• A dual-species co-cultivation system to study the interactions between Roseobacters and dinoflagellates

      Wang, Hui; Tomasch, Jürgen; Jarek, Michael; Wagner-Döbler, Irene (2014-07-11)
    • The Alternative Sigma Factor SigX Controls Bacteriocin Synthesis and Competence, the Two Quorum Sensing Regulated Traits in Streptococcus mutans.

      Reck, Michael; Tomasch, Jürgen; Wagner-Döbler, Irene; Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2015-07)
      Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat.
    • Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay.

      Vilchez, Ramiro; Lemme, André; Thiel, Verena; Schulz, Stefan; Sztajer, Helena; Wagner-Döbler, Irene (2007-01-01)
      Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe(3+) within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 microM. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of >120 with a standard deviation of approximately 30% in a reliable and reproducible way.
    • Autoinducer-2-regulated genes in Streptococcus mutans UA159 and global metabolic effect of the luxS mutation.

      Sztajer, Helena; Lemme, André; Vilchez, Ramiro; Schulz, Stefan; Geffers, Robert; Yip, Cindy Ying Yin; Levesque, Celine M; Cvitkovitch, Dennis G; Wagner-Döbler, Irene; Helmholtz-Center for Infection Research, Division of Cell Biology, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2008-01)
      Autoinducer 2 (AI-2) is the only species-nonspecific autoinducer known in bacteria and is produced by both gram-negative and gram-positive organisms. Consequently, it is proposed to function as a universal quorum-sensing signal for interaction between bacterial species. AI-2 is produced as the by-product of a metabolic transformation carried out by the LuxS enzyme. To separate the metabolic function of the LuxS enzyme from the signaling role of AI-2, we carried out a global transcriptome analysis of a luxS null mutant culture of Streptococcus mutans UA159, an important cariogenic bacterium and a crucial component of the dental plaque biofilm community, in comparison to a luxS null mutant culture supplemented with chemically pure 4,5-dihydroxy-2,3-pentanedione, the precursor of AI-2. The data revealed fundamental changes in gene expression affecting 585 genes (30% of the genome) which could not be restored by the signal molecule AI-2 and are therefore not caused by quorum sensing but by lack of the transformation carried out by the LuxS enzyme in the activated methyl cycle. All functional classes of enzymes were affected, including genes known to be important for biofilm formation, bacteriocin synthesis, competence, and acid tolerance. At the same time, 59 genes were identified whose transcription clearly responded to the addition of AI-2. Some of them were related to protein synthesis, stress, and cell division. Three membrane transport proteins were upregulated which are not related to any of the known AI-2 transporters. Three transcription factors were identified whose transcription was stimulated repeatedly by AI-2 addition during growth. Finally, a global regulatory protein, the delta subunit of the RNA polymerase (rpoE), was induced 147-fold by AI-2, representing the largest differential gene expression observed. The data show that many phenotypes related to the luxS mutation cannot be ascribed to quorum sensing and have identified for the first time regulatory proteins potentially mediating AI-2-based signaling in gram-positive bacteria.
    • Bacterioplankton Biogeography of the Atlantic Ocean: A Case Study of the Distance-Decay Relationship.

      Milici, Mathias; Tomasch, Jürgen; Wos-Oxley, Melissa L; Decelle, Johan; Jáuregui, Ruy; Wang, Hui; Deng, Zhi-Luo; Plumeier, Iris; Giebel, Helge-Ansgar; Badewien, Thomas H; et al. (2016)
      In order to determine the influence of geographical distance, depth, and Longhurstian province on bacterial community composition and compare it with the composition of photosynthetic micro-eukaryote communities, 382 samples from a depth-resolved latitudinal transect (51°S-47°N) from the epipelagic zone of the Atlantic ocean were analyzed by Illumina amplicon sequencing. In the upper 100 m of the ocean, community similarity decreased toward the equator for 6000 km, but subsequently increased again, reaching similarity values of 40-60% for samples that were separated by ~12,000 km, resulting in a U-shaped distance-decay curve. We conclude that adaptation to local conditions can override the linear distance-decay relationship in the upper epipelagial of the Atlantic Ocean which is apparently not restrained by barriers to dispersal, since the same taxa were shared between the most distant communities. The six Longhurstian provinces covered by the transect were comprised of distinct microbial communities; ~30% of variation in community composition could be explained by province. Bacterial communities belonging to the deeper layer of the epipelagic zone (140-200 m) lacked a distance-decay relationship altogether and showed little provincialism. Interestingly, those biogeographical patterns were consistently found for bacteria from three different size fractions of the plankton with different taxonomic composition, indicating conserved underlying mechanisms. Analysis of the chloroplast 16S rRNA gene sequences revealed that phytoplankton composition was strongly correlated with both free-living and particle associated bacterial community composition (R between 0.51 and 0.62, p < 0.002). The data show that biogeographical patterns commonly found in macroecology do not hold for marine bacterioplankton, most likely because dispersal and evolution occur at drastically different rates in bacteria.
    • The Binding Site of the V-ATPase Inhibitor Apicularen Is in the Vicinity of Those for Bafilomycin and Archazolid.

      Osteresch, Christin; Bender, Tobias; Grond, Stephanie; von Zezschwitz, Paultheo; Kunze, Brigitte; Jansen, Rolf; Huss, Markus; Wieczorek, Helmut; From the Fachbereich Biologie/Chemie, Abteilung Tierphysiologie, Universität Osnabrück, Barbarastrasse 11, 49069 Osnabrück. (2012-09-14)
      The investigation of V-ATPases as potential therapeutic drug targets and hence of their specific inhibitors is a promising approach in osteoporosis and cancer treatment because the occurrence of these diseases is interrelated to the function of the V-ATPase. Apicularen belongs to the novel inhibitor family of the benzolactone enamides, which are highly potent but feature the unique characteristic of not inhibiting V-ATPases from fungal sources. In this study we specify, for the first time, the binding site of apicularen within the membrane spanning V(O) complex. By photoaffinity labeling using derivatives of apicularen and of the plecomacrolides bafilomycin and concanamycin, each coupled to (14)C-labeled 4-(3-trifluoromethyldiazirin-3-yl)benzoic acid, we verified that apicularen binds at the interface of the V(O) subunits a and c. The binding site is in the vicinity to those of the plecomacrolides and of the archazolids, a third family of V-ATPase inhibitors. Expression of subunit c homologues from Homo sapiens and Manduca sexta, both species sensitive to benzolactone enamides, in a Saccharomyces cerevisiae strain lacking the corresponding intrinsic gene did not transfer this sensitivity to yeast. Therefore, the binding site of benzolactone enamides cannot be formed exclusively by subunit c. Apparently, subunit a substantially contributes to the binding of the benzolactone enamides.
    • The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli.

      Donner, Jannik; Reck, Michael; Bunk, Boyke; Jarek, Michael; App, Constantin Benjamin; Meier-Kolthoff, Jan P; Overmann, Jörg; Müller, Rolf; Kirschning, Andreas; Wagner-Döbler, Irene; et al. (2017-11-01)
      The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
    • The biofilm inhibitor Carolacton inhibits planktonic growth of virulent pneumococci via a conserved target.

      Donner, Jannik; Reck, Michael; Bergmann, Simone; Kirschning, Andreas; Müller, Rolf; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      New antibacterial compounds, preferentially exploiting novel cellular targets, are urgently needed to fight the increasing resistance of pathogens against conventional antibiotics. Here we demonstrate that Carolacton, a myxobacterial secondary metabolite previously shown to damage Streptococcus mutans biofilms, inhibits planktonic growth of Streptococcus pneumoniae TIGR4 and multidrug-resistant clinical isolates of serotype 19A at nanomolar concentrations. A Carolacton diastereomer is inactive in both streptococci, indicating a highly specific interaction with a conserved cellular target. S. mutans requires the eukaryotic-like serine/threonine protein kinase PknB and the cysteine metabolism regulator CysR for susceptibility to Carolacton, whereas their homologues are not needed in S. pneumoniae, suggesting a specific function for S. mutans biofilms only. A bactericidal effect of Carolacton was observed for S. pneumoniae TIGR4, with a reduction of cell numbers by 3 log units. The clinical pneumonia isolate Sp49 showed immediate growth arrest and cell lysis, suggesting a bacteriolytic effect of Carolacton. Carolacton treatment caused a reduction in membrane potential, but not membrane integrity, and transcriptome analysis revealed compensatory reactions of the cell. Our data show that Carolacton might have potential for treating pneumococcal infections.
    • Biofilm transplantation in the deep sea.

      Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-05)
      A gold rush is currently going on in microbial ecology, which is powered by the possibility to determine the full complexity of microbial communities through next-generation sequencing. Accordingly, enormous efforts are underway to describe microbiomes worldwide, in humans, animals, plants, soil, air and the ocean. While much can be learned from these studies, only experiments will finally unravel mechanisms. One of the key questions is how a microbial community is assembled from a pool of bacteria in the environment, and how it responds to change - be it the increase in CO2 concentration in the ocean, or antibiotic treatment of the gut microbiome. The study by Zhang et al. () in this issue is one of the very few that approaches this problem experimentally in the natural environment. The authors selected a habitat which is both extremely interesting and difficult to access. They studied the Thuwal Seep in the Red Sea at 850 m depth and used a remotely operated vehicle (ROV) to place a steel frame carrying substrata for biofilm growth into the brine pool and into the adjacent normal bottom water (NBW). Biofilms were allowed to develop for 3 days, and then those that had been growing in the brine pool were transported to normal bottom water and stayed there for another 3 days, and vice versa. The 'switched' biofilms were then compared with their source communities by metagenome sequencing. Strikingly, both 'switched' biofilms were now dominated by the same two species. These species were able to cope with conditions in both source ecosystems, as shown by assembly of their genomes and detection of expression of key genes. The biofilms had adapted to environmental change, rather than to brine pools or NBW. The study shows both the resilience and adaptability of biofilm communities and has implications for microbial ecology in general and even for therapeutic approaches such as transplantation of faecal microbiomes.
    • Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria.

      Brinkhoff, Thorsten; Fischer, Doreen; Vollmers, John; Voget, Sonja; Beardsley, Christine; Thole, Sebastian; Mussmann, Marc; Kunze, Brigitte; Wagner-Döbler, Irene; Daniel, Rolf; et al. (2012-06)
      Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.
    • Biological activity of volatiles from marine and terrestrial bacteria.

      Schulz, Stefan; Dickschat, Jeroen S; Kunze, Brigitte; Wagner-Döbler, Irene; Diestel, Randi; Sasse, Florenz; Institute of Organic Chemistry, University of Braunschweig-Institute of Technology, Hagenring 30, Braunschweig, Germany. Stefan.Schulz@tu-bs.de (2010)
      The antiproliferative activity of 52 volatile compounds released from bacteria was investigated in agar diffusion assays against medically important microorganisms and mouse fibroblasts. Furthermore, the activity of these compounds to interfere with the quorum-sensing-systems was tested with two different reporter strains. While some of the compounds specific to certain bacteria showed some activity in the antiproliferative assay, the compounds common to many bacteria were mostly inactive. In contrast, some of these compounds were active in the quorum-sensing-tests. γ-Lactones showed a broad reactivity, while pyrazines seem to have only low intrinsic activity. A general discussion on the ecological importance of these findings is given.
    • Carolacton Treatment Causes Delocalization of the Cell Division Proteins PknB and DivIVa in Streptococcus mutans in vivo.

      Reck, Michael; Wagner-Döbler, Irene; Helmholtzzentrum für Infektionsforschung, 38124 Braunschweig (2016)
      The small inhibitory molecule Carolacton has been shown to cause chain formation and bulging in Streptococci, suggesting a defect in cell division, but it is not known how cell division is impaired on a molecular level. Fluorescent fusion proteins have successfully been applied to visualize protein localization and dynamics in vivo and have revolutionized our understanding of cell wall growth, cell division, chromosome replication and segregation. However, in Streptococci the required vectors are largely lacking. We constructed vectors for chromosomal integration and inducible expression of fluorescent fusion proteins based on GFP+ in S. mutans. Their applicability was verified using four proteins with known localization in the cell. We then determined the effect of Carolacton on the subcellular localization of GFP+ fusions of the cell division protein DivIVa and the serine-threonine protein kinase PknB. Carolacton caused a significant delocalization of these proteins from midcell, in accordance with a previous study demonstrating the Carolacton insensitive phenotype of a pknB deletion strain. Carolacton treated cells displayed an elongated phenotype, increased septum formation and a severe defect in daughter cell separation. GFP+ fusions of two hypothetical proteins (SMU_503 and SMU_609), that had previously been shown to be the most strongly upregulated genes after Carolacton treatment, were found to be localized at the septum in midcell, indicating their role in cell division. These findings highlight the importance of PknB as a key regulator of cell division in streptococci and indicate a profound impact of Carolacton on the coordination between peripheral and septal cell wall growth. The established vector system represents a novel tool to study essential steps of cellular metabolism.
    • Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

      Lemme, André; Sztajer, Helena; Wagner-Döbler, Irene (2010-02-23)
      Abstract Background One of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far. Results Here we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation. Conclusion This study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate.
    • Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans.

      Lemme, André; Sztajer, Helena; Wagner-Döbler, Irene; Helmholtz-Centre for Infection Research, Division of Cell Biology, Braunschweig, Germany. ale05@helmholtz-hzi.de (2010)
      BACKGROUND: One of the key virulence determinants of Streptococcus mutans, the primary etiological agent of human dental caries, is its strong acid tolerance. The acid tolerance response (ATR) of S. mutans comprises several mechanisms that are induced at low pH and allow the cells to quickly adapt to a lethal pH environment. Malolactic fermentation (MLF) converts L-malate to L-lactate and carbon dioxide and furthermore regenerates ATP, which is used to translocate protons across the membrane. Thus, MLF may contribute to the aciduricity of S. mutans but has not been associated with the ATR so far. RESULTS: Here we show that the malolactic fermentation (mle) genes are under the control of acid inducible promoters which are induced within the first 30 minutes upon acid shock in the absence of malate. Thus, MLF is part of the early acid tolerance response of S. mutans. However, acidic conditions, the presence of the regulator MleR and L-malate were required to achieve maximal expression of all genes, including mleR itself. Deletion of mleR resulted in a decreased capacity to carry out MLF and impaired survival at lethal pH in the presence of L-malate. Gel retardation assays indicated the presence of multiple binding sites for MleR. Differences in the retardation patterns occurred in the presence of L-malate, thus demonstrating its role as co-inducer for transcriptional regulation. CONCLUSION: This study shows that the MLF gene cluster is part of the early acid tolerance response in S. mutans and is induced by both low pH and L-malate.
    • Co-occurrence Analysis of Microbial Taxa in the Atlantic Ocean Reveals High Connectivity in the Free-Living Bacterioplankton.

      Milici, Mathias; Deng, Zhi-Luo; Tomasch, Jürgen; Decelle, Johan; Wos-Oxley, Melissa L; Wang, Hui; Jáuregui, Ruy; Plumeier, Iris; Giebel, Helge-Ansgar; Badewien, Thomas H; et al. (2016)
      We determined the taxonomic composition of the bacterioplankton of the epipelagic zone of the Atlantic Ocean along a latitudinal transect (51°S-47°N) using Illumina sequencing of the V5-V6 region of the 16S rRNA gene and inferred co-occurrence networks. Bacterioplankon community composition was distinct for Longhurstian provinces and water depth. Free-living microbial communities (between 0.22 and 3 μm) were dominated by highly abundant and ubiquitous taxa with streamlined genomes (e.g., SAR11, SAR86, OM1, Prochlorococcus) and could clearly be separated from particle-associated communities which were dominated by Bacteroidetes, Planktomycetes, Verrucomicrobia, and Roseobacters. From a total of 369 different communities we then inferred co-occurrence networks for each size fraction and depth layer of the plankton between bacteria and between bacteria and phototrophic micro-eukaryotes. The inferred networks showed a reduction of edges in the deepest layer of the photic zone. Networks comprised of free-living bacteria had a larger amount of connections per OTU when compared to the particle associated communities throughout the water column. Negative correlations accounted for roughly one third of the total edges in the free-living communities at all depths, while they decreased with depth in the particle associated communities where they amounted for roughly 10% of the total in the last part of the epipelagic zone. Co-occurrence networks of bacteria with phototrophic micro-eukaryotes were not taxon-specific, and dominated by mutual exclusion (~60%). The data show a high degree of specialization to micro-environments in the water column and highlight the importance of interdependencies particularly between free-living bacteria in the upper layers of the epipelagic zone.
    • Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

      Wang, Wei; Hollmann, Rajan; Deckwer, Wolf-Dieter (2006)
      High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available.High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses.The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ.In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.
    • Comparing the cariogenic species Streptococcus sobrinus and S. mutans on whole genome level.

      Conrads, Georg; de Soet, Johannes J; Song, Lifu; Henne, Karsten; Sztajer, Helena; Wagner-Döbler, Irene; Zeng, An-Ping (2014)
      Two closely related species of mutans streptococci, namely Streptococcus mutans and Streptococcus sobrinus, are associated with dental caries in humans. Their acidogenic and aciduric capacity is directly associated with the cariogenic potential of these bacteria. To survive acidic and temporarily harsh conditions in the human oral cavity with hundreds of other microbial co-colonizers as competitors, both species have developed numerous mechanisms for adaptation.
    • Complete Genome Sequences of Three Multidrug-Resistant Clinical Isolates of Streptococcus pneumoniae Serotype 19A with Different Susceptibilities to the Myxobacterial Metabolite Carolacton.

      Donner, Jannik; Bunk, Boyke; Schober, Isabel; Spröer, Cathrin; Bergmann, Simone; Jarek, Michael; Overmann, Jörg; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-16)
      The full-genome sequences of three drug- and multidrug-resistant Streptococcus pneumoniae clinical isolates of serotype 19A were determined by PacBio single-molecule real-time sequencing, in combination with Illumina MiSeq sequencing. A comparison to the genomes of other pneumococci indicates a high nucleotide sequence identity to strains Hungary19A-6 and TCH8431/19A.
    • Complete sequence of the suicide vector pJP5603.

      Riedel, Thomas; Rohlfs, Meike; Buchholz, Ina; Wagner-Döbler, Irene; Reck, Michael; Helmholtz-Centre for Infection Research, Group Microbial Communication, Braunschweig, Germany. tri07@helmholtz-hzi.de (2013-01)
      We have sequenced the complete R6K-based and mobilizable suicide vector pJP5603. For the replication of the vector a trans supply of the pir-encoded π protein of plasmid R6K is essential. The 3.126 kb plasmid encodes a kanamycin resistance cassette for selection and contains a lacZ-α-system that allows a blue-white selection of cloned fragments.
    • Construction and verification of the transcriptional regulatory response network of Streptococcus mutans upon treatment with the biofilm inhibitor carolacton.

      Sudhakar, Padhmanand; Reck, Michael; Wang, Wei; He, Feng Q; Dobler, Irene W; Zeng, An-Ping (2014)
      Carolacton is a newly identified secondary metabolite causing altered cell morphology and death of Streptococcus mutans biofilm cells. To unravel key regulators mediating these effects, the transcriptional regulatory response network of S. mutans biofilms upon carolacton treatment was constructed and analyzed. A systems biological approach integrating time-resolved transcriptomic data, reverse engineering, transcription factor binding sites, and experimental validation was carried out.