - 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. KW - signal-transduction systems KW - biosynthetic gene-cluster KW - escherichia coli KW - response regulator KW - oviedomycin KW - expression KW - organization KW - integration KW - bacteria KW - sequence Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115151 SN - 1932-6203 VL - 9 IS - 10 ER -