• Lack of the delta subunit of RNA polymerase increases virulence related traits of Streptococcus mutans.

      Xue, Xiaoli; Sztajer, Helena; Buddruhs, Nora; Petersen, Jörn; Rohde, Manfred; Talay, Susanne R; Wagner-Döbler, Irene; Research Group Microbial Communication, Division of Cell Biology, Helmholtz Centre for Infection Research GmbH, Braunschweig, Germany. (2011)
      The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased.
    • Low diversity of planktonic bacteria in the tropical ocean.

      Milici, Mathias; Tomasch, Jürgen; Wos-Oxley, Melissa L; Wang, Hui; Jáuregui, Ruy; Camarinha-Silva, Amelia; Deng, Zhi-Luo; Plumeier, Iris; Giebel, Helge-Ansgar; Wurst, Mascha; Pieper, Dietmar H; Simon, Meinhard; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2016)
      The diversity of macro-organisms increases towards the equator, with almost no exceptions. It is the most conserved biogeographical pattern on earth and is thought to be related to the increase of temperature and productivity in the tropics. The extent and orientation of a latitudinal gradient of marine bacterioplankton diversity is controversial. Here we studied the euphotic zone of the Atlantic Ocean based on a transect covering ~12.000 km from 51°S to 47 °N. Water samples were collected at 26 stations at five depths between 20 and 200 m and sequentially filtered through 8 μm, 3 μm and 0,22 μm filters, resulting in a total of 359 samples. Illumina sequencing of the V5-V6 region of the 16S rRNA gene revealed a clear biogeographic pattern with a double inverted latitudinal gradient. Diversity was higher in mid-latitudinal regions of the Atlantic Ocean and decreased towards the equator. This pattern was conserved for bacteria from all three planktonic size fractions. Diversity showed a non-linear relationship with temperature and was negatively correlated with bacterial cell numbers in the upper depth layers (<100 m). The latitudinal gradients of marine bacterial diversity and the mechanisms that govern them are distinct from those found in macro-organisms.
    • The luxS mutation causes loosely-bound biofilms in Shewanella oneidensis

      Bodor, Agnes M; Jänsch, Lothar; Wissing, Josef; Wagner-Döbler, Irene (2011-06-10)
      Abstract Background The luxS gene in Shewanella oneidensis was shown to encode an autoinducer-2 (AI-2)-like molecule, the postulated universal bacterial signal, but the impaired biofilm growth of a luxS deficient mutant could not be restored by AI-2, indicating it might not have a signalling role in this organism. Findings Here, we provide further evidence regarding the metabolic role of a luxS mutation in S. oneidensis. We constructed a luxS mutant and compared its phenotype to a wild type control with respect to its ability to remove AI-2 from the medium, expression of secreted proteins and biofilm formation. We show that S. oneidensis has a cell-dependent mechanism by which AI-2 is depleted from the medium by uptake or degradation at the end of the exponential growth phase. As AI-2 depletion is equally active in the luxS mutant and thus does not require AI-2 as an inducer, it appears to be an unspecific mechanism suggesting that AI-2 for S. oneidensis is a metabolite which is imported under nutrient limitation. Secreted proteins were studied by iTraq labelling and liquid chromatography mass spectrometry (LC-MS) detection. Differences between wild type and mutant were small. Proteins related to flagellar and twitching motility were slightly up-regulated in the luxS mutant, in accordance with its loose biofilm structure. An enzyme related to cysteine metabolism was also up-regulated, probably compensating for the lack of the LuxS enzyme. The luxS mutant developed an undifferentiated, loosely-connected biofilm which covered the glass surface more homogenously than the wild type control, which formed compact aggregates with large voids in between. Conclusions The data confirm the role of the LuxS enzyme for biofilm growth in S. oneidensis and make it unlikely that AI-2 has a signalling role in this organism.
    • Metabolic fluxes in the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis, two members of the marine Roseobacter clade

      Fürch, Tobias; Preusse, Matthias; Tomasch, Jürgen; Zech, Hajo; Wagner-Döbler, Irene; Rabus, Ralf; Wittmann, Christoph (2009-09-29)
      Abstract Background In the present work the central carbon metabolism of Dinoroseobacter shibae and Phaeobacter gallaeciensis was studied at the level of metabolic fluxes. These two strains belong to the marine Roseobacter clade, a dominant bacterial group in various marine habitats, and represent surface-associated, biofilm-forming growth (P. gallaeciensis) and symbiotic growth with eukaryotic algae (D. shibae). Based on information from recently sequenced genomes, a rich repertoire of pathways has been identified in the carbon core metabolism of these organisms, but little is known about the actual contribution of the various reactions in vivo. Results Using 13C labelling techniques in specifically designed experiments, it could be shown that glucose-grown cells of D. shibae catabolise the carbon source exclusively via the Entner-Doudoroff pathway, whereas alternative routes of glycolysis and the pentose phosphate pathway are obviously utilised for anabolic purposes only. Enzyme assays confirmed this flux pattern and link the lack of glycolytic flux to the absence of phosphofructokinase activity. The previously suggested formation of phosphoenolpyruvate from pyruvate during mixotrophic CO2 assimilation was found to be inactive under the conditions studied. Moreover, it could be shown that pyruvate carboxylase is involved in CO2 assimilation and that the cyclic respiratory mode of the TCA cycle is utilised. Interestingly, the use of intracellular pathways was highly similar for P. gallaeciensis. Conclusion The present study reveals the first insight into pathway utilisation within the Roseobacter group. Fluxes through major intracellular pathways of the central carbon metabolism, which are closely linked to the various important traits found for the Roseobacter clade, could be determined. The close similarity of fluxes between the two physiologically rather different species might provide the first indication of more general key properties among members of the Roseobacter clade which may explain their enormous success in the marine realm.
    • Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

      Deng, Zhi-Luo; Gottschick, Cornelia; Bhuju, Sabin; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-27)
      Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the
    • Mixotrophic growth of bacteriochlorophyll a-containing members of the OM60/NOR5 clade of marine gammaproteobacteria is carbon-starvation independent and correlates with the type of carbon source and oxygen availability.

      Spring, Stefan; Riedel, Thomas; Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures, Inhoffenstr, 7B, Braunschweig 38124, Germany. ssp@dsmz.de (2013)
      Populations of aerobic anoxygenic photoheterotrophic bacteria in marine environments are dominated by members of the Roseobacter lineage within the Alphaproteobacteria and the OM60/NOR5 clade of gammaproteobacteria. A wealth of information exists about the regulation of pigment production and mixotrophic growth in various members of the Roseobacter clade, but a detailed knowledge about aerobic bacteriochlorophyll a-containing gammaproteobacteria is still limited to one strain of the species Congregibacter litoralis.
    • The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD.

      Fu, Chengzhang; Sikandar, Asfandyar; Donner, Jannik; Zaburannyi, Nestor; Herrmann, Jennifer; Reck, Michael; Wagner-Döbler, Irene; Koehnke, Jesko; Müller, Rolf; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124Braunschweig, Germany. (2017-11-16)
      The natural product carolacton is a macrolide keto-carboxylic acid produced by the myxobacterium Sorangium cellulosum, and was originally described as an antibacterial compound. Here we show that carolacton targets FolD, a key enzyme from the folate-dependent C1 metabolism. We characterize the interaction between bacterial FolD and carolacton biophysically, structurally and biochemically. Carolacton binds FolD with nanomolar affinity, and the crystal structure of the FolD-carolacton complex reveals the mode of binding. We show that the human FolD orthologs, MTHFD1 and MTHFD2, are also inhibited in the low nM range, and that micromolar concentrations of carolacton inhibit the growth of cancer cell lines. As mitochondrial MTHFD2 is known to be upregulated in cancer cells, it may be possible to use carolacton as an inhibitor tool compound to assess MTHFD2 as an anti-cancer target.
    • Oxidative stress and starvation in Dinoroseobacter shibae: the role of extrachromosomal elements.

      Soora, Maya; Tomasch, Jürgen; Wang, Hui; Michael, Victoria; Petersen, Jörn; Engelen, Bert; Wagner-Döbler, Irene; Cypionka, Heribert; HZI Helmholtzzentrum für Infektionsforschung (2015)
      Aerobic anoxygenic phototrophic bacteria (AAP) are abundant in the photic zone of the marine environment. Dinoroseobacter shibae, a representative of the Roseobacter group, converts light into additional energy that enhances its survival especially under starvation. However, light exposure results in the production of cytotoxic reactive oxygen species in AAPs. Here we investigated the response of D. shibae to starvation and oxidative stress, focusing on the role of extrachromosomal elements (ECRs). D. shibae possessing five ECRs (three plasmids and two chromids) was starved for 4 weeks either in the dark or under light/dark cycles and the survival was monitored. Transcriptomics showed that on the chromosome genes with a role in oxidative stress response and photosynthesis were differentially expressed during the light period. Most extrachromosomal genes in contrast showed a general loss of transcriptional activity, especially in dark-starved cells. The observed decrease of gene expression was not due to plasmid loss, as all five ECRs were maintained in the cells. Interestingly, the genes on the 72-kb chromid were the least downregulated, and one region with genes of the oxygen stress response and a light-dependent protochlorophyllide reductase of cyanobacterial origin was strongly activated under the light/dark cycle. A Δ72-kb curing mutant lost the ability to survive under starvation in a light/dark cycle demonstrating the essential role of this chromid for adaptation to starvation and oxidative stress. Our data moreover suggest that the other four ECRs of D. shibae have no vital function under the investigated conditions and therefore were transcriptionally silenced.
    • Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

      Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene; Helmholtz-Zentrum for Infektion Research GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.
    • Phylogenomics of Rhodobacteraceae reveals evolutionary adaptation to marine and non-marine habitats.

      Simon, Meinhard; Scheuner, Carmen; Meier-Kolthoff, Jan P; Brinkhoff, Thorsten; Wagner-Döbler, Irene; Ulbrich, Marcus; Klenk, Hans-Peter; Schomburg, Dietmar; Petersen, Jörn; Göker, Markus; Helmholtz Centre for infection research, Ihoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-20)
      Marine Rhodobacteraceae (Alphaproteobacteria) are key players of biogeochemical cycling, comprise up to 30% of bacterial communities in pelagic environments and are often mutualists of eukaryotes. As 'Roseobacter clade', these 'roseobacters' are assumed to be monophyletic, but non-marine Rhodobacteraceae have not yet been included in phylogenomic analyses. Therefore, we analysed 106 genome sequences, particularly emphasizing gene sampling and its effect on phylogenetic stability, and investigated relationships between marine versus non-marine habitat, evolutionary origin and genomic adaptations. Our analyses, providing no unequivocal evidence for the monophyly of roseobacters, indicate several shifts between marine and non-marine habitats that occurred independently and were accompanied by characteristic changes in genomic content of orthologs, enzymes and metabolic pathways. Non-marine Rhodobacteraceae gained high-affinity transporters to cope with much lower sulphate concentrations and lost genes related to the reduced sodium chloride and organohalogen concentrations in their habitats. Marine Rhodobacteraceae gained genes required for fucoidan desulphonation and synthesis of the plant hormone indole 3-acetic acid and the compatible solutes ectoin and carnitin. However, neither plasmid composition, even though typical for the family, nor the degree of oligotrophy shows a systematic difference between marine and non-marine Rhodobacteraceae. We suggest the operational term 'Roseobacter group' for the marine Rhodobacteraceae strains.The ISME Journal advance online publication, 20 January 2017; doi:10.1038/ismej.2016.198.
    • Plasmid Transfer in the Ocean - A Case Study from the Roseobacter Group.

      Petersen, Jörn; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017)
      Plasmid mediated horizontal gene transfer (HGT) has been speculated to be one of the prime mechanisms for the adaptation of roseobacters (Rhodobacteraceae) to their ecological niches in the marine habitat. Their plasmids contain ecologically crucial functional modules of up to ∼40-kb in size, e.g., for aerobic anoxygenic photosynthesis, flagellar formation and the biosynthesis of the antibiotic tropodithietic acid. Furthermore, the widely present type four secretion system (T4SS) of roseobacters has been shown to mediate conjugation across genus barriers, albeit in the laboratory. Here we discovered that Confluentimicrobium naphthalenivorans NS6(T), a tidal flat bacterium isolated in Korea, carries a 185-kb plasmid, which exhibits a long-range synteny with the conjugative 126-kb plasmid of Dinoroseobacter shibae DFL12(T). Both replicons are stably maintained by RepABC operons of the same compatibility group (-2) and they harbor a homologous T4SS. Principal component analysis of the codon usage shows a large similarity between the two plasmids, while the chromosomes are very distinct, showing that neither of the two bacterial species represents the original host of those RepABC-2 type plasmids. The two species do not share a common habitat today and they are phylogenetically only distantly related. Our finding demonstrates the first clear-cut evidence for conjugational plasmid transfer across biogeographical and phylogenetic barriers in Rhodobacteraceae and documents the importance of conjugative HGT in the ocean.
    • Potential for luxS related signalling in marine bacteria and production of autoinducer-2 in the genus Shewanella.

      Bodor, Agnes; Elxnat, Bettina; Thiel, Verena; Schulz, Stefan; Wagner-Döbler, Irene; Helmholtz-Center for Infection Research, Group Microbial Communication, Division of Cell Biology, Inhoffenstr, 7, 38124 Braunschweig, Germany. agb@gbf.de (2008)
      BACKGROUND: The autoinducer-2 (AI-2) group of signalling molecules are produced by both Gram positive and Gram negative bacteria as the by-product of a metabolic transformation carried out by the LuxS enzyme. They are the only non species-specific quorum sensing compounds presently known in bacteria. The luxS gene coding for the AI-2 synthase enzyme was found in many important pathogens. Here, we surveyed its occurrence in a collection of 165 marine isolates belonging to abundant marine phyla using conserved degenerated PCR primers and sequencing of selected positive bands to determine if the presence of the luxS gene is phylogenetically conserved or dependent on the habitat. RESULTS: The luxS gene was not present in any of the Alphaproteobacteria (n = 71) and Bacteroidetes strains (n = 29) tested; by contrast, these bacteria harboured the sahH gene, coding for an alternative enzyme for the detoxification of S-adenosylhomocysteine (SAH) in the activated methyl cycle. Within the Gammaproteobacteria (n = 76), luxS was found in all Shewanella, Vibrio and Alteromonas isolates and some Pseudoalteromonas and Halomonas species, while sahH was detected in Psychrobacter strains. A number of Gammaproteobacteria (n = 27) appeared to have neither the luxS nor the sahH gene. We then studied the production of AI-2 in the genus Shewanella using the Vibrio harveyi bioassay. All ten species of Shewanella tested produced a pronounced peak of AI-2 towards the end of the exponential growth phase in several media investigated. The maximum of AI-2 activity was different in each Shewanella species, ranging from 4% to 46% of the positive control. CONCLUSION: The data are consistent with those of fully sequenced bacterial genomes and show that the potential for luxS related signalling is dependent on phylogenetic affiliation rather than ecological niche and is largest in certain groups of Gammaproteobacteria in the marine environment. This is the first report on AI-2 production in Shewanella species; its signalling role in these organisms remains to be elucidated.
    • Quorum sensing of Streptococcus mutans is activated by Aggregatibacter actinomycetemcomitans and by the periodontal microbiome.

      Szafrański, Szymon P; Deng, Zhi-Luo; Tomasch, Jürgen; Jarek, Michael; Bhuju, Sabin; Rohde, Manfred; Sztajer, Helena; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-20)
      The oral cavity is inhabited by complex microbial communities forming biofilms that can cause caries and periodontitis. Cell-cell communication might play an important role in modulating the physiologies of individual species, but evidence so far is limited.
    • Screening for inhibitors of mutacin synthesis in Streptococcus mutans using fluorescent reporter strains.

      Premnath, Priyanka; Reck, Michael; Wittstein, Kathrin; Stadler, Marc; Wagner-Döbler, Irene; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (BMC, 2018-03-27)
      Within the polymicrobial dental plaque biofilm, bacteria kill competitors by excreting mixtures of bacteriocins, resulting in improved fitness and survival. Inhibiting their bacteriocin synthesis might therefore be a useful strategy to eliminate specific pathogens. We used Streptococcus mutans, a highly acidogenic inhabitant of dental plaque, as a model and searched for natural products that reduced mutacin synthesis. To this end we fused the promoter of mutacin VI to the GFP+ gene and integrated the construct into the genome of S. mutans UA159 by single homologous recombination. The resulting reporter strain 423p - gfp + was used to screen 297 secondary metabolites from different sources, mainly myxobacteria and fungi, for their ability to reduce the fluorescence of the fully induced reporter strain by > 50% while growth was almost unaffected (> 90% of control). Seven compounds with different chemical structures and different modes of action were identified. Erinacine C was subsequently validated and shown to inhibit transcription of all three mutacins of S. mutans. The areas of the inhibition zones of the sensor strains S. sanguinis and Lactococcus lactis were reduced by 35% to 61% in comparison to controls in the presence of erinacine C, demonstrating that the amount of active mutacins in the culture supernatants of S. mutans was reduced. Erinacines are cyathane diterpenes that were extracted from cultures of the edible mushroom Hericium erinaceus. They have anti-inflammatory, antimicrobial and neuroprotective effects. For erinacine C, a new biological activity was found here. We demonstrate the successful development of a whole-cell fluorescent reporter for the screening of natural compounds and report that erinacine C suppresses mutacin synthesis in S. mutans without affecting cell viability.
    • Screening of Compounds against Gardnerella vaginalis Biofilms.

      Gottschick, Cornelia; Szafranski, Szymon P; Kunze, Brigitte; Sztajer, Helena; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      Bacterial vaginosis (BV) is a common infection in reproductive age woman and is characterized by dysbiosis of the healthy vaginal flora which is dominated by Lactobacilli, followed by growth of bacteria like Gardnerella vaginalis. The ability of G. vaginalis to form biofilms contributes to the high rates of recurrence that are typical for BV and which unfortunately make repeated antibiotic therapy inevitable. Here we developed a biofilm model for G. vaginalis and screened a large spectrum of compounds for their ability to prevent biofilm formation and to resolve an existing G. vaginalis biofilm. The antibiotics metronidazole and tobramycin were highly effective in preventing biofilm formation, but had no effect on an established biofilm. The application of the amphoteric tenside sodium cocoamphoacetate (SCAA) led to disintegration of existing biofilms, reducing biomass by 51% and viability by 61% and it was able to increase the effect of metronidazole by 40% (biomass) and 61% (viability). Our data show that attacking the biofilm and the bacterial cells by the combination of an amphoteric tenside with the antibiotic metronidazole might be a useful strategy against BV.
    • Sociodemographic determinants of spatial disparities in early childhood caries: An ecological analysis in Braunschweig, Germany.

      Meyer, Frederic; Karch, André; Schlinkmann, Kristin Maria; Dreesman, Johannes; Horn, Johannes; Rübsamen, Nicole; Sudradjat, Henny; Schubert, Rainer; Mikolajczyk, Rafael; Helmholtz Centre for infection research, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-05-26)
      To identify spatial disparities in dental caries experience (measured by dmft (decayed missing filled teeth) index) of children in the city of Braunschweig and to evaluate whether these disparities can be explained by sociodemographic characteristics.
    • Stool metatranscriptomics: A technical guideline for mRNA stabilisation and isolation

      Reck, Michael; Tomasch, Jürgen; Deng, Zhiluo; Jarek, Michael; Husemann, Peter; Wagner-Döbler, Irene (2015-07-04)
      Abstract Background 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. Results Comparison of three commercially available RNA extraction kits with the method of Zoetendal revealed that the Powermicrobiome Kit (MoBio) performed best with respect to RNA yield and purity. Next, the influence of the stabilization reagent during sample storage for up to 15 days was studied. RIN analysis and qRT-PCR of spiked-in and indigenous genes revealed that RNA Later preserved mRNA integrity most efficiently, while samples conserved in RNA Protect showed substantial mRNA decay. Using the optimized pipeline developed here, recovery rates for spiked-in E.coli cells expressing fluorescing proteins were 8.7-9.7 % for SuperfolderGFP and 14.7-17.8 % for mCherry. The mRNA of stabilized stool samples as well as of snap-frozen controls was sequenced with Illumina Hiseq, yielding on average 74 million reads per sample. PCoA analysis, taxonomic classification using Kraken and functional classification using bwa showed that the transcriptomes of samples conserved in RNA Later were unchanged for up to 6 days even at room temperature, while RNA Protect was inefficient for storage durations exceeding 24 h. However, our data indicate that RNA Later introduces a bias which is then maintained throughout storage, while RNA Protect conserved samples are initially more similar to the snap frozen controls. RNA Later conserved samples had a reduced abundance of e.g. Prevotellaceae transcripts and were depleted for e.g. COG category “Carbohydrate transport and metabolism”. Conclusion Since the overall similarity between all stool transcriptional profiles studied here was >0.92, these differences are unlikely to affect global comparisons, but should be taken into account when rare but critically important members of the stool microbiome are being studied.
    • 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; Gutierrez, Maximiliano Gabriel; Bigi, Fabiana (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.