Integrated application of genomic, biochemical and cultivation approaches to characterize 1,2-dichloropropane dichloroelemination in organohalide respiring Chloroflexi

Chlorinated solvents are among the most encountered groundwater pollutants. These toxic compounds cause harm to ecosystem functioning and human health. 1,2-Dichloropropane (1,2-D) was used in a variety of industrial and agricultural applications until it was banned in the U.S. in the 1970s. Only a handful of bacteria have been described to reductively dechlorinate 1,2-D to innocuous propene and inorganic chloride, among these the Dehalococcoides (Dhc) strains RC and KS. In order to shed light into the genetic basis of 1,2-D dechlorination, efforts focused in identifying the gene encoding the enzyme system (i.e., reductive dehalogenase) responsible for 1,2-D to propene transformation. To accomplish this goal, a multiple lines of evidence approach combining gene cloning, transcriptional studies, and enzyme activity assays implicated the dcpA gene in 1,2-D reductive dechlorination in Dhc strains RC and KS. This gene was also identified in Dehalogenimonas lykanthroporepellens (Dhgm) strain BL-DC-9, another member of the organohalide-respiring Chloroflexi group, and also capable of growth with 1,2-D as electron acceptor. Propene-producing enrichment cultures were derived from a variety of environments and the presence of dcpA correlated with 1,2-D reductive dechlorination observed in situ and/or in microcosms. Nested PCR and qPCR assays were designed and validated to detected and quantify the gene in laboratory cultures and in environmental samples. These surveys shed light into the distribution of this gene in diverse environments including pristine environments. Genomic and bioinformatics tools explored the gene neighborhood of dcpA and revealed a genomic island shared between Dhc and Dhgm indicative of a horizontal gene transfer event. Metagenome analysis of consortia RC and KS enabled the draft genome assemblies of these two Dhc strains. This analysis revealed that the Dhc strain RC and strain KS harbor at least 34 and 31 reductive dehalogenase genes, including genes implicated in PCB reductive dechlorination. These findings reveal broad reductive dechlorination potential and emphasize that such dedicated dechlorinators (i.e., Dhc strain FL2) occur in pristine environments and are members of natural microbial assemblages that have not been exposed to anthropogenic contamination

Methylation and loss of Imprinting: Unending rivalries unleashed between “kneaded erasers†and “fate writersâ€Â

Genome is a complex barcode that is interpreted at molecular level. There are various proteins which are modulating the expression or repression of the genes. Miscellaneous proteins work in collaboration to stimulate or repress the gene expression. Chromatin remodeling factors are the artists which chisel, carve and mould the sculpture of genome. In this review we will emphasize on exemptions and extensions which trigger genomic instability in broad range of molecular anomalies. Doubtlessly therapeutic interventions have shown tremendous promise in cancer therapy, but the selectivity profiles of these compounds have largely relied on serendipity or 'off-target' activities rather than rational drug design. Purposefully designed compounds with activity against methyltransferase, demethylase and HDAC will bring us a step closer to personalized medicine

Molecular biological and biochemical approaches to expand the spectrum of fungal natural products

At least 3.5 billion years ago, the first life on earth arose. This was the starting point of the evolutionary development of numerous living beings. According to current estimations, there are 1012 different species on our planet. Most of this enormous biodiversity originates from the kingdom of bacteria and archaea. Based on these estimations, only 0.001 % of all species are known to this day. The omnipresent competition between living beings led to the development of secondary metabolism. The metabolites derived from this metabolism are not essential for survival, yet their production offers the organism various selection advantages. Plants, bacteria, and fungi are the main producers of secondary metabolites. The more than 2,140,000 million known secondary metabolites can be divided into five large groups: (1) non-ribosomal polypeptides, (2) polyketides, (3) alkaloids, (4) terpenoids and steroids and (5) enzyme cofactors. Many of these natural compounds show a biological or pharmaceutical activity and were used for the development of drugs. The large number of not yet identified microorganisms harbors an enormous, mostly unused genetic potential to produce further new natural compounds. Such compounds may be suitable for the development of urgently needed new drugs. Various approaches, such as heterologous expression in suitable host organisms, are being investigated to make this potential accessible. Additionally, through synthetic biology approaches, the diversity of natural substances can be further extended, and new natural substances can be discovered or produced. In the context of research on secondary metabolites, this work focuses on three main topics: 1. The extension of the spectrum of possible substrates for prenyltransferases, by using a database to predict new substrates. 2. The identification and characterization of previously unknown biosynthetic gene clusters, as well as the investigation of a possible application of the enzymes involved to produce new natural substances. 3. The generation of a host for the heterologous expression of secondary metabolite genes and investigation of their unknown products. Prenyltransferases catalyze the transfer of prenyl units (n × C5) to their target substrates. This is of importance, as an increase in the biological activity of prenylated compounds compared to their unprenylated counterparts has been observed for many compounds. A special property of prenyltransferases is their promiscuity with respect to the substrates. This makes them suitable candidates to produce pharmaceutically active substances. However, in practice, it is difficult to identify new substrates for prenyltransferases. In order to address this problem, a database, PrenDB, was developed for the prediction of such substrates. The predictive power of this database was experimentally tested with 38 predicted substrates by their acceptance with the prenyltransferases FtmPT1, FgapT2, and CdpNPT. For 27 of the 38 substrates, prenylation by at least one of the three tested enzymes was observed, 17 with conversion yields of more than 50 %. This proved the predictive power of the developed database and enabled the targeted selection of new potential substrates and the identification of new substrate classes. The identification of biosynthetic gene clusters and the subsequent biochemical characterization of the enzymes involved in the biosynthetic pathways form the basis for synthetic biology approaches to produce natural products. Based on the cyclic dipeptide echinulin, a possible procedure for the identification of the responsible gene cluster and the use of the involved enzymes for the biosynthesis of new substances was described. The enzymatic prerequisites for the biosynthesis of echinulin were determined based on the structural peculiarities of echinulin. Potential candidate gene clusters must encode one non-ribosomal peptide synthetase and several prenyltransferases. In the genome of the echinulin producer Aspergillus ruber, a gene cluster with these prerequisites was identified. Enzyme assays with the echinulin precursor cyclo-L-tryptophanyl-L-alaninyl and the heterologously produced prenyltransferases EchPT1 and EchPT2 led to a well-founded biosynthetic hypothesis and confirmed the involvement of this cluster in the biosynthesis of echinulin. The combination of EchPT1 and EchPT2 with cyclo-L-tryptophanyl-L-alaninyl as a substrate led to the formation of 7 products with different degrees of prenylation. This special property was subsequently used to prenylate further cyclic dipeptides. The stereoisomers of cyclo-tryptophanyl-alaninyl and cyclo-tryptophanyl-prolinyl were used for this purpose. Analogous to the biosynthesis of echinulin, this led to the formation of triprenylated main products prenylated at position C2, C5 and C7, as well as further di-, tri- and tetraprenylated side products. Another possibility to investigate and produce secondary metabolites is the heterologous expression in a suitable host. A potential new host for heterologous expression, Penicillium crustosum, was examined in this thesis. The genome of the fungus was sequenced and the involvement of the polyketide synthase Pcr4401 in the biosynthesis of the melanin precursor YWA1 was confirmed by deletion and expression experiments. Successful integration of foreign genes in the pcr4401 gene locus can easily be recognized by the occurrence of an albino phenotype. For better use as an expression host, a pyrG deficient strain and two plasmids were generated to integrate foreign genes into the pcr4401 gene locus. The applicability as an expression host was subsequently verified by the successful expression of three PKS genes and the structural elucidation of the formed products

Modeling of flexible side chains for protein-ligand docking

This work comprises new approaches that are developed to support structure-based drug design in cases where side-chain conformations are uncertain, be it through exibility or the devised modeling procedure. A knowledge-based scoring function ROTA is derived that can successfully identify correct rotamers and near-native ligand placements. ROTA is also able to reliably estimate the binding anity of a protein-ligand complex, even if the conformations of one or both binding partners contain small errors. The side-chain prediction algorithm IRECS is developed for generating protein models that contain ensembles of rotamers for flexible side chains. IRECS is guided by ROTA and can accurately predict single and multiple side-chain conformations that represent the exibility and conformational space of the respective side chains. IRECS is also able to include knowledge of side-chain conformations from a homologous protein used as a template directly in its optimization procedure. A modeling and docking pipeline is constructed that comprises IRECS, ROTA and the docking program FlexE. This pipeline is tested on 40 targets of the screening database DUD, where it is shown that the application of ROTA and IRECS can signicantly increase the performance of screening experiments in cases in which side chains are exible or were modeled.Diese Arbeit stellt neue Methoden vor, die die strukturbasierte Suche nach Wirkstoffen in solchen Fällen unterstützen soll, in denen Seitenkettenkonformationen durch Flexibilität der Seitenketten oder durch die verwendete Modellierungstechnik nicht sicher bestimmt werden können. Die Bewertungsfunktion ROTA wurde abgeleitet um richtige Rotamere und Ligandplazierungen zu erkennen. ROTA ist außerdem in der Lage die Bindungsaffinität eines Protein-Ligand-Komplexes zuverlässig zu bestimmen, auch wenn die Konformationen der Bindungspartner geringe Fehler aufweisen. Das Programm IRECS wurde entwickelt um Proteinmodelle zu erzeugen, die Ensembles von Rotameren für flexible Seitenketten enthalten. IRECS verwendet ROTA zur Bewertung von Proteinkonformationen und kann zuverlässig Ensembles von Rotameren bestimmen, die die Flexibilität und den konformellen Raum der jeweiligen Seitenketten repräsentieren. IRECS ist auch in der Lage zusätzliche Informationen über Seitenketten eines homologen Proteins, das der Modellierung als Vorlage diente, während seiner Optimierungsprozedur zu nutzen. IRECS, ROTA und das Dockingprogramm FlexE wurden zu einer Modellierungs- und Dockingpipeline vereinigt und auf den 40 Proteinen der Screening-Datenbank DUD getestet. Es konnte gezeigt werden, dass in Fällen mit flexiblen oder modellierten Seitenketten die Anwendung von ROTA und IRECS die Leistung von Screening-Experimenten deutlich steigern kann

An object-oriented database for the compilation of signal transduction pathways

Transpath ist ein Informationssystem fuer Signaltransduktionsnetze. Der Fokus liegt auf Signalpfaden und -kaskaden, die an der Regulation von Transkriptionsfaktoren beteiligt sind. Molekuele und Reaktionenen werden als Knoten in einem Signalgraphen aufgefasst und, zusammen mit Informationen ueber ihre Lokalisation, Qualitaet, Familienhierarchien und Signalmotive, in einer objekt-orientierten Databank gespeichert. Weiterhin werden Verweise zu anderen Datenbanken und der Originalliteratur gespeichert. Transpath unterscheidet zwischen den Zuständen eines Signalmoleküles und kann die Reaktionsmechanismen der Signalinteraktionen angemessen beschreiben. Transpath is über das World Wide Web (http://transpath.gbf.de) verfügbar durch ein Servlet-basiertes Interface, das dynamische Sichten direkt aus dem Inhalt der Datenbank erzeugt. Signalpfad-Abfragen und verschiedene Arten der Visualisierung werden zusammen mit textbasierenden Abfragen und Detailinformationen zu einzelnen Eintraegen unterstuetzt. Es wird gezeigt dass die Datenbank zur Analyse des Signalnetzes von Nutzen ist und die Grundlage fuer Simulationen liefern kann.Transpath is an information system on signal-transduction networks. It focuses on pathways involved in the regulation of transcription factors. Molecules and reactions are the nodes in a signaling graph. They are stored in an object-oriented database, together with information about their location, quality, family relationships and signaling motifs. Also stored are links to other databases and references to the original literature. Transpath differentiates between the states of a signal molecule, and can adequately describe the reaction mechanisms of signaling interactions. It is available over the web (http://transpath.gbf.de) through a Servlet-based interface, that creates dynamic content directly from the contents of the database. Pathway query mechanisms and several kinds of display are provided for the database in addition to text-based queries and information on single entries. We show that the database is useful for analysis of the signaling network and can provide the basis for simulations

DEVELOPMENT OF A HIGH-THROUGHPUT ASSAY TO MEASURE DNA MISMATCH REPAIR EFFICIENCY IN VIVO

Whether due to mutagens or DNA replication errors, mismatches arise spontaneously in vivo. If left unrepaired, accumulation of mutations at a high rate can be detrimental to the survival of the organism. Cells recognize the mismatches and repair them via a dedicated mismatch repair system. Although its efficiency has been shown to depend on the type and the sequence context of the mismatch, only a small subset of possible mismatched sequences could be examined thus far. In this work, I describe a high-throughput sequencing based approach that can assess the repair efficiency of many different mismatches in parallel, enabling a systematic analysis of the sequence effect on mismatch repair. In this scheme, an in vitro synthesized plasmid containing a single mismatch is introduced to an E. coli cell. If the mismatch is repaired prior to replication, all of the descendants will share the same sequence. If, however, replication precedes mismatch repair, the descendants have a mixture of two different sequences, and therefore the event frequencies of these two types provide information on the repair efficiency. Use of DNA barcodes enables obtaining single-molecule level information regarding the fate of each mismatch carrying molecules, through which the repair of 4434 different mismatches and 1300 insertion loops were monitored in vivo under various conditions. The results showed that CC mismatches are always poorly repaired whereas local sequence context is a strong determinant of the highly heterogeneous repair efficiency of TT, AG and CT mismatches. In contrast, most of the insertion loops were repaired with a high efficiency without an appreciable sequence context dependence. The measurement of the repair efficiency in mutant cell strains of different mismatch repair pathway mutants also showed a residual repair capability, potentially an indication of side-processes that lead to an apparent repair of mismatch bearing plasmids

Computer Aided Verification

This open access two-volume set LNCS 13371 and 13372 constitutes the refereed proceedings of the 34rd International Conference on Computer Aided Verification, CAV 2022, which was held in Haifa, Israel, in August 2022. The 40 full papers presented together with 9 tool papers and 2 case studies were carefully reviewed and selected from 209 submissions. The papers were organized in the following topical sections: Part I: Invited papers; formal methods for probabilistic programs; formal methods for neural networks; software Verification and model checking; hyperproperties and security; formal methods for hardware, cyber-physical, and hybrid systems. Part II: Probabilistic techniques; automata and logic; deductive verification and decision procedures; machine learning; synthesis and concurrency. This is an open access book