• Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation.

      Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015)
      The complex microbiome of the gut has an enormous impact on human health. Analysis of the transcriptional activity of microorganisms through mRNA sequencing (metatranscriptomics) opens a completely new window into their activity in vivo, but it is highly challenging due to numerous technical and bioinformatical obstacles. Here we present an optimized pipeline for extraction of high quality mRNA from stool samples.
    • Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis.

      Forrellad, Marina Andrea; Bianco, María Verónica; Blanco, Federico Calos; Nuñez, Javier; Klepp, Laura Inés; Vazquez, Cristina Lourdes; de la Santangelo, María Paz; Rocha, Rosana Valeria; Soria, Marcelo; Golby, Paul; et al. (2013-09-05)
      Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor.
    • Subpopulation-specific transcriptome analysis of competence-stimulating-peptide-induced Streptococcus mutans.

      Lemme, André; Gröbe, Lothar; Reck, Michael; Tomasch, Jürgen; Wagner-Döbler, Irene; Helmholtz Centre for Infection Research, Research Group Microbial Communication, Braunschweig, Germany. Andre.Lemme@helmholtz-hzi.de (2011-04)
      Competence-stimulating-peptide (CSP)-mediated competence development in Streptococcus mutans is a transient and biphasic process, since only a subpopulation induces the expression of ComX in the presence of CSP, and the activation of the DNA uptake machinery in this fraction shuts down ~3 to 4 h postinduction. Here, we combine for the first time, to our knowledge, the bacterial flow-cytometric sorting of cells and subpopulation-specific transcriptome analysis of both the competent and noncompetent fraction of CSP-treated S. mutans cells. Sorting was guided by a ComX-green fluorescent protein (ComX-GFP) reporter, and the transcriptome analysis demonstrated the successful combination of both methods, because a strong enrichment of transcripts for comX and its downstream genes was achieved. Three two-component systems were expressed in the competent fraction, and among them was ComDE. Moreover, the recently identified regulator system ComR/S was expressed exclusively in the competent fraction. In contrast, the expression of bacteriocin-related genes was at the same level in all cells. GFP reporter strains for ComE and CipB (mutacin V) confirmed this expression pattern on the single-cell level. Fluorescence microscopy revealed that some ComX-expressing cells committed autolysis in an early stage of competence initiation. In viable ComX-expressing cells, the uptake of DNA could be shown on the single-cell level. This study demonstrates that all cells in the population respond to CSP through the activation of bacteriocin-related genes. Some of these cells start to activate ComX expression but then segregate into two subpopulations, one becoming competent and another one that lyses, resulting in intrapopulation diversity.
    • Taxonomy and evolution of bacteriochlorophyll a-containing members of the OM60/NOR5 clade of marine gammaproteobacteria: description of Luminiphilus syltensis gen. nov., sp. nov., reclassification of Haliea rubra as Pseudohaliea rubra gen. nov., comb. nov., and emendation of Chromatocurvus halotolerans

      Spring, Stefan; Riedel, Thomas; Spröer, Cathrin; Yan, Shi; Harder, Jens; Fuchs, Bernhard M (2013-05-24)
      Abstract Background Aerobic gammaproteobacteria affiliated to the OM60/NOR5 clade are widespread in saline environments and of ecological importance in several marine ecosystems, especially the euphotic zone of coastal areas. Within this group a close relationship between aerobic anoxygenic photoheterotrophs and non-phototrophic members has been found. Results Several strains of aerobic red-pigmented bacteria affiliated to the OM60/NOR5 clade were obtained from tidal flat sediment samples at the island of Sylt (North Sea, Germany). Two of the novel isolates, Rap1red and Ivo14T, were chosen for an analysis in detail. Strain Rap1red shared a 16S rRNA sequence identity of 99% with the type strain of Congregibacter litoralis and was genome-sequenced to reveal the extent of genetic microheterogeneity among closely related strains within this clade. In addition, a draft genome sequence was obtained from the isolate Ivo14T, which belongs to the environmental important NOR5-1 lineage that contains so far no cultured representative with a comprehensive description. Strain Ivo14T was characterized using a polyphasic approach and compared with other red-pigmented members of the OM60/NOR5 clade, including Congregibacter litoralis DSM 17192T, Haliea rubra DSM 19751T and Chromatocurvus halotolerans DSM 23344T. All analyzed strains contained bacteriochlorophyll a and spirilloxanthin as photosynthetic pigments. Besides a detailed phenotypic characterization including physiological and chemotaxonomic traits, sequence information based on protein-coding genes and a comparison of draft genome data sets were used to identify possible features characteristic for distinct taxa within this clade. Conclusions Comparative sequence analyses of the pufLM genes of genome-sequenced representatives of the OM60/NOR5 clade indicated that the photosynthetic apparatus of these species was derived from a common ancestor and not acquired by multiple horizontal gene transfer from phylogenetically distant species. An affiliation of the characterized bacteriochlorophyll a-containing strains to different genera was indicated by significant phenotypic differences and pufLM nucleotide sequence identity values below 82%. The revealed high genotypic and phenotypic diversity of closely related strains within this phylogenetic group reflects a rapid evolution and frequent niche separation in the OM60/NOR5 clade, which is possibly driven by the necessities of an adaptation to oligotrophic marine habitats.
    • Taxonomy and evolution of bacteriochlorophyll a-containing members of the OM60/NOR5 clade of marine gammaproteobacteria: description of Luminiphilus syltensis gen. nov., sp. nov., reclassification of Haliea rubra as Pseudohaliea rubra gen. nov., comb. nov., and emendation of Chromatocurvus halotolerans.

      Spring, Stefan; Riedel, Thomas; Spröer, Cathrin; Yan, Shi; Harder, Jens; Fuchs, Bernhard M; Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures, Inhoffenstr, 7B, Braunschweig 38124, Germany. ssp@dsmz.de. (2013)
      Aerobic gammaproteobacteria affiliated to the OM60/NOR5 clade are widespread in saline environments and of ecological importance in several marine ecosystems, especially the euphotic zone of coastal areas. Within this group a close relationship between aerobic anoxygenic photoheterotrophs and non-phototrophic members has been found.
    • Tracking gene expression and oxidative damage of O-stressed Clostridioides difficile by a multi-omics approach.

      Neumann-Schaal, Meina; Metzendorf, Nicole G; Troitzsch, Daniel; Nuss, Aaron Mischa; Hofmann, Julia Danielle; Beckstette, Michael; Dersch, Petra; Otto, Andreas; Sievers, Susanne; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-05-31)
      Clostridioides difficile is the major pathogen causing diarrhea following antibiotic treatment. It is considered to be a strictly anaerobic bacterium, however, previous studies have shown a certain and strain-dependent oxygen tolerance. In this study, the model strain C. difficile 630Δerm was shifted to micro-aerobiosis and was found to stay growing to the same extent as anaerobically growing cells with only few changes in the metabolite pattern. However, an extensive change in gene expression was determined by RNA-Seq. The most striking adaptation strategies involve a change in the reductive fermentation pathways of the amino acids proline, glycine and leucine. But also a far-reaching restructuring in the carbohydrate metabolism was detected with changes in the phosphotransferase system (PTS) facilitated uptake of sugars and a repression of enzymes of glycolysis and butyrate fermentation. Furthermore, a temporary induction in the synthesis of cofactor riboflavin was detected possibly due to an increased demand for flavin mononucleotid (FMN) and flavin adenine dinucleotide (FAD) in redox reactions. However, biosynthesis of the cofactors thiamin pyrophosphate and cobalamin were repressed deducing oxidation-prone enzymes and intermediates in these pathways. Micro-aerobically shocked cells were characterized by an increased demand for cysteine and a thiol redox proteomics approach revealed a dramatic increase in the oxidative state of cysteine in more than 800 peptides after 15 min of micro-aerobic shock. This provides not only a catalogue of oxidation-prone cysteine residues in the C. difficile proteome but also puts the amino acid cysteine into a key position in the oxidative stress response. Our study suggests that tolerance of C. difficile towards O
    • Treatment of biofilms in bacterial vaginosis by an amphoteric tenside pessary-clinical study and microbiota analysis.

      Gottschick, Cornelia; Deng, Zhi-Luo; Vital, Marius; Masur, Clarissa; Abels, Christoph; Pieper, Dietmar H; Rohde, Manfred; Mendling, Werner; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09-13)
      Bacterial vaginosis (BV) is the most common vaginal syndrome among women in their reproductive years. It is associated with an increased risk of acquiring sexually transmitted infections and complications like preterm labor. BV is characterized by a high recurrence rate for which biofilms frequently found on vaginal epithelial cells may be a reason.
    • A unique mechanism for methyl ester formation via an amide intermediate found in myxobacteria.

      Müller, Inga; Weinig, Stefan; Steinmetz, Heinrich; Kunze, Birgitte; Veluthoor, Sheeba; Mahmud, Taifo; Müller, Rolf (2006-08-01)
      Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.
    • An Unprecedented Medium-Chain Diunsaturated -acylhomoserine Lactone from Marine Group Bacteria.

      Ziesche, Lisa; Wolter, Laura; Wang, Hui; Brinkhoff, Thorsten; Pohlner, Marion; Engelen, Bert; Wagner-Döbler, Irene; Schulz, Stefan; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2018-12-31)
      N-acylhomoserine lactones (AHLs), bacterial signaling compounds involved in quorum-sensing, are a structurally diverse group of compounds. We describe here the identification, synthesis, occurrence and biological activity of a new AHL, N-((2E,5Z)-2,5-dodecadienoyl)homoserine lactone (11) and its isomer N-((3E,5Z)-3,5-dodecadienoyl)homoserine lactone (13), occurring in several Roseobacter group bacteria (Rhodobacteraceae). The analysis of 26 strains revealed the presence of 11 and 13 in six of them originating from the surface of the macroalgae Fucus spiralis or sediments from the North Sea. In addition, 18 other AHLs were detected in 12 strains. Compound identification was performed by GC/MS. Mass spectral analysis revealed a diunsaturated C12 homoserine lactone as structural element of the new AHL. Synthesis of three likely candidate compounds, 11, 13 and N-((2E,4E)-2,4-dodecadienoyl)homoserine lactone (5), revealed the former to be the natural AHLs. Bioactivity test with quorum-sensing reporter strains showed high activity of all three compounds. Therefore, the configuration and stereochemistry of the double bonds in the acyl chain seemed to be unimportant for the activity, although the chains have largely different shapes, solely the chain length determining activity. In combination with previous results with other Roseobacter group bacteria, we could show that there is wide variance between AHL composition within the strains. Furthermore, no association of certain AHLs with different habitats like macroalgal surfaces or sediment could be detected. View Full-Text
    • The urinary microbiota of men and women and its changes in women during bacterial vaginosis and antibiotic treatment.

      Gottschick, Cornelia; Deng, Zhi-Luo; Vital, Marius; Masur, Clarissa; Abels, Christoph; Pieper, Dietmar H; Wagner-Döbler, Irene; Helmholtz Centre for infection researchGmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-14)
      The urinary microbiota is similarly complex as the vaginal and penile microbiota, yet its role as a reservoir for pathogens and for recurrent polymicrobial biofilm diseases like bacterial vaginosis (BV) is not clear.
    • Use of Single-Frequency Impedance Spectroscopy to Characterize the Growth Dynamics of Biofilm Formation in Pseudomonas aeruginosa.

      van Duuren, Jozef B J H; Müsken, Mathias; Karge, Bianka; Tomasch, Jürgen; Wittmann, Christoph; Häussler, Susanne; Brönstrup, Mark (2017-07-12)
      Impedance spectroscopy has been applied in prokaryotic and eukaryotic cytometry as a label-free method for the investigation of adherent cells. In this paper, its use for characterizing the growth dynamics of P. aeruginosa biofilms is described and compared to crystal violet staining and confocal microscopy. The method allows monitoring the growth of biofilm-forming P. aeruginosa in a continuous and label-free manner over a period of 72 h in a 96 well plate format. Impedance curves obtained for P. aeruginosa PA14 wild type and mutant strains with a transposon insertion in pqsA and pelA genes exhibited distinct phases. We propose that the slope of the declining curve following a maximum at ca. 35-40 h is a measure of biofilm formation. Transplant experiments with P. aeruginosa biofilms and paraffin suggest that the impedance also reflects pellicle formation at the liquid-air interface, a barely considered contributor to impedance. Finally, the impairment of biofilm formation upon treatment of cultures with L-arginine and with ciprofloxacin, tobramycin and meropenem was studied by single frequency impedance spectroscopy. We suggest that these findings qualify impedance spectroscopy as an additional technique to characterize biofilm formation and its modulation by small molecule drugs.