• Salmonella Typhimurium discreet-invasion of the murine gut absorptive epithelium.

      Fattinger, Stefan A; Böck, Desirée; Di Martino, Maria Letizia; Deuring, Sabrina; Samperio Ventayol, Pilar; Ek, Viktor; Furter, Markus; Kreibich, Saskia; Bosia, Francesco; Müller-Hauser, Anna A; et al. (PLOS, 2020-05-04)
      Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.
    • SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G.

      Bergmann, Simone; Eichhorn, Inga; Kohler, Thomas P; Hammerschmidt, Sven; Goldmann, Oliver; Rohde, M; Fulde, Marcus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.
    • The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon.

      Frantz, Renate; Teubner, Lisa; Schultze, Tilman; La Pietra, Luigi; Müller, Christin; Gwozdzinski, Konrad; Pillich, Helena; Hain, Torsten; Weber-Gerlach, Michaela; Panagiotidis, Georgios-Dimitrios; et al. (ASM, 2019-10-08)
      Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response.IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.
    • Sequencing and Characterization of Pseudomonas aeruginosa phage JG004

      Garbe, Julia; Bunk, Boyke; Rohde, Manfred; Schobert, Max (2011-05-14)
      Abstract Background Phages could be an important alternative to antibiotics, especially for treatment of multiresistant bacteria as e.g. Pseudomonas aeruginosa. For an effective use of bacteriophages as antimicrobial agents, it is important to understand phage biology but also genes of the bacterial host essential for phage infection. Results We isolated and characterized a lytic Pseudomonas aeruginosa phage, named JG004, and sequenced its genome. Phage JG004 is a lipopolysaccharide specific broad-host-range phage of the Myoviridae phage family. The genome of phage JG004 encodes twelve tRNAs and is highly related to the PAK-P1 phage genome. To investigate phage biology and phage-host interactions, we used transposon mutagenesis of the P. aeruginosa host and identified P. aeruginosa genes, which are essential for phage infection. Analysis of the respective P. aeruginosa mutants revealed several characteristics, such as host receptor and possible spermidine-dependance of phage JG004. Conclusions Whole genome sequencing of phage JG004 in combination with identification of P. aeruginosa host genes essential for infection, allowed insights into JG004 biology, revealed possible resistance mechanisms of the host bacterium such as mutations in LPS and spermidine biosynthesis and can also be used to characterize unknown gene products in P. aeruginosa.
    • The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2

      Krause-Gruszczynska, Malgorzata; Boehm, Manja; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Buday, Laszlo; Oyarzabal, Omar A; Backert, Steffen (2011-12-28)
      Abstract Background Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake. Results Using high-resolution scanning electron microscopy, GTPase pulldowns, G-Lisa and gentamicin protection assays we found that each studied host factor is necessary for induction of Cdc42-GTP and efficient invasion. Interestingly, filopodia formation and associated membrane dynamics linked to invasion were only seen during infection of wild-type but not in knockout cells. Infection of cells stably expressing integrin-beta1 variants with well-known defects in fibronectin fibril formation or FAK signaling also exhibited severe deficiencies in Cdc42 activation and bacterial invasion. We further demonstrated that infection of wild-type cells induces increasing amounts of phosphorylated FAK and growth factor receptors (EGFR and PDGFR) during the course of infection, correlating with accumulating Cdc42-GTP levels and C. jejuni invasion over time. In studies using pharmacological inhibitors, silencing RNA (siRNA) and dominant-negative expression constructs, EGFR, PDGFR and PI3-kinase appeared to represent other crucial components upstream of Cdc42 and invasion. siRNA and the use of Vav1/2-/- knockout cells further showed that the guanine exchange factor Vav2 is required for Cdc42 activation and maximal bacterial invasion. Overexpression of certain mutant constructs indicated that Vav2 is a linker molecule between Cdc42 and activated EGFR/PDGFR/PI3-kinase. Using C. jejuni mutant strains we further demonstrated that the fibronectin-binding protein CadF and intact flagella are involved in Cdc42-GTP induction, indicating that the bacteria may directly target the fibronectin/integrin complex for inducing signaling leading to its host cell entry. Conclusion Collectively, our findings led us propose that C. jejuni infection triggers a novel fibronectin→integrin-beta1→FAK/Src→EGFR/PDGFR→PI3-kinase→Vav2 signaling cascade, which plays a crucial role for Cdc42 GTPase activity associated with filopodia formation and enhances bacterial invasion.
    • The signaling pathway of Campylobacter jejuni-induced Cdc42 activation: Role of fibronectin, integrin beta1, tyrosine kinases and guanine exchange factor Vav2.

      Krause-Gruszczynska, Malgorzata; Boehm, Manja; Rohde, Manfred; Tegtmeyer, Nicole; Takahashi, Seiichiro; Buday, Laszlo; Oyarzabal, Omar A; Backert, Steffen; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2011)
      Host cell invasion by the foodborne pathogen Campylobacter jejuni is considered as one of the primary reasons of gut tissue damage, however, mechanisms and key factors involved in this process are widely unclear. It was reported that small Rho GTPases, including Cdc42, are activated and play a role during invasion, but the involved signaling cascades remained unknown. Here we utilised knockout cell lines derived from fibronectin-/-, integrin-beta1-/-, focal adhesion kinase (FAK)-/- and Src/Yes/Fyn-/- deficient mice, and wild-type control cells, to investigate C. jejuni-induced mechanisms leading to Cdc42 activation and bacterial uptake.
    • Silvanigrella aquatica gen. nov., sp. nov., isolated from a freshwater lake, description of Silvanigrellaceae fam. nov. and Silvanigrellales ord. nov., reclassification of the order Bdellovibrionales in the class Oligoflexia, reclassification of the families Bacteriovoracaceae and Halobacteriovoraceae in the new order Bacteriovoracales ord. nov., and reclassification of the family Pseudobacteriovoracaceae in the order Oligoflexales.

      Hahn, Martin W; Schmidt, Johanna; Koll, Ulrike; Rohde, M; Verbarg, Susanne; Pitt, Alexandra; Nakai, Ryosuke; Naganuma, Takeshi; Lang, Elke; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08)
      The unusual chemo-organoheterotrophic proteobacterial strain MWH-Nonnen-W8redT was isolated from a lake located in the Black Forest (Schwarzwald), Germany, by using the filtration-acclimatization method. Phylogenetic analyses based on the 16S rRNA gene sequence of the strain could not provide clear hints on classification of the strain in one of the current classes of the phylum Proteobacteria. Whole-genome sequencing resulted in a genome size of 3.5 Mbp and revealed a quite low DNA G+C content of 32.6 mol%. In-depth phylogenetic analyses based on alignments of 74 protein sequences of a phylogenetically broad range of taxa suggested assignment of the strain to a new order of the class Oligoflexia. These analyses also suggested that the order Bdellovibrionales should be transferred from the class Deltaproteobacteria to the class Oligoflexia, that this order should be split into two orders, and that the family Pseudobacteriovoracaceae should be transferred from the order Bdellovibrionales to the order Oligoflexales. We propose to establish for strain MWH-Nonnen-W8redT (=DSM 23856T=CCUG 58639T) the novel species and genus Silvanigrella aquatica gen. nov., sp. nov. to be placed in the new family Silvanigrellaceae fam. nov. of the new order Silvanigrellales ord. nov.
    • Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

      David, Florian; Berger, Antje; Hänsch, Robert; Rohde, Manfred; Franco-Lara, Ezequiel (2011-04-15)
      Abstract Background Single cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively. Results Here different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied. Conclusions It was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.
    • Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia.

      Kumaraswamy, Monika; Lin, Leo; Olson, Joshua; Sun, Ching-Fang; Nonejuie, Poochit; Corriden, Ross; Döhrmann, Simon; Ali, Syed Raza; Amaro, Deirdre; Rohde, M; et al. (2016-05)
      The Gram-negative bacillus Stenotrophomonas maltophilia (SM) is an emerging MDR opportunistic pathogen. Recent studies identify a potentially relevant activity of azithromycin against Gram-negative bacteria overlooked in standard bacteriological testing. We investigated azithromycin activity against SM in testing conditions incorporating mammalian tissue culture medium and host defence factors.
    • Stieleria varia sp. nov., isolated from wood particles in the Baltic Sea, constitutes a novel species in the family Pirellulaceae within the phylum Planctomycetes.

      Surup, Frank; Wiegand, Sandra; Boedeker, Christian; Heuer, Anja; Peeters, Stijn H; Jogler, Mareike; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian; Kallscheuer, Nicolai; et al. (Springer, 2020-08-14)
      Species belonging to the bacterial phylum Planctomycetes are ubiquitous members of the microbial communities in aquatic environments and are frequently isolated from various biotic and abiotic surfaces in marine and limnic water bodies. Planctomycetes have large genomes of up to 12.4 Mb, follow complex lifestyles and display an uncommon cell biology; features which motivate the investigation of members of this phylum in greater detail. As a contribution to the current collection of axenic cultures of Planctomycetes, we here describe strain Pla52T isolated from wood particles in the Baltic Sea. Phylogenetic analysis places the strain in the family Pirellulaceae and suggests two species of the recently described genus Stieleria as current closest neighbours. Strain Pla52nT shows typical features of members of the class Planctomycetia, including division by polar budding and the presence of crateriform structures. Colonies of strain Pla52nT have a light orange colour, which is an unusual pigmentation compared to the majority of members in the phylum, which show either a pink to red pigmentation or entirely lack pigmentation. Optimal growth of strain Pla52nT at 33 °C and pH 7.5 indicates a mesophilic (i.e. with optimal growth between 20 and 45 °C) and neutrophilic growth profile. The strain is an aerobic heterotroph with motile daughter cells. Its genome has a size of 9.6 Mb and a G + C content of 56.0%. Polyphasic analyses justify delineation of the strain from described species within the genus Stieleria. Therefore, we conclude that strain Pla52nT = LMG 29463T = VKM B-3447T should be classified as the type strain of a novel species, for which we propose the name Stieleria varia sp. nov.
    • Still Something to Discover: Novel Insights into Phage Diversity and Taxonomy.

      Korf, Imke H E; Meier-Kolthoff, Jan P; Adriaenssens, Evelien M; Kropinski, Andrew M; Nimtz, Manfred; Rohde, Manfred; van Raaij, Mark J; Wittmann, Johannes; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2019-05-17)
      The aim of this study was to gain further insight into the diversity of Escherichia coli phagesfollowed by enhanced work on taxonomic issues in that field. Therefore, we present the genomiccharacterization and taxonomic classification of 50 bacteriophages against E. coli isolated fromvarious sources, such as manure or sewage. All phages were examined for their host range on a setof different E. coli strains, originating, e.g., from human diagnostic laboratories or poultry farms.Transmission electron microscopy revealed a diversity of morphotypes (70% Myo-, 22% Sipho-, and8% Podoviruses), and genome sequencing resulted in genomes sizes from ~44 to ~370 kb.Annotation and comparison with databases showed similarities in particular to T4- and T5-likephages, but also to less-known groups. Though various phages against E. coli are already describedin literature and databases, we still isolated phages that showed no or only few similarities to otherphages, namely phages Goslar, PTXU04, and KWBSE43-6. Genome-based phylogeny andclassification of the newly isolated phages using VICTOR resulted in the proposal of new generaand led to an enhanced taxonomic classification of E. coli phages.
    • Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells.

      Skive, Bolette; Rohde, M; Molinari, Gabriella; Braunstein, Thomas Hartig; Bojesen, Anders M; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017)
      Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a phagolysosome. The results indicate that an intracellular phase may be one way S. zooepidemicus survives in the host, and could in part explain how S. zooepidemicus can cause recurrent/persistent infections. Future studies should reveal the ability of S. zooepidemicus to internalize and survive in primary equine endometrial cells and during in vivo conditions.
    • Streptococcus pyogenes adhesion and colonization.

      Brouwer, Stephan; Barnett, Timothy C; Rivera-Hernandez, Tania; Rohde, M; Walker, Mark J; Helmholtz Centre for infection research, Inhoffenstr. 7,38124 Braunschweig, Germany. (2016-06-17)
      Streptococcus pyogenes (group A Streptococcus, GAS) is a human-adapted pathogen responsible for a wide spectrum of disease. GAS can cause relatively mild illnesses, such as strep throat or impetigo, and less frequent but severe life-threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. GAS is an important public health problem causing significant morbidity and mortality worldwide. The main route of GAS transmission between humans is through close or direct physical contact, and particularly via respiratory droplets. The upper respiratory tract and skin are major reservoirs for GAS infections. The ability of GAS to establish an infection in the new host at these anatomical sites primarily results from two distinct physiological processes, namely bacterial adhesion and colonization. These fundamental aspects of pathogenesis rely upon a variety of GAS virulence factors, which are usually under strict transcriptional regulation. Considerable progress has been made in better understanding these initial infection steps. This review summarizes our current knowledge of the molecular mechanisms of GAS adhesion and colonization.
    • Sulfate-Reducing Bacteria That Produce Exopolymers Thrive in the Calcifying Zone of a Hypersaline Cyanobacterial Mat.

      Spring, Stefan; Sorokin, Dimitry Y; Verbarg, Susanne; Rohde, M; Woyke, Tanja; Kyrpides, Nikos C; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (Frontiers, 2019-01-01)
      Calcifying microbial mats in hypersaline environments are important model systems for the study of the earliest ecosystems on Earth that started to appear more than three billion years ago and have been preserved in the fossil record as laminated lithified structures known as stromatolites. It is believed that sulfate-reducing bacteria play a pivotal role in the lithification process by increasing the saturation index of calcium minerals within the mat. Strain L21-Syr-ABT was isolated from anoxic samples of a several centimeters-thick microbialite-forming cyanobacterial mat of a hypersaline lake on the Kiritimati Atoll (Kiribati, Central Pacific). The novel isolate was assigned to the family Desulfovibrionaceae within the Deltaproteobacteria. Available 16S rRNA-based population surveys obtained from discrete layers of the mat indicate that the occurrence of a species-level clade represented by strain L21-Syr-ABT is restricted to a specific layer of the suboxic zone, which is characterized by the presence of aragonitic spherulites. To elucidate a possible function of this sulfate-reducing bacterium in the mineral formation within the mat a comprehensive phenotypic characterization was combined with the results of a comparative genome analysis. Among the determined traits of strain L21-Syr-ABT, several features were identified that could play a role in the precipitation of calcium carbonate: (i) the potential deacetylation of polysaccharides and consumption of substrates such as lactate and sulfate could mobilize free calcium; (ii) under conditions that favor the utilization of formate and hydrogen, the alkalinity engine within the mat is stimulated, thereby increasing the availability of carbonate; (iii) the production of extracellular polysaccharides could provide nucleation sites for calcium mineralization. In addition, our data suggest the proposal of the novel species and genus Desulfohalovibrio reitneri represented by the type strain L21-Syr-ABT (=DSM 26903T = JCM 18662T).
    • Targeting bioenergetics is key to counteracting the drug-tolerant state of biofilm-grown bacteria.

      Donnert, Monique; Elsheikh, Sarah; Arce-Rodriguez, Alejandro; Pawar, Vinay; Braubach, Peter; Jonigk, Danny; Haverich, Axel; Weiss, Siegfried; Müsken, Mathias; Häussler, Susanne; et al. (PLOS, 2020-12-22)
      Embedded in an extracellular matrix, biofilm-residing bacteria are protected from diverse physicochemical insults. In accordance, in the human host the general recalcitrance of biofilm-grown bacteria hinders successful eradication of chronic, biofilm-associated infections. In this study, we demonstrate that upon addition of promethazine, an FDA approved drug, antibiotic tolerance of in vitro biofilm-grown bacteria can be abolished. We show that following the addition of promethazine, diverse antibiotics are capable of efficiently killing biofilm-residing cells at minimal inhibitory concentrations. Synergistic effects could also be observed in a murine in vivo model system. PMZ was shown to increase membrane potential and interfere with bacterial respiration. Of note, antibiotic killing activity was elevated when PMZ was added to cells grown under environmental conditions that induce low intracellular proton levels. Our results imply that biofilm-grown bacteria avoid antibiotic killing and become tolerant by counteracting intracellular alkalization through the adaptation of metabolic and transport functions. Abrogation of antibiotic tolerance by interfering with the cell's bioenergetics promises to pave the way for successful eradication of biofilm-associated infections. Repurposing promethazine as a biofilm-sensitizing drug has the potential to accelerate the introduction of new treatments for recalcitrant, biofilm-associated infections into the clinic.
    • TatBC-Independent TatA/Tat Substrate Interactions Contribute to Transport Efficiency.

      Taubert, Johannes; Hou, Bo; Risselada, H Jelger; Mehner, Denise; Lünsdorf, Heinrich; Grubmüller, Helmut; Brüser, Thomas (2015)
      The Tat system can transport folded, signal peptide-containing proteins (Tat substrates) across energized membranes of prokaryotes and plant plastids. A twin-arginine motif in the signal peptide of Tat substrates is recognized by TatC-containing complexes, and TatA permits the membrane passage. Often, as in the model Tat systems of Escherichia coli and plant plastids, a third component - TatB - is involved that resembles TatA but has a higher affinity to TatC. It is not known why most TatA dissociates from TatBC complexes in vivo and distributes more evenly in the membrane. Here we show a TatBC-independent substrate-binding to TatA from Escherichia coli, and we provide evidence that this binding enhances Tat transport. First hints came from in vivo cross-linking data, which could be confirmed by affinity co-purification of TatA with the natural Tat substrates HiPIP and NrfC. Two positions on the surface of HiPIP could be identified that are important for the TatA interaction and transport efficiency, indicating physiological relevance of the interaction. Distributed TatA thus may serve to accompany membrane-interacting Tat substrates to the few TatBC spots in the cells.
    • Taxonomic analyses of members of the Streptomyces cinnabarinus cluster, description of Streptomyces cinnabarigriseus sp. nov. and Streptomyces davaonensis sp. nov.

      Landwehr, Wiebke; Kämpfer, Peter; Glaeser, Stefanie P; Rückert, Christian; Kalinowski, Jörn; Blom, Jochen; Goesmann, Alexander; Mack, Matthias; Schumann, Peter; Atasayar, Ewelina; et al. (2017-12-11)
      Roseoflavin is the only known riboflavin (vitamin B2) analog with antibiotic properties. It is actively taken up by many micro-organisms and targets flavinmononucleotide riboswitches and flavoproteins. It is described as the product of the tentatively named 'Streptomyces davawensis' JCM 4913. Taxonomic analysis of this strain with a polyphasic approach showed that it is very closely related to Streptomyces cinnabarinus (DSM 40467). The two Streptomyces isolates were obtained from different geographical locations (the Philippines and the Kamchatka Peninsula, respectively), their genomes have been sequenced and the question was whether or not the two isolates were representatives of the same species. As we also worked with another isolate of Streptomyces cinnabarinus JS 360, the producer of the cinnabaramides, we wanted to clarify the taxonomic position of the three isolates by using a polyphasic approach. After analysis of the 16S rRNA gene sequence, we found in total 23 species of the genus Streptomyces that showed a similarity higher than 98.5 % to the three strains. We showed that 'S. davawensis' JCM 4913 and S. cinnabarinus DSM 40467 were very closely related but belong to two different species. Hence, we validate 'S. davawensis' as Streptomyces davaonensis sp. nov. with the type strain JCM 4913T (=DSM 101723T). In addition, the cinnabaramide producer can be clearly differentiated from S. davaonensis and this isolate is described as Streptomyces cinnabarigriseus sp. nov. with strain JS360T (=NCCB 100590T=DSM 101724T) as the type strain.
    • Terricaulis silvestris gen. Nov., sp. nov., a novel prosthecate, budding member of the family caulobacteraceae isolated from forest soil

      Vieira, Selma; Pascual, Javier; Boedeker, Christian; Geppert, Alicia; Riedel, Thomas; Rohde, Manfred; Overmann, Jörg; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Microbiology Society, 2020-08-07)
      The family Caulobacteraceae comprises prosthecate bacteria with a dimorphic cell cycle and also non-prosthecate bacteria. Cells of all described species divide by binary fission. Strain 0127_4T was isolated from forest soil in Baden Württemberg (Germany) and determined to be the first representative of the family Caulobacteraceae which divided by budding. Cells of strain 0127_4T were Gram-negative, rod-shaped, prosthecate, motile by means of a polar flagellum, non-spore-forming and non-capsulated. The strain formed small white colonies and grew aerobically and chemo-organotrophically utilizing organic acids, amino acids and proteinaceous substrates. 16S rRNA gene sequence analysis indicated that this bacterium was related to Aquidulcibacter paucihalophilus TH1-2T and Asprobacter aquaticus DRW22-8T with 91.3 and 89.7% sequence similarity, respectively. Four unidentified glycolipids were detected as the major polar lipids and, unlike all described members of the family Caulobacteraceae, phosphatidylglycerol was absent. The major fatty acids were summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), summed feature 9 (iso-C17 : 1ω9c/C16 : 0 10-methyl), C16 : 0 and summed feature 3 (C16 : 1ω6c/C16 : 1ω7c). The major respiratory quinone was Q-10. The G+C content of the genomic DNA was 63.5 %. Based on the present taxonomic characterization, strain 0127_4T represents a novel species of a new genus, Terricaulis silvestris gen. nov., sp. nov. The type strain of Terricaulis silvestris is 0127_4T (=DSM 104635T=CECT 9243T).
    • Thalassoglobus polymorphus sp. nov., a novel Planctomycete isolated close to a public beach of Mallorca Island.

      Rivas-Marin, Elena; Wiegand, Sandra; Kallscheuer, Nicolai; Jogler, Mareike; Peeters, Stijn H; Heuer, Anja; Jetten, Mike S M; Boedeker, Christian; Rohde, Manfred; Devos, Damien P; et al. (Springer, 2020-06-24)
      Access to axenic cultures is crucial to extend the knowledge of the biology, lifestyle or metabolic capabilities of bacteria from different phyla. The phylum Planctomycetes is an excellent example since its members display an unusual cell biology and complex lifestyles. As a contribution to the current collection of axenic planctomycete cultures, here we describe strain Mal48T isolated from phytoplankton material sampled at the coast of S'Arenal close to Palma de Mallorca (Spain). The isolated strain shows optimal growth at pH 7.0-7.5 and 30 °C and exhibits typical features of Planctomycetes. Cells of the strain are spherical to pear-shaped, divide by polar budding with daughter cells showing the same shape as the mother cell, tend to aggregate, display a stalk and produce matrix or fimbriae. Strain Mal48T showed 95.8% 16S rRNA gene sequence similarity with the recently described Thalassoglobus neptunius KOR42T. The genome sequence of the novel isolate has a size of 6,357,355 bp with a G+C content of 50.3%. A total of 4874 protein-coding genes, 41 tRNA genes and 2 copies of the 16S rRNA gene are encoded in the genome. Based on phylogenetic, morphological and physiological analyses, we conclude that strain Mal48T (= DSM 100737T = LMG 29019T) should be classified as the type strain of a new species in the genus Thalassoglobus, for which the name Thalassoglobus polymorphus sp. nov. is proposed.
    • Three glycosylated serine-rich repeat proteins play a pivotal role in adhesion and colonization of the pioneer commensal bacterium, Streptococcus salivarius.

      Couvigny, Benoit; Lapaque, Nicolas; Rigottier-Gois, Lionel; Guillot, Alain; Chat, Sophie; Meylheuc, Thierry; Kulakauskas, Saulius; Rohde, M; Mistou, Michel-Yves; Renault, Pierre; et al. (Wiley-Blackwell, 2017-01-01)
      Bacterial adhesion is a critical step for colonization of the host. The pioneer colonizer and commensal bacterium of the human gastrointestinal tract, Streptococcus salivarius, has strong adhesive properties but the molecular determinants of this adhesion remain uncharacterized. Serine‐rich repeat (SRR) glycoproteins are a family of adhesins that fulfil an important role in adhesion. In general, Gram‐positive bacterial genomes have a unique SRR glycoprotein‐encoding gene. We demonstrate that S. salivarius expresses three large and glycosylated surface‐exposed proteins – SrpA, SrpB and SrpC – that show characteristics of SRR glycoproteins and are secreted through the accessory SecA2/Y2 system. Two glycosyltransferases – GtfE/F – encoded outside of the secA2/Y2 locus, unusually, perform the first step of the sequential glycosylation process, which is crucial for SRR activity. We show that SrpB and SrpC play complementary adhesive roles involved in several steps of the colonization process: auto‐aggregation, biofilm formation and adhesion to a variety of host epithelial cells and components. We also show that at least one of the S. salivarius SRR glycoproteins is important for colonization in mice. SrpA, SrpB and SrpC are the main factors underlying the multifaceted adhesion of S. salivarius and, therefore, play a major role in host colonization.