• A short story about a big magic bug.

      Bunk, Boyke; Schulz, Arne; Stammen, Simon; Münch, Richard; Warren, Martin J; Rohde, Manfred; Jahn, Dieter; Biedendieck, Rebekka; Institute of Microbiology; Technische Universität Braunschweig; Braunschweig, Germany. (2011-03-02)
      Bacillus megaterium, the "big beast," is a Gram-positive bacterium with a size of 4 × 1.5 µm. During the last years, it became more and more popular in the field of biotechnology for its recombinant protein production capacity. For the purpose of intra- as well as extracellular protein synthesis several vectors were constructed and commercialized (MoBiTec GmbH, Germany). On the basis of two compatible vectors, a T7 RNA polymerase driven protein production system was established. Vectors for chromosomal integration enable the direct manipulation of the genome. The vitamin B(12) biosynthesis of B. megaterium served as a model for the systematic development of a production strain using these tools. For this purpose, the overexpression of chromosomal and plasmid encoded genes and operons, the synthesis of anti-sense RNA for gene silencing, the removal of inhibitory regulatory elements in combination with the utilization of strong promoters, directed protein design, and the recombinant production of B(12) binding proteins to overcome feedback inhibition were successfully employed. For further system biotechnology based optimization strategies the genome sequence will provide a closer look into genomic capacities of B. megaterium. DNA arrays are available. Proteome, fluxome and metabolome analyses are possible. All data can be integrated by using a novel bioinformatics platform. Finally, the size of the "big beast" B. megaterium invites for cell biology research projects. All these features provide a solid basis for challenging biotechnological approaches.
    • Adherence and invasion of streptococci to eukaryotic cells and their role in disease pathogenesis.

      Rohde, Manfred; Chhatwal, G Singh; Department of Medical Microbiology, Helmholtz Centre for Infection Research, Braunschweig, Germany. manfred.rohde@helmholtz-hzi.de (2013)
      Streptococcal adhesion, invasion, intracellular trafficking, dissemination, and persistence in eukaryotic cells have a variety of implications in the infection pathogenesis. While cell adhesion establishes the initial host contact, adhering bacteria exploit the host cell for their own benefit. Internalization into the host cell is an essential step for bacterial survival and subsequent dissemination and persistence, thus playing a key role in the course of infection. This chapter summarizes the current knowledge about the diverse mechanisms of streptococcal adhesion to and invasion into different eukaryotic cells and the impact on dissemination and persistence which is reflected by consequences for the pathogenesis of streptococcal infections.
    • Bacillus megaterium--from simple soil bacterium to industrial protein production host.

      Vary, Patricia S; Biedendieck, Rebekka; Fuerch, Tobias; Meinhardt, Friedhelm; Rohde, Manfred; Deckwer, Wolf-Dieter; Jahn, Dieter; Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA. (2007-10)
      Bacillus megaterium has been industrially employed for more than 50 years, as it possesses some very useful and unusual enzymes and a high capacity for the production of exoenzymes. It is also a desirable cloning host for the production of intact proteins, as it does not possess external alkaline proteases and can stably maintain a variety of plasmid vectors. Genetic tools for this species include transducing phages and several hundred mutants covering the processes of biosynthesis, catabolism, division, sporulation, germination, antibiotic resistance, and recombination. The seven plasmids of B. megaterium strain QM B1551 contain several unusual metabolic genes that may be useful in bioremediation. Recently, several recombinant shuttle vectors carrying different strong inducible promoters and various combinations of affinity tags for simple protein purification have been constructed. Leader sequences-mediated export of affinity-tagged proteins into the growth medium was made possible. These plasmids are commercially available. For a broader application of B. megaterium in industry, sporulation and protease-deficient as well as UV-sensitive mutants were constructed. The genome sequence of two different strains, plasmidless DSM319 and QM B1551 carrying seven natural plasmids, is now available. These sequences allow for a systems biotechnology optimization of the production host B. megaterium. Altogether, a "toolbox" of hundreds of genetically characterized strains, genetic methods, vectors, hosts, and genomic sequences make B. megaterium an ideal organism for industrial, environmental, and experimental applications.
    • Bifidobacterial enolase, a cell surface receptor for human plasminogen involved in the interaction with the host.

      Candela, Marco; Biagi, Elena; Centanni, Manuela; Turroni, Silvia; Vici, Manuela; Musiani, Francesco; Vitali, Beatrice; Bergmann, Simone; Hammerschmidt, Sven; Brigidi, Patrizia; et al. (2009-10)
      The interaction with the host plasminogen/plasmin system represents a novel component in the molecular cross-talk between bifidobacteria and human host. Here, we demonstrated that the plasminogen-binding bifidobacterial species B. longum, B. bifidum, B. breve and B. lactis share the key glycolytic enzyme enolase as a surface receptor for human plasminogen. Enolase was visualized on the cell surface of the model strain B. lactis BI07. The His-tagged recombinant protein showed a high affinity for human plasminogen, with an equilibrium dissociation constant in the nanomolar range. By site-directed mutagenesis we demonstrated that the interaction between the B. lactis BI07 enolase and human plasminogen involves an internal plasminogen-binding site homologous to that of pneumococcal enolase. According to our data, the positively charged residues Lys-251 and Lys-255, as well as the negatively charged Glu-252, of the B. lactis BI07 enolase are crucial for plasminogen binding. Acting as a human plasminogen receptor, the bifidobacterial surface enolase is suggested to play an important role in the interaction process with the host.
    • Biofilm formation by Salmonella enterica serovar Typhimurium colonizing solid tumours.

      Crull, Katja; Rohde, Manfred; Westphal, Kathrin; Loessner, Holger; Wolf, Kathrin; Felipe-López, Alfonso; Hensel, Michael; Weiss, Siegfried (2011-08)
      Systemic administration of Salmonella enterica serovar Typhimurium to tumour bearing mice results in preferential colonization of the tumours and retardation of tumour growth. Although the bacteria are able to invade the tumour cells in vitro, in tumours they were never detected intracellularly. Ultrastructural analysis of Salmonella-colonized tumours revealed that the bacteria had formed biofilms. Interestingly, depletion of neutrophilic granulocytes drastically reduced biofilm formation. Obviously, bacteria form biofilms in response to the immune reactions of the host. Importantly, we tested Salmonella mutants that were no longer able to form biofilms by deleting central regulators of biofilm formation. Such bacteria could be observed intracellularly in immune cells of the host or in tumour cells. Thus, tumour colonizing S. typhimurium might form biofilms as protection against phagocytosis. Since other bacteria are behaving similarly, solid murine tumours might represent a unique model to study biofilm formation in vivo.
    • Biological functions of GCS3, a novel plasminogen-binding protein of Streptococcus dysgalactiae ssp. equisimilis.

      Bergmann, René; Dinkla, Katrin; Nitsche-Schmitz, D Patric; Graham, Rikki M A; Lüttge, Melanie; Sanderson-Smith, Martina L; Nerlich, Andreas; Rohde, Manfred; Chhatwal, Gursharan S; Dept. of Medical Microbiology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2011-02)
      Increasing awareness of the relevance of Streptococcus dysgalactiae ssp. equisimilis as a human pathogen motivates the analysis of its pathomechanisms. One of the mechanisms that increases infectivity and dissemination of several streptococcal species is the recruitment and subsequent activation of host plasminogen on the streptococcal surface. This study identified GCS3 as a novel plasminogen-binding M protein of S. dysgalactiae ssp. equisimilis and revealed a difference in the mode of binding as compared to the plasminogen-binding protein PAM of S. pyogenes. In contrast to PAM, GCS3 did not bind to the kringle 1-3 region of plasminogen. Despite this difference, GCS3 exerts the same function of recruiting plasminogen to the streptococcal surface, which can be activated by streptokinase and host plasminogen activators to serve as a spreading factor. Moreover, we demonstrate a role of GCS3 in plasminogen-dependent streptococcal adherence to human pharyngeal cells (cell line Detroit 562) that indicates an additional function of the protein as an adhesin in the oral cavity.
    • Caveolin limits membrane microdomain mobility and integrin-mediated uptake of fibronectin-binding pathogens.

      Hoffmann, Christine; Berking, Anne; Agerer, Franziska; Buntru, Alexander; Neske, Florian; Chhatwal, G Singh; Ohlsen, Knut; Hauck, Christof R; Lehrstuhl Zellbiologie X908, Universität Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany. (2010-12-15)
      Staphylococcus aureus, which is a leading cause of hospital-acquired infections, binds via fibronectin to integrin α5β1, a process that can promote host colonization in vivo. Integrin engagement induces actin cytoskeleton rearrangements that result in the uptake of S. aureus by non-professional phagocytic cells. Interestingly, we found that fibronectin-binding S. aureus trigger the redistribution of membrane microdomain components. In particular, ganglioside GM1 and GPI-linked proteins were recruited upon integrin β1 engagement, and disruption of membrane microdomains blocked bacterial internalization. Several membrane-microdomain-associated proteins, such as flotillin-1 and flotillin-2, as well as caveolin, were recruited to sites of bacterial attachment. Whereas dominant-negative versions of flotillin-2 did not affect bacterial attachment or internalization, cells deficient for caveolin-1 (Cav1(-/-)) showed increased uptake of S. aureus and other Fn-binding pathogens. Recruitment of membrane microdomains to cell-associated bacteria was unaltered in Cav1(-/-) cells. However, fluorescence recovery after photobleaching (FRAP) revealed an enhanced mobility of membrane-microdomain-associated proteins in the absence of caveolin-1. Enhanced membrane microdomain mobility and increased uptake of S. aureus was repressed by expression of wild-type caveolin-1, but not caveolin-1 G83S, which harbors a point mutation in the caveolin scaffolding domain. Similarly, chemical or physical stimulation of membrane fluidity led to increased uptake of S. aureus. These results highlight a crucial role for caveolin-1 in negative regulation of membrane microdomain mobility, thereby affecting endocytosis of bacteria-engaged integrins. This process might not only limit host cell invasion by integrin-binding bacterial pathogens, but might also be physiologically relevant for integrin-mediated cell adhesion.
    • Cellular aspects of the distinct M protein and SfbI anchoring pathways in Streptococcus pyogenes.

      Raz, Assaf; Talay, Susanne R; Fischetti, Vincent A; Bacterial Pathogenesis and Immunology, Rockefeller University, New York, USA. araz@rockefeller.edu (2012-05)
      Wall-anchored surface proteins are critical for the in vivo survival of Streptococcus pyogenes. Cues in the signal sequence direct the membrane translocation of surface proteins: M protein to the septum, and SfbI to the poles. Both proteins are subsequently anchored to the wall by the membrane bound enzyme sortase A. However, the cellular features of these pathways are not fully understood. Here we show that M protein and SfbI are anchored simultaneously throughout the cell cycle. M protein is rapidly anchored at the septum, and in part of the cell cycle, is anchored simultaneously at the mother and daughter septa. Conversely, SfbI accumulates gradually on peripheral peptidoglycan, resulting in a polar distribution. Sortase is not required for translocation of M protein or SfbI at their respective locations. Methicillin-induced unbalanced peptidoglycan synthesis diminishes surface M protein but not SfbI. Furthermore, overexpression of the division regulator DivIVA also diminishes surface M protein but increases SfbI. These results demonstrate a close connection between the regulation of cell division and protein anchoring. Better understanding of the spatial regulation of surface anchoring may lead to the identification of novel targets for the development of anti-infective agents, given the importance of surface molecules for pathogenesis.
    • Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus.

      Sugareva, Venelina; Härtl, Albert; Brock, Matthias; Hübner, Katrin; Rohde, Manfred; Heinekamp, Thorsten; Brakhage, Axel A (2006-11-01)
      Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Deltaabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Deltaabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Deltaabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.
    • Characterization of JG024, a pseudomonas aeruginosa PB1-like broad host range phage under simulated infection conditions.

      Garbe, Julia; Wesche, Andrea; Bunk, Boyke; Kazmierczak, Marlon; Selezska, Katherina; Rohde, Christine; Sikorski, Johannes; Rohde, Manfred; Jahn, Dieter; Schobert, Max (2010)
      Pseudomonas aeruginosa causes lung infections in patients suffering from the genetic disorder Cystic Fibrosis (CF). Once a chronic lung infection is established, P. aeruginosa cannot be eradicated by antibiotic treatment. Phage therapy is an alternative to treat these chronic P. aeruginosa infections. However, little is known about the factors which influence phage infection of P. aeruginosa under infection conditions and suitable broad host range phages.
    • Clinical and microbiologic characteristics of invasive Streptococcus pyogenes infections in north and south India.

      Haggar, Axana; Nerlich, Andreas; Kumar, Rajesh; Abraham, Vinod J; Brahmadathan, Kootallur N; Ray, Pallab; Dhanda, Vanita; Joshua, John Melbin Jose; Mehra, Narinder; Bergmann, Rene; et al. (2012-05)
      The lack of epidemiologic data on invasive Streptococcus pyogenes infections in many developing countries is concerning, as S. pyogenes infections are commonly endemic in these areas. Here we present the results of the first prospective surveillance study of invasive Streptococcus pyogenes infections in India. Fifty-four patients with invasive S. pyogenes infections were prospectively enrolled at two study sites, one in the north and one in the south of India. Sterile-site isolates were collected, and clinical information was documented using a standardized questionnaire. Available acute-phase sera were tested for their ability to inhibit superantigens produced by the patient's own isolate using a cell-based neutralizing assay. The most common clinical presentations were bacteremia without focus (30%), pneumonia (28%), and cellulitis (17%). Only two cases of streptococcal toxic shock syndrome and no cases of necrotizing fasciitis were identified. Characterization of the isolates revealed great heterogeneity, with 32 different emm subtypes and 29 different superantigen gene profiles being represented among the 49 sterile-site isolates. Analyses of acute-phase sera showed that only 20% of the cases in the north cohort had superantigen-neutralizing activity in their sera, whereas 50% of the cases from the south site had neutralizing activity. The results demonstrate that there are important differences in both clinical presentation and strain characteristics between invasive S. pyogenes infections in India and invasive S. pyogenes infections in Western countries. The findings underscore the importance of epidemiologic studies on streptococcal infections in India and have direct implications for current vaccine developments.
    • Complete genome sequence of Acetohalobium arabaticum type strain (Z-7288).

      Sikorski, Johannes; Lapidus, Alla; Chertkov, Olga; Lucas, Susan; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Tice, Hope; Cheng, Jan-Fang; Han, Cliff; et al. (2010)
      Acetohalobium arabaticum Zhilina and Zavarzin 1990 is of special interest because of its physiology and its participation in the anaerobic C(1)-trophic chain in hypersaline environments. This is the first completed genome sequence of the family Halobacteroidaceae and only the second genome sequence in the order Halanaerobiales. The 2,469,596 bp long genome with its 2,353 protein-coding and 90 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Acidaminococcus fermentans type strain (VR4).

      Chang, Yun-Juan; Pukall, Rüdiger; Saunders, Elizabeth; Lapidus, Alla; Copeland, Alex; Nolan, Matt; Glavina Del Rio, Tijana; Lucas, Susan; Chen, Feng; Tice, Hope; et al. (2010)
      Acidaminococcus fermentans (Rogosa 1969) is the type species of the genus Acidaminococcus, and is of phylogenetic interest because of its isolated placement in a genomically little characterized region of the Firmicutes. A. fermentans is known for its habitation of the gastrointestinal tract and its ability to oxidize trans-aconitate. Its anaerobic fermentation of glutamate has been intensively studied and will now be complemented by the genomic basis. The strain described in this report is a nonsporulating, nonmotile, Gram-negative coccus, originally isolated from a pig alimentary tract. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the family Acidaminococcaceae, and the 2,329,769 bp long genome with its 2,101 protein-coding and 81 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Actinosynnema mirum type strain (101).

      Land, Miriam; Lapidus, Alla; Mayilraj, Shanmugam; Chen, Feng; Copeland, Alex; Del Rio, Tijana Glavina; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; et al. (2009)
      Actinosynnema mirum Hasegawa et al. 1978 is the type species of the genus, and is of phylogenetic interest because of its central phylogenetic location in the Actino-synnemataceae, a rapidly growing family within the actinobacterial suborder Pseudo-nocardineae. A. mirum is characterized by its motile spores borne on synnemata and as a producer of nocardicin antibiotics. It is capable of growing aerobically and under a moderate CO(2) atmosphere. The strain is a Gram-positive, aerial and substrate mycelium producing bacterium, originally isolated from a grass blade collected from the Raritan River, New Jersey. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Actinosynnemataceae, and only the second sequence from the actinobacterial suborder Pseudonocardineae. The 8,248,144 bp long single replicon genome with its 7100 protein-coding and 77 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Alicyclobacillus acidocaldarius type strain (104-IA).

      Mavromatis, Konstantinos; Sikorski, Johannes; Lapidus, Alla; Glavina Del Rio, Tijana; Copeland, Alex; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; et al. (2010)
      Alicyclobacillus acidocaldarius (Darland and Brock 1971) is the type species of the larger of the two genera in the bacillal family 'Alicyclobacillaceae'. A. acidocaldarius is a free-living and non-pathogenic organism, but may also be associated with food and fruit spoilage. Due to its acidophilic nature, several enzymes from this species have since long been subjected to detailed molecular and biochemical studies. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first completed genome sequence of the family 'Alicyclobacillaceae'. The 3,205,686 bp long genome (chromosome and three plasmids) with its 3,153 protein-coding and 82 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Aminobacterium colombiense type strain (ALA-1).

      Chertkov, Olga; Sikorski, Johannes; Brambilla, Evelyne; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; et al. (2010)
      Aminobacterium colombiense Baena et al. 1999 is the type species of the genus Aminobacterium. This genus is of large interest because of its isolated phylogenetic location in the family Synergistaceae, its strictly anaerobic lifestyle, and its ability to grow by fermentation of a limited range of amino acids but not carbohydrates. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member of the family Synergistaceae and the first genome sequence of a member of the genus Aminobacterium. The 1,980,592 bp long genome with its 1,914 protein-coding and 56 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Arcanobacterium haemolyticum type strain (11018).

      Yasawong, Montri; Teshima, Hazuki; Lapidus, Alla; Nolan, Matt; Lucas, Susan; Glavina Del Rio, Tijana; Tice, Hope; Cheng, Jan-Fang; Bruce, David; Detter, Chris; et al. (2010)
      Arcanobacterium haemolyticum (ex MacLean et al. 1946) Collins et al. 1983 is the type species of the genus Arcanobacterium, which belongs to the family Actinomycetaceae. The strain is of interest because it is an obligate parasite of the pharynx of humans and farm animal; occasionally, it causes pharyngeal or skin lesions. It is a Gram-positive, nonmotile and non-sporulating bacterium. The strain described in this study was isolated from infections amongst American soldiers of certain islands of the North and West Pacific. This is the first completed sequence of a member of the genus Arcanobacterium and the ninth type strain genome from the family Actinomycetaceae. The 1,986,154 bp long genome with its 1,821 protein-coding and 64 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Archaeoglobus profundus type strain (AV18).

      von Jan, Mathias; Lapidus, Alla; Del Rio, Tijana Glavina; Copeland, Alex; Tice, Hope; Cheng, Jan-Fang; Lucas, Susan; Chen, Feng; Nolan, Matt; Goodwin, Lynne; et al. (2010)
      Archaeoglobus profundus (Burggraf et al. 1990) is a hyperthermophilic archaeon in the euryarchaeal class Archaeoglobi, which is currently represented by the single family Archaeoglobaceae, containing six validly named species and two strains ascribed to the genus 'Geoglobus' which is taxonomically challenged as the corresponding type species has no validly published name. All members were isolated from marine hydrothermal habitats and are obligate anaerobes. Here we describe the features of the organism, together with the complete genome sequence and annotation. This is the second completed genome sequence of a member of the class Archaeoglobi. The 1,563,423 bp genome with its 1,858 protein-coding and 52 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.
    • Complete genome sequence of Arcobacter nitrofigilis type strain (CI).

      Pati, Amrita; Gronow, Sabine; Lapidus, Alla; Copeland, Alex; Glavina Del Rio, Tijana; Nolan, Matt; Lucas, Susan; Tice, Hope; Cheng, Jan-Fang; Han, Cliff; et al. (2010)
      Arcobacter nitrofigilis (McClung et al. 1983) Vandamme et al. 1991 is the type species of the genus Arcobacter in the family Campylobacteraceae within the Epsilonproteobacteria. The species was first described in 1983 as Campylobacter nitrofigilis [1] after its detection as a free-living, nitrogen-fixing Campylobacter species associated with Spartina alterniflora Loisel roots [2]. It is of phylogenetic interest because of its lifestyle as a symbiotic organism in a marine environment in contrast to many other Arcobacter species which are associated with warm-blooded animals and tend to be pathogenic. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a type stain of the genus Arcobacter. The 3,192,235 bp genome with its 3,154 protein-coding and 70 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.