• Complete genome sequence of Planctomyces brasiliensis type strain (DSM 5305T), phylogenomic analysis and reclassification of Planctomycetes including the descriptions of Gimesia gen. nov., Planctopirus gen. nov. and Rubinisphaera gen. nov. and emended descriptions of the order Planctomycetales and the family Planctomycetaceae

      Scheuner, Carmen; Tindall, Brian J; Lu, Megan; Nolan, Matt; Lapidus, Alla; Cheng, Jan-Fang; Goodwin, Lynne; Pitluck, Sam; Huntemann, Marcel; Liolios, Konstantinos; et al. (2014-12-08)
      Abstract Planctomyces brasiliensis Schlesner 1990 belongs to the order Planctomycetales, which differs from other bacterial taxa by several distinctive features such as internal cell compartmentalization, multiplication by forming buds directly from the spherical, ovoid or pear-shaped mother cell and a cell wall consisting of a proteinaceous layer rather than a peptidoglycan layer. The first strains of P. brasiliensis, including the type strain IFAM 1448T, were isolated from a water sample of Lagoa Vermelha, a salt pit near Rio de Janeiro, Brasil. This is the second completed genome sequence of a type strain of the genus Planctomyces to be published and the sixth type strain genome sequence from the family Planctomycetaceae. The 6,006,602 bp long genome with its 4,811 protein-coding and 54 RNA genes is a part of the G enomic E ncyclopedia of Bacteria and Archaea project. Phylogenomic analyses indicate that the classification within the Planctomycetaceae is partially in conflict with its evolutionary history, as the positioning of Schlesneria renders the genus Planctomyces paraphyletic. A re-analysis of published fatty-acid measurements also does not support the current arrangement of the two genera. A quantitative comparison of phylogenetic and phenotypic aspects indicates that the three Planctomyces species with type strains available in public culture collections should be placed in separate genera. Thus the genera Gimesia, Planctopirus and Rubinisphaera are proposed to accommodate P. maris, P. limnophilus and P. brasiliensis, respectively. Pronounced differences between the reported G + C content of Gemmata obscuriglobus, Singulisphaera acidiphila and Zavarzinella formosa and G + C content calculated from their genome sequences call for emendation of their species descriptions. In addition to other features, the range of G + C values reported for the genera within the Planctomycetaceae indicates that the descriptions of the family and the order should be emended.
    • First genome sequences of Achromobacter phages reveal new members of the N4 family

      Wittmann, Johannes; Dreiseikelmann, Brigitte; Rohde, Manfred; Meier-Kolthoff, Jan P; Bunk, Boyke; Rohde, Christine (2014-01-27)
      Abstract Background Multi-resistant Achromobacter xylosoxidans has been recognized as an emerging pathogen causing nosocomially acquired infections during the last years. Phages as natural opponents could be an alternative to fight such infections. Bacteriophages against this opportunistic pathogen were isolated in a recent study. This study shows a molecular analysis of two podoviruses and reveals first insights into the genomic structure of Achromobacter phages so far. Methods Growth curve experiments and adsorption kinetics were performed for both phages. Adsorption and propagation in cells were visualized by electron microscopy. Both phage genomes were sequenced with the PacBio RS II system based on single molecule, real-time (SMRT) technology and annotated with several bioinformatic tools. To further elucidate the evolutionary relationships between the phage genomes, a phylogenomic analysis was conducted using the genome Blast Distance Phylogeny approach (GBDP). Results In this study, we present the first detailed analysis of genome sequences of two Achromobacter phages so far. Phages JWAlpha and JWDelta were isolated from two different waste water treatment plants in Germany. Both phages belong to the Podoviridae and contain linear, double-stranded DNA with a length of 72329 bp and 73659 bp, respectively. 92 and 89 putative open reading frames were identified for JWAlpha and JWDelta, respectively, by bioinformatic analysis with several tools. The genomes have nearly the same organization and could be divided into different clusters for transcription, replication, host interaction, head and tail structure and lysis. Detailed annotation via protein comparisons with BLASTP revealed strong similarities to N4-like phages. Conclusions Analysis of the genomes of Achromobacter phages JWAlpha and JWDelta and comparisons of different gene clusters with other phages revealed that they might be strongly related to other N4-like phages, especially of the Escherichia group. Although all these phages show a highly conserved genomic structure and partially strong similarities at the amino acid level, some differences could be identified. Those differences, e.g. the existence of specific genes for replication or host interaction in some N4-like phages, seem to be interesting targets for further examination of function and specific mechanisms, which might enlighten the mechanism of phage establishment in the host cell after infection.
    • Genomic analysis reveals key aspects of prokaryotic symbiosis in the phototrophic consortium “Chlorochromatium aggregatum”

      Liu, Zhenfeng; Müller, Johannes; Li, Tao; Alvey, Richard M; Vogl, Kajetan; Frigaard, Niels-Ulrik; Rockwell, Nathan C; Boyd, Eric S; Tomsho, Lynn P; Schuster, Stephan C; et al. (2013-11-22)
      Abstract Background ‘Chlorochromatium aggregatum’ is a phototrophic consortium, a symbiosis that may represent the highest degree of mutual interdependence between two unrelated bacteria not associated with a eukaryotic host. ‘Chlorochromatium aggregatum’ is a motile, barrel-shaped aggregate formed from a single cell of ‘Candidatus Symbiobacter mobilis”, a polarly flagellated, non-pigmented, heterotrophic bacterium, which is surrounded by approximately 15 epibiont cells of Chlorobium chlorochromatii, a non-motile photolithoautotrophic green sulfur bacterium. Results We analyzed the complete genome sequences of both organisms to understand the basis for this symbiosis. Chl. chlorochromatii has acquired relatively few symbiosis-specific genes; most acquired genes are predicted to modify the cell wall or function in cell-cell adhesion. In striking contrast, ‘Ca. S. mobilis’ appears to have undergone massive gene loss, is probably no longer capable of independent growth, and thus may only reproduce when consortia divide. A detailed model for the energetic and metabolic bases of the dependency of ‘Ca. S. mobilis’ on Chl. chlorochromatii is described. Conclusions Genomic analyses suggest that three types of interactions lead to a highly sophisticated relationship between these two organisms. Firstly, extensive metabolic exchange, involving carbon, nitrogen, and sulfur sources as well as vitamins, occurs from the epibiont to the central bacterium. Secondly, ‘Ca. S. mobilis’ can sense and move towards light and sulfide, resources that only directly benefit the epibiont. Thirdly, electron cycling mechanisms, particularly those mediated by quinones and potentially involving shared protonmotive force, could provide an important basis for energy exchange in this and other symbiotic relationships.
    • 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.
    • 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.