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1. A cryptic oxidoreductase safeguards oxidative protein folding in Corynebacterium diphtheriae.

Author

Reardon-Robinson, Melissa, Nguyen, Minh, Sanchez, Belkys, Osipiuk, Jerzy, Rückert, Christian, Chang, Chungyu, Chen, Bo, Nagvekar, Rahul, Joachimiak, Andrzej, Tauch, Andreas, Das, Asis, and Ton-That, Hung

Subjects
Corynebacterium diphtheriae, diphtheria toxin, disulfide bond, gram-positive bacteria, pili, Bacterial Proteins, Corynebacterium diphtheriae, Oxidative Stress, Protein Disulfide Reductase (Glutathione), Protein Folding
Abstract

In many gram-positive Actinobacteria, including Actinomyces oris and Corynebacterium matruchotii, the conserved thiol-disulfide oxidoreductase MdbA that catalyzes oxidative folding of exported proteins is essential for bacterial viability by an unidentified mechanism. Intriguingly, in Corynebacterium diphtheriae, the deletion of mdbA blocks cell growth only at 37 °C but not at 30 °C, suggesting the presence of alternative oxidoreductase enzyme(s). By isolating spontaneous thermotolerant revertants of the mdbA mutant at 37 °C, we obtained genetic suppressors, all mapped to a single T-to-G mutation within the promoter region of tsdA, causing its elevated expression. Strikingly, increased expression of tsdA-via suppressor mutations or a constitutive promoter-rescues the pilus assembly and toxin production defects of this mutant, hence compensating for the loss of mdbA. Structural, genetic, and biochemical analyses demonstrated TsdA is a membrane-tethered thiol-disulfide oxidoreductase with a conserved CxxC motif that can substitute for MdbA in mediating oxidative folding of pilin and toxin substrates. Together with our observation that tsdA expression is upregulated at nonpermissive temperature (40 °C) in wild-type cells, we posit that TsdA has evolved as a compensatory thiol-disulfide oxidoreductase that safeguards oxidative protein folding in C. diphtheriae against thermal stress.

Published
2023

2. Safeguarding the Evidential Value of Forensic Cryptocurrency Investigations

Author

Fröwis, Michael, Gottschalk, Thilo, Haslhofer, Bernhard, Rückert, Christian, and Pesch, Paulina

Subjects
Computer Science - Computers and Society
Abstract

Analyzing cryptocurrency payment flows has become a key forensic method in law enforcement and is nowadays used to investigate a wide spectrum of criminal activities. However, despite its widespread adoption, the evidential value of obtained findings in court is still largely unclear. In this paper, we focus on the key ingredients of modern cryptocurrency analytics techniques, which are clustering heuristics and attribution tags. We identify internationally accepted standards and rules for substantiating suspicions and providing evidence in court and project them onto current cryptocurrency forensics practices. By providing an empirical analysis of CoinJoin transactions, we illustrate possible sources of misinterpretation in algorithmic clustering heuristics. Eventually, we derive a set of legal key requirements and translate them into a technical data sharing framework that fosters compliance with existing legal and technical standards in the realm of cryptocurrency forensics. Integrating the proposed framework in modern cryptocurrency analytics tools could allow more efficient and effective investigations, while safeguarding the evidential value of the analysis and the fundamental rights of affected persons.

Published
2019

17. The Subsystems Approach to Genome Annotation and its Use in the Project to Annotate 1000 Genomes

Author

Overbeek, Ross, Begley, Tadhg, Butler, Ralph M, Choudhuri, Jomuna V, Chuang, Han-Yu, Cohoon, Matthew, de Crécy-Lagard, Valérie, Diaz, Naryttza, Disz, Terry, Edwards, Robert, Fonstein, Michael, Frank, Ed D, Gerdes, Svetlana, Glass, Elizabeth M, Goesmann, Alexander, Hanson, Andrew, Iwata-Reuyl, Dirk, Jensen, Roy, Jamshidi, Neema, Krause, Lutz, Kubal, Michael, Larsen, Niels, Linke, Burkhard, McHardy, Alice C, Meyer, Folker, Neuweger, Heiko, Olsen, Gary, Olson, Robert, Osterman, Andrei, Portnoy, Vasiliy, Pusch, Gordon D, Rodionov, Dmitry A, Rückert, Christian, Steiner, Jason, Stevens, Rick, Thiele, Ines, Vassieva, Olga, Ye, Yuzhen, Zagnitko, Olga, and Vonstein, Veronika

Subjects
Biotechnology, Acyl Coenzyme A, Coenzyme A, Computational Biology, Genome, Archaeal, Genome, Bacterial, Genomics, Internet, Leucine, Ribosomal Proteins, Software, Terminology as Topic, Vocabulary, Controlled, Environmental Sciences, Biological Sciences, Information and Computing Sciences, Developmental Biology
Abstract

The release of the 1000th complete microbial genome will occur in the next two to three years. In anticipation of this milestone, the Fellowship for Interpretation of Genomes (FIG) launched the Project to Annotate 1000 Genomes. The project is built around the principle that the key to improved accuracy in high-throughput annotation technology is to have experts annotate single subsystems over the complete collection of genomes, rather than having an annotation expert attempt to annotate all of the genes in a single genome. Using the subsystems approach, all of the genes implementing the subsystem are analyzed by an expert in that subsystem. An annotation environment was created where populated subsystems are curated and projected to new genomes. A portable notion of a populated subsystem was defined, and tools developed for exchanging and curating these objects. Tools were also developed to resolve conflicts between populated subsystems. The SEED is the first annotation environment that supports this model of annotation. Here, we describe the subsystem approach, and offer the first release of our growing library of populated subsystems. The initial release of data includes 180 177 distinct proteins with 2133 distinct functional roles. This data comes from 173 subsystems and 383 different organisms.

Published
2005

35. Genome Assembly and Genetic Traits of the Pleuromutilin-Producer Clitopilus passeckerianus DSM1602

Author

Schafhauser, Thomas, Wibberg, Daniel, Binder, Antonia, Rückert, Christian, Busche, Tobias, Wohlleben, Wolfgang, and Kalinowski, Jörn

Subjects
660.6
Abstract

The gilled mushroom Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known to produce the terpenoid pleuromutilin, which is the biotechnological basis for medically important antibiotics such as lefamulin and retapamulin. Their unique mode of action and good tolerance entails an increasing demand of pleuromutilin-derived antibiotics in veterinary and human health care. Surprisingly, despite their pharmaceutical importance, no genome sequence is available of any pleuromutilin-producing fungus. Here, we present the high-quality draft genome sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including functional genome annotation. More precisely, we employed a hybrid assembly strategy combining Illumina sequencing and Nanopore sequencing to assemble the mitochondrial genome as well as the nuclear genome. In accordance with the dikaryotic state of the fungus, the nuclear genome has a diploid character. Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis revealed a versatile secondary metabolism with an emphasis on terpenoid biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles of biosynthetic gene clusters for pleuromutilin were found in the genome of C. passeckerianus. The pleuromutilin genes were reassembled with yeast-specific elements for heterologous expression in Saccharomyces cerevisiae. Our work lays the foundation for metabolic strain engineering towards higher yields of the valuable compound pleuromutilin.

Published
2022

36. Bioassay-Guided Fractionation Leads to the Detection of Cholic Acid Generated by the Rare Thalassomonas sp.

Author

Pheiffer, Fazlin, Schneider, Yannik K.-H., Hansen, Espen Holst, Andersen, Jeanette Hammer, Isaksson, Johan, Busche, Tobias, Rückert, Christian, Kalinowski, Jörn, Zyl, Leonardo van, and Trindade, Marla

Abstract

Bacterial symbionts of marine invertebrates are rich sources of novel, pharmaceutically relevant natural products that could become leads in combatting multidrug-resistant pathogens and treating disease. In this study, the bioactive potential of the marine invertebrate symbiont Thalassomonas actiniarum was investigated. Bioactivity screening of the strain revealed Gram-positive specific antibacterial activity as well as cytotoxic activity against a human melanoma cell line (A2058). The dereplication of the active fraction using HPLC-MS led to the isolation and structural elucidation of cholic acid and 3-oxo cholic acid. T. actiniarum is one of three type species belonging to the genus Thalassomonas. The ability to generate cholic acid was assessed for all three species using thin-layer chromatography and was confirmed by LC-MS. The re-sequencing of all three Thalassomonas type species using long-read Oxford Nanopore Technology (ONT) and Illumina data produced complete genomes, enabling the bioinformatic assessment of the ability of the strains to produce cholic acid. Although a complete biosynthetic pathway for cholic acid synthesis in this genus could not be determined based on sequence-based homology searches, the identification of putative penicillin or homoserine lactone acylases in all three species suggests a mechanism for the hydrolysis of conjugated bile acids present in the growth medium, resulting in the generation of cholic acid and 3-oxo cholic acid. With little known currently about the bioactivities of this genus, this study serves as the foundation for future investigations into their bioactive potential as well as the potential ecological role of bile acid transformation, sterol modification and quorum quenching by Thalassomonas sp. in the marine environment. [ABSTRACT FROM AUTHOR]

Published
2023
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37. Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining

Author

Oberhofer, Martina, primary, Malfent, Fabian, additional, Zehl, Martin, additional, Urban, Ernst, additional, Wackerlig, Judith, additional, Reznicek, Gottfried, additional, Vignolle, Gabriel A., additional, Rückert, Christian, additional, Busche, Tobias, additional, Wibberg, Daniel, additional, and Zotchev, Sergey B., additional

Published
2022
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39. A universal approach to gene expression engineering

Author

Lale, Rahmi, primary, Tietze, Lisa, additional, Fages-Lartaud, Maxime, additional, Nesje, Jenny, additional, Onsager, Ingerid, additional, Engelhardt, Kerstin, additional, Wong, Che Fai Alex, additional, Akan, Madina, additional, Hummel, Niklas, additional, Kalinowski, Jörn, additional, Rückert, Christian, additional, and Hohmann-Marriott, Martin Frank, additional

Published
2022
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45. Extensive Reannotation of the Genome of the Model Streptomycete Streptomyces lividans TK24 Based on Transcriptome and Proteome Information

Author

Droste, Julian, Rückert, Christian, Kalinowski, Jörn, Hamed, Mohamed Belal, Anné, Jozef, Simoens, Kenneth, Bernaerts, Kristel, Economou, Anastassios, and Busche, Tobias

Subjects
promoter, reannotation, secondary metabolite gene clusters, cis-regulatory elements, RNA sequencing (RNA-seq), Streptomyces lividans TK24, Microbiology, attenuation, 660.6, Original Research
Abstract

Streptomyces lividans TK24 is a relevant Gram-positive soil inhabiting bacterium and one of the model organisms of the genus Streptomyces. It is known for its potential to produce secondary metabolites, antibiotics, and other industrially relevant products. S. lividans TK24 is the plasmid-free derivative of S. lividans 66 and a close genetic relative of the strain Streptomyces coelicolor A3(2). In this study, we used transcriptome and proteome data to improve the annotation of the S. lividans TK24 genome. The RNA-seq data of primary 5'-ends of transcripts were used to determine transcription start sites (TSS) in the genome. We identified 5,424 TSS, of which 4,664 were assigned to annotated CDS and ncRNAs, 687 to antisense transcripts distributed between 606 CDS and their UTRs, 67 to tRNAs, and 108 to novel transcripts and CDS. Using the TSS data, the promoter regions and their motifs were analyzed in detail, revealing a conserved -10 (TAnnnT) and a weakly conserved -35 region (nTGACn). The analysis of the 5' untranslated region (UTRs) of S. lividans TK24 revealed 17% leaderless transcripts. Several cis-regulatory elements, like riboswitches or attenuator structures could be detected in the 5'-UTRs. The S. lividans TK24 transcriptome contains at least 929 operons. The genome harbors 27 secondary metabolite gene clusters of which 26 could be shown to be transcribed under at least one of the applied conditions. Comparison of the reannotated genome with that of the strain Streptomyces coelicolor A3(2) revealed a high degree of similarity. This study presents an extensive reannotation of the S. lividans TK24 genome based on transcriptome and proteome analyses. The analysis of TSS data revealed insights into the promoter structure, 5'-UTRs, cis-regulatory elements, attenuator structures and novel transcripts, like small RNAs. Finally, the repertoire of secondary metabolite gene clusters was examined. These data provide a basis for future studies regarding gene characterization, transcriptional regulatory networks, and usage as a secondary metabolite producing strain. ispartof: FRONTIERS IN MICROBIOLOGY vol:12 ispartof: location:Switzerland status: published

Published
2021

49. Additional file 1 of Adaptive laboratory evolution accelerated glutarate production by Corynebacterium glutamicum

Author

Prell, Carina, Busche, Tobias, Rückert, Christian, Nolte, Lea, Brandenbusch, Christoph, and Wendisch, Volker F.

Abstract

Additional file 1: Figure S1. (A) DNA region of the plasmid pEC-XT99A-gabTDP134L with the 21 bp sequence deleted in ALE strain GluA T7 (boxed in red). (B) Determination of the minimum inhibitory concentration for tetracycline for strain GluA T0 with the orginal plasmid pEC-XT99A-gabTDP134L and GluA RG1 carrying this plasmid with the 21 bp deletion. Cells were grown in Duetz cultivation system using BHIS supplemented with increasing concentrations of tetracycline (0–80 µg µL−1). The ΔOD600 values were determined after 48 hours and are shown as means with standard deviations (n = 3 cultivations). Figure S2. Dependence of the specific acitvity of GOGAT on the glutamine concentration determined for GRLys1ΔgltB overexpressing the native version of gltBD (A) or the mutated gltBE686QD (B). Crude extracts of GRLys1ΔgltB (pEKEx3-gltBD) and GRLys1ΔgltB (pEKEx3-gltBE686QD) were assayed for specific activity of the glutamate synthase (GOGAT) in the presence of increasing glutamine concentrations (0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 2 mM, 5 mM, 10 mM). Values represent means and standard deviations of triplicate measurements. Figure S3. Glutarate production by GluA T7 in chloride-free medium. Cells were grown in Duetz cultivation system using either standard CGXII minimal medium or CGXII minimal medium without chloride supplemented with 40 g L−1 glucose, induced with 1 mM IPTG, and harvested after 48 h. Values and error bars represent means and standard deviations of glutarate (green) and 5AVA concentrations (red) in the culture supernatants (n = 3 cultivations).

Published
2021
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