63 research outputs found

    Development of a read mapping analysis software and computational pan genome analysis of 20 Pseudomonas aeruginosa strains

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    Hilker R. Development of a read mapping analysis software and computational pan genome analysis of 20 Pseudomonas aeruginosa strains. Bielefeld: Bielefeld University; 2015.In times of multi-resistant pathogenic bacteria, their detailed study is of utmost importance. Their comparative analysis can even aid the emerging field of personalized medicine by enabling optimized treatment depending on the presence of virulence factors and antibiotic resistances in the infection concerned. The weaknesses and functionality of these pathogenic bacteria can be investigated using modern computer science and novel sequencing technologies. One of these methods is the bioinformatics evaluation of high-throughput sequencing data. A pathogenic bacterium posing severe health care issues is the ubiquitous Pseudomonas aeruginosa. It is involved in a wide range of infections mainly affecting the pulmonary or urinary tract, open wounds and burns. The prevalence of chronic obstructive pulmonary disease cases with P. aeruginosa in Germany alone is ~600,000 per year. Within the framework of this dissertation, computational comparative genomics experiments were conducted with a panel of 20 of the most abundant Pseudomonas aeruginosa strains. 15 of these strains were isolated from clinical cases, while the remaining 5 were strains without a known infection history isolated from the environment. This division was chosen to enable direct comparison of the pathogenic potential of clinical and environmental strains and identification of their possible characteristic differences. When designing the bioinformatics experiments and searching for an efficient visualization and automatic analysis platform for read alignment (mapping) data, it became evident that no adequate solution was available that included all required functionalities. On these grounds, the decision was made to define two main subjects for this dissertation. Besides the P. aeruginosa pan genome analysis, a novel read mapping visualization and analysis software was developed and published in the journal Bioinformatics. This software - ReadXplorer - is partly based upon a prototype, which was developed during a preceding master's thesis at the Center for Biotechnology of the Bielefeld University under the name VAMP. The software was developed into a comprehensive user-friendly platform augmented with several newly developed and implemented automatic bioinformatics read mapping analyses. Two examples of these are the transcription start site detection and the single nucleotide polymorphism detection. Moreover, new intuitive visualizations were added to the existent ones and existing visualizations were greatly enhanced. ReadXplorer is designed to support not only DNA-seq data as accrued in the P. aeruginosa experiments, but also any kind of standard read mapping data as obtained from RNA-seq or ChIP-seq experiments. The data management was designed to comply with the latest performance and efficiency needs emerging from the large next generation sequencing data sets. Finally, ReadXplorer was empowered to deal with eukaryotic read mapping data as well. Amongst other software, ReadXplorer was then used to analyze different comparative genomics aspects of P. aeruginosa and to draw conclusions regarding the development of their pathogenicity. The list of conducted experiments includes phylogeny and gene set determination, analysis of regions of genomic plasticity and identification of single nucleotide polymorphisms. The achieved results were published in the journal Environmental Biology

    ReadXplorer 2 - detailed read mapping analysis and visualization from one single source

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    Hilker R, Stadermann KB, Schwengers O, et al. ReadXplorer 2 - detailed read mapping analysis and visualization from one single source. Bioinformatics. 2016;32(24):3702-3708.Motivation: The vast amount of already available and currently generated read mapping data re-quires comprehensive visualization, and should benefit from bioinformatics tools offering a wide spec-trum of analysis functionality from just one source. Appropriate handling of multiple mapped reads during mapping analyses remains an issue that demands improvement. Results: The capabilities of the read mapping analysis and visualization tool ReadXplorer were vastly enhanced. Here, we present an even finer granulated read mapping classification, improving the level of detail for analyses and visualizations. The spectrum of automatic analysis functions has been broadened to include genome rearrangement detection as well as correlation analysis between two mapping data sets. Existing functions were refined and enhanced, namely the computation of differ-entially expressed genes, the read count and normalization analysis and the transcription start site (TSS) detection. Additionally, ReadXplorer 2 features a highly improved support for large eukaryotic data sets and a command line version, enabling its integration into workflows. Finally, the new version is now able to display any kind of tabular results from other bioinformatics tools. Availability: http://www.readxplorer.or

    Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress.

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    Hillion M, Bernhardt J, Busche T, et al. Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress. Sci Rep. 2017;7(1): 1195.Mycothiol (MSH) is the major low molecular weight (LMW) thiol in Actinomycetes. Here, we used shotgun proteomics, OxICAT and RNA-seq transcriptomics to analyse protein S-mycothiolation, reversible thiol-oxidations and their impact on gene expression in Mycobacterium smegmatis under hypochlorite stress. In total, 58 S-mycothiolated proteins were identified under NaOCl stress that are involved in energy metabolism, fatty acid and mycolic acid biosynthesis, protein translation, redox regulation and detoxification. Protein S-mycothiolation was accompanied by MSH depletion in the thiol-metabolome. Quantification of the redox state of 1098 Cys residues using OxICAT revealed that 381 Cys residues (33.6%) showed >10% increased oxidations under NaOCl stress, which overlapped with 40 S-mycothiolated Cys-peptides. The absence of MSH resulted in a higher basal oxidation level of 338 Cys residues (41.1%). The RseA and RshA anti-sigma factors and the Zur and NrdR repressors were identified as NaOCl-sensitive proteins and their oxidation resulted in an up-regulation of the SigH, SigE, Zur and NrdR regulons in the RNA-seq transcriptome. In conclusion, we show here that NaOCl stress causes widespread thiol-oxidation including protein S-mycothiolation resulting in induction of antioxidant defense mechanisms in M. smegmatis. Our results further reveal that MSH is important to maintain the reduced state of protein thiols

    Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation

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    Wittchen M, Busche T, Gaspar AH, et al. Transcriptome sequencing of the human pathogen Corynebacterium diphtheriae NCTC 13129 provides detailed insights into its transcriptional landscape and into DtxR-mediated transcriptional regulation. BMC Genomics. 2018;19(1): 82.Background The human pathogen Corynebacterium diphtheriae is the causative agent of diphtheria. In the 1990s a large diphtheria outbreak in Eastern Europe was caused by the strain C. diphtheriae NCTC 13129. Although the genome was sequenced more than a decade ago, not much is known about its transcriptome. Our aim was to use transcriptome sequencing (RNA-Seq) to close this knowledge gap and gain insights into the transcriptional landscape of a C. diphtheriae tox+ strain. Results We applied two different RNA-Seq techniques, one to retrieve 5′-ends of primary transcripts and the other to characterize the whole transcriptional landscape in order to gain insights into various features of the C. diphtheriae NCTC 13129 transcriptome. By examining the data we identified 1656 transcription start sites (TSS), of which 1202 were assigned to genes and 454 to putative novel transcripts. By using the TSS data promoter regions recognized by the housekeeping sigma factor σA and its motifs were analyzed in detail, revealing a well conserved −10 but an only weakly conserved −35 motif, respectively. Furthermore, with the TSS data 5’-UTR lengths were explored. The observed 5’-UTRs range from zero length (leaderless transcripts), which make up 20% of all genes, up to over 450 nt long leaders, which may harbor regulatory functions. The C. diphtheriae transcriptome consists of 471 operons which are further divided into 167 sub-operon structures. In a differential expression analysis approach, we discovered that genetic disruption of the iron-sensing transcription regulator DtxR, which controls expression of diphtheria toxin (DT), causes a strong influence on general gene expression. Nearly 15% of the genome is differentially transcribed, indicating that DtxR might have other regulatory functions in addition to regulation of iron metabolism and DT. Furthermore, our findings shed light on the transcriptional landscape of the DT encoding gene tox and present evidence for two tox antisense RNAs, which point to a new way of transcriptional regulation of toxin production. Conclusions This study presents extensive insights into the transcriptome of C. diphtheriae and provides a basis for future studies regarding gene characterization, transcriptional regulatory networks, and regulation of the tox gene in particular

    Deciphering the genome repertoire of Pseudomonas sp. M1 toward β-Myrcene biotransformation

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    Pseudomonas sp. M1 is able to mineralize several unusual substrates of natural and xenobiotic origin, contributing to its competence to thrive in different ecological niches. In this work, the genome of M1 strain was resequenced by Illumina MiSeq to refine the quality of a published draft by resolving the majority of repeat-rich regions. In silico genome analysis led to the prediction of metabolic pathways involved in biotransformation of several unusual substrates (e.g., plant-derived volatiles), providing clues on the genomic complement required for such biodegrading/biotransformation functionalities. Pseudomonas sp. M1 exhibits a particular sensory and biotransformation/biocatalysis potential toward β-myrcene, a terpene vastly used in industries worldwide. Therefore, the genomic responsiveness of M1 strain toward β-myrcene was investigated, using an RNA sequencing approach. M1 cells challenged with β-myrcene(compared with cells grown in lactate) undergo an extensive alteration of the transcriptome expression profile, including 1,873 genes evidencing at least 1.5-fold of altered expression (627 upregulated and 1,246 downregulated), toward β-myrcene-imposed molecular adaptation and cellular specialization. A thorough data analysis identified a novel 28-kb genomic island, whose expression was strongly stimulated in β-myrcene-supplemented medium, that is essential for β-myrcene catabolism. This island includes β-myrcene-induced genes whose products are putatively involved in 1) substrate sensing, 2) gene expression regulation, and 3) β-myrcene oxidation and bioconversion of β-myrcene derivatives into central metabolism intermediates. In general, this locus does not show high homology with sequences available in databases and seems to have evolved through the assembly of several functional blocks acquired from different bacteria, probably, at different evolutionary stages.Acknowledgments This work was supported by FEDER through POFC— COMPETE and by national funds from Foundation for Science and Technology (Portugal) through the projects PEst-C/BIA/UI4050/2011, PTDC/EBB-BIO/104980/2008 and PTDC/BIA-MIC/113733/09, and through a PhD grant (grant number SFRH/BD/76894/2011) to P.S.-C.info:eu-repo/semantics/publishedVersio

    A detailed view of the intracellular transcriptome of Listeria monocytogenes in murine macrophages using RNA-seq

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    Listeria monocytogenes is a bacterial pathogen and causative agent for the foodborne infection listeriosis, which is mainly a threat for pregnant, elderly or immunocompromised individuals. Due to its ability to invade and colonize diverse eukaryotic cell types including cells from invertebrates, L. monocytogenes has become a well-established model organism for intracellular growth. Almost ten years ago, we and others presented the first whole-genome microarray-based intracellular transcriptome of L. monocytogenes. With the advent of newer technologies addressing transcriptomes in greater detail, we revisit this work, and analyze the intracellular transcriptome of L. monocytogenes during growth in murine macrophages using a deep sequencing based approach.We detected 656 differentially expressed genes of which 367 were upregulated during intracellular growth in macrophages compared to extracellular growth in BHI. This study confirmed ~64% of all regulated genes previously identified by microarray analysis. Many of the regulated genes that were detected in the current study involve transporters for various metals, ions as well as complex sugars such as mannose. We also report changes in antisense transcription, especially upregulations during intracellular bacterial survival. A notable finding was the detection of regulatory changes for a subset of temperate A118-like prophage genes, thereby shedding light on the transcriptional profile of this bacteriophage during intracellular growth. In total, our study provides an updated genome-wide view of the transcriptional landscape of L. monocytogenes during intracellular growth and represents a rich resource for future detailed analysis

    Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032

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    Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σC, σD, σE, σH, and σM are mostly involved in stress responses. The role of σD consists in the control of cell wall integrity. The σD regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in C. glutamicum. RNA sequencing of the transcriptome from a strain overexpressing the sigD gene provided 29 potential σD-controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both in vitro transcription and the in vivo two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σD-dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAACA/G and -10 GAT. Surprisingly, nearly all of these σD-dependent promoters were also active to a much lower extent with σHin vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the σH-dependent promoters is different (-35 GGAAT/C and -10 GTT). In addition to the activity of σH at the σD-controlled promoters, we discovered separated or overlapping σA- or σB-regulated or σH-regulated promoters within the upstream region of 8 genes of the σD-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σD-dependent genes. Computer modeling revealed that σH binds to the promoter DNA in a similar manner as σD to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σD and Lys 53 in σH, bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the σD-controlled genes also belong to the σH-regulon and are also transcribed from the overlapping or closely located housekeeping (σA-regulated) and/or general stress (σB-regulated) promoters
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