• Additions to the genus Gimesia: description of Gimesia alba sp. nov., Gimesia algae sp. nov., Gimesia aquarii sp. nov., Gimesia aquatilis sp. nov., Gimesia fumaroli sp. nov. and Gimesia panareensis sp. nov., isolated from aquatic habitats of the Northern Hemisphere.

      Wiegand, Sandra; Jogler, Mareike; Boedeker, Christian; Heuer, Anja; Rast, Patrick; Peeters, Stijn H; Jetten, Mike S M; Kaster, Anne-Kristin; Rohde, Manfred; Kallscheuer, Nicolai; et al. (Springer, 2020-11-24)
      Thirteen novel planctomycetal strains were isolated from five different aquatic sampling locations. These comprise the hydrothermal vent system close to Panarea Island (Italy), a biofilm on the surface of kelp at Monterey Bay (CA, USA), sediment and algae on Mallorca Island (Spain) and Helgoland Island (Germany), as well as a seawater aquarium in Braunschweig, Germany. All strains were shown to belong to the genus Gimesia. Their genomes cover a size range from 7.22 to 8.29 Mb and have a G+C content between 45.1 and 53.7%. All strains are mesophilic (Topt 26-33 °C) with generation times between 12 and 32 h. Analysis of fatty acids yielded palmitic acid (16:0) and a fatty acid with the equivalent chain length of 15.817 as major compounds. While five of the novel strains belong to the already described species Gimesia maris and Gimesia chilikensis, the other strains belong to novel species, for which we propose the names Gimesia alba (type strain Pan241wT = DSM 100744T = LMG 31345T = CECT 9841T = VKM B-3430T), Gimesia algae (type strain Pan161T = CECT 30192T = STH00943T = LMG 29130T), Gimesia aquarii (type strain V144T = DSM 101710T = VKM B-3433T), Gimesia fumaroli (type strain Enr17T = DSM 100710T = VKM B-3429T) and Gimesia panareensis (type strain Enr10T = DSM 100416T = LMG 29082T). STH numbers refer to the Jena Microbial Resource Collection (JMRC).
    • Adenosine Triphosphate Neutralizes Pneumolysin-induced Neutrophil Activation.

      Cuypers, Fabian; Klabunde, Björn; Gesell Salazar, Manuela; Surabhi, Surabhi; Skorka, Sebastian B; Burchhardt, Gerhard; Michalik, Stephan; Thiele, Thomas; Rohde, Manfred; Völker, Uwe; et al. (Oxford University Press, 2020-05-23)
      Background: In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyper-inflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. Methods: Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. Results: Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. Conclusions: Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin induced neutrophil activation.
    • Analysis of bacterial communities in a municipal duck pond during a phytoplankton bloom and isolation of Anatilimnocola aggregata gen. nov., sp. nov., Lacipirellula limnantheis sp. nov. and Urbifossiella limnaea gen. nov., sp. nov. belonging to the phylum Planctomycetes.

      Kallscheuer, Nicolai; Rast, Patrick; Jogler, Mareike; Wiegand, Sandra; Kohn, Timo; Boedeker, Christian; Jeske, Olga; Heuer, Anja; Quast, Christian; Glöckner, Frank Oliver; et al. (Wiley & Sond Ltd., 2021-01-12)
      Waterbodies such as lakes and ponds are fragile environments affected by human influences. Suitable conditions can result in massive growth of phototrophs, commonly referred to as phytoplankton blooms. Such events benefit heterotrophic bacteria able to use compounds secreted by phototrophs or their biomass as major nutrient source. One example of such bacteria are Planctomycetes, which are abundant on the surfaces of marine macroscopic phototrophs; however, less data are available on their ecological roles in limnic environments. In this study, we followed a cultivation-independent deep sequencing approach to study the bacterial community composition during a cyanobacterial bloom event in a municipal duck pond. In addition to cyanobacteria, which caused the bloom event, members of the phylum Planctomycetes were significantly enriched in the cyanobacteria-attached fraction compared to the free-living fraction. Separate datasets based on isolated DNA and RNA point towards considerable differences in the abundance and activity of planctomycetal families, indicating different activity peaks of these families during the cyanobacterial bloom. Motivated by the finding that the sampling location harbours untapped bacterial diversity, we included a complementary cultivation-dependent approach and isolated and characterized three novel limnic strains belonging to the phylum Planctomycetes.
    • Aridibacter famidurans gen. nov., sp. nov. and Aridibacter kavangonensis sp. nov., two novel members of subdivision 4 of the Acidobacteria isolated from semiarid savannah soil.

      Huber, Katharina J; Wüst, Pia K; Rohde, Manfred; Overmann, Jörg; Foesel, Bärbel U; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2014-06)
      Acidobacteria constitute an abundant fraction of the soil microbial community and are currently divided into 26 subdivisions. Most cultivated members of the Acidobacteria are affiliated with subdivision 1, while only a few representatives of subdivisions 3, 4, 8, 10 and 23 have been isolated and described so far. Two novel isolates of subdivision 4 of the Acidobacteria were isolated from subtropical savannah soils and are characterized in the present work. Cells of strains A22_HD_4H(T) and Ac_23_E3(T) were immotile rods that divided by binary fission. Colonies were pink and white, respectively. The novel strains A22_HD_4H(T) and Ac_23_E3(T) were aerobic mesophiles with a broad range of tolerance towards pH (4.0-9.5 and 3.5-10.0, respectively) and temperature (15-44 and 12-47 °C, respectively). Both showed chemo-organoheterotrophic growth on some sugars, the amino sugar N-acetylgalactosamine, a few amino acids, organic acids and various complex protein substrates. Major fatty acids of A22_HD_4H(T) and Ac_23_E3(T) were iso-C(15 : 0), summed feature 1 (C(13 : 0) 3-OH/iso-C(15 : 1) H), summed feature 3 (C(16 : 1)ω7c/C(16 : 1)ω6c) and anteiso-C(17 : 0). The major quinone was MK-8; in addition, MK-7 occurred in small amounts. The DNA G+C contents of A22_HD_4H(T) and Ac_23_E3(T) were 53.2 and 52.6 mol%, respectively. The closest described relative was Blastocatella fastidiosa A2-16(T), with 16S rRNA gene sequence identity of 93.2 and 93.3%, respectively. Strains A22_HD_4H(T) and Ac_23_E3(T) displayed 16S rRNA gene sequence similarity of 97.4% to each other. On the basis of the low DNA-DNA hybridization value, the two isolates represent different species. Based on morphological, physiological and molecular characteristics, the new genus Aridibacter gen. nov. is proposed, with two novel species, the type species Aridibacter famidurans sp. nov. (type strain A22_HD_4H(T) = DSM 26555(T) = LMG 27985(T)) and a second species, Aridibacter kavangonensis sp. nov. (type strain Ac_23_E3(T) = DSM 26558(T) = LMG 27597(T)).
    • Aureliella helgolandensis gen. nov., sp. nov., a novel Planctomycete isolated from a jellyfish at the shore of the island Helgoland.

      Kallscheuer, Nicolai; Wiegand, Sandra; Boedeker, Christian; Peeters, Stijn H; Jogler, Mareike; Rast, Patrick; Heuer, Anja; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian; et al. (Springer, 2020-03-27)
      A novel planctomycetal strain, designated Q31aT, was isolated from a jellyfish at the shore of the island Helgoland in the North Sea. The strain forms lucid white colonies on solid medium and displays typical characteristics of planctomycetal strains, such as division by budding, formation of rosettes, presence of crateriform structures, extracellular matrix or fibre and a holdfast structure. Q31aT is mesophilic (temperature optimum 27 °C), neutrophilic (pH optimum 7.5), aerobic and heterotrophic. A maximal growth rate of 0.017 h- 1 (generation time of 41 h) was observed. Q31aT has a genome size of 8.44 Mb and a G + C content of 55.3%. Phylogenetically, the strain represents a novel genus and species in the recently introduced family Pirellulaceae, order Pirellulales, class Planctomycetia. We propose the name Aureliella helgolandensis gen. nov., sp. nov. for the novel species, represented by Q31aT (= DSM 103537T = LMG 29700T) as the type strain.
    • Azithromycin Synergizes with Cationic Antimicrobial Peptides to Exert Bactericidal and Therapeutic Activity Against Highly Multidrug-Resistant Gram-Negative Bacterial Pathogens

      Lin, Leo; Nonejuie, Poochit; Munguia, Jason; Hollands, Andrew; Olson, Joshua; Dam, Quang; Kumaraswamy, Monika; Rivera, Heriberto; Corriden, Ross; Rohde, Manfred; et al. (2015-06)
    • Bacterial microcompartment-directed polyphosphate kinase promotes stable polyphosphate accumulation in E. coli.

      Liang, Mingzhi; Frank, Stefanie; Lünsdorf, Heinrich; Warren, Martin J; Prentice, Michael B; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (Wiley-Blackwell, 2017-03-01)
      Processes for the biological removal of phosphate from wastewater rely on temporary manipulation of bacterial polyphosphate levels by phased environmental stimuli. In E. coli polyphosphate levels are controlled via the polyphosphate-synthesizing enzyme polyphosphate kinase (PPK1) and exopolyphosphatases (PPX and GPPA), and are temporarily enhanced by PPK1 overexpression and reduced by PPX overexpression. We hypothesised that partitioning PPK1 from cytoplasmic exopolyphosphatases would increase and stabilise E. coli polyphosphate levels. Partitioning was achieved by co-expression of E. coli PPK1 fused with a microcompartment-targeting sequence and an artificial operon of Citrobacter freundii bacterial microcompartment genes. Encapsulation of targeted PPK1 resulted in persistent phosphate uptake and stably increased cellular polyphosphate levels throughout cell growth and into the stationary phase, while PPK1 overexpression alone produced temporary polyphosphate increase and phosphate uptake. Targeted PPK1 increased polyphosphate in microcompartments 8-fold compared with non-targeted PPK1. Co-expression of PPX polyphosphatase with targeted PPK1 had little effect on elevated cellular polyphosphate levels because microcompartments retained polyphosphate. Co-expression of PPX with non-targeted PPK1 reduced cellular polyphosphate levels. Thus, subcellular compartmentalisation of a polymerising enzyme sequesters metabolic products from competing catabolism by preventing catabolic enzyme access. Specific application of this process to polyphosphate is of potential application for biological phosphate removal.
    • Batrachochytrium salamandrivorans in the ruhr district, germany: History, distribution, decline dynamics and disease symptoms of the salamander plague

      Schulz, Vanessa; Schulz, Alina; Klamke, Marine; Preissler, Kathleen; Sabino-Pinto, Joana; Müsken, Mathias; Schlüpmann, Martin; Heldt, Lorenz; Kamprad, Felix; Enss, Julian; et al. (Deutsche Gesellschaft für Herpetologie und Terrarienkunde e.V. (DGHT), 2020-08-15)
      he chytrid fungus Batrachochytrium salamandrivorans (Bsal), recently introduced from Asia to Europe, causes mortality in numerous species of salamanders and newts and has led to catastrophic declines and local extinctions of the European fire salamander (Salamandra salamandra) in the Netherlands, Belgium, and Germany. Due to the continuous spread of the pathogen, Germany can be considered as the current ‘hotspot’ of Bsal-driven salamander declines. The pathogen was detected in 2015 in the Eifel Mountains where it probably has been present at least since 2004. Moreover, Bsal was found in 2017 in the Ruhr District where it also might occur since 2004. The Ruhr District is a heavily urbanized and industrialized region in western Germany, which offers an unprecedented opportunity to monitor range expansion and infection dynamics of Bsal in an area affected by intense human activities. We here review the current knowledge on Bsal in the Ruhr District where the pathogen by now has been recorded based on qPCR data from 18 sites distributed over eight cities. Transect counts (adult salamanders) and larval removal-sampling at two sites where Bsal was recorded in 2017 and 2018, confirm that fire salamander populations at the affected sites have declined dramatically. However, single negative-tested individuals were still observed and reproduction could be ascertained. Moreover, we successfully detected Bsal by analysing environmental DNA (eDNA) from samples obtained from a standing water body as well as a stream. Detailed monitoring of a site in Essen (Kruppwald) from January to May 2019 provided data on infection and disease dynamics during an acute Bsal-outbreak in a population of European fire salamanders. After initial observation of single dead infected salamanders in January and February 2019, the maximum Bsal loads in the population ranged from 7.90E+03 ITS copies in early March to 2.29E+09 ITS copies at the end of March. Prevalence of infection ranged from 4% to 50% and significantly increased over time; prevalence of externally visible disease symptoms peaked on May 2 and May 8. Single dead salamanders were encountered throughout the monitoring period. Recaptures of two infected salamanders indicated an increase of Bsal load by about one order of magnitude within one week. Infected salamanders showed small-sized regular round ulcerations usually of 0.25–1 mm but sometimes up to 2.5 mm in diameter, which gave the impression of outward growth from the centre of each ulceration. Among salamander individuals monitored in the Kruppwald, such ulcerations were only found in infected salamanders, but we found no significant correlation between the intensity of the ulcerations and Bsal load. Heat treatment proved effective to cure even deep ulcerations when salamanders were kept for 10 days at 25–27°C or 14 days at 25°C, but infection persisted and ulcerations reappeared six weeks after the end of the treatment; only heat treatment at 25°C for 21 days proved effective to reliably clear the infection in three tested salamanders. Key words. Amphibia, Caudata, Salamandra salamandra, European fire salamander, Bsal, chytridiomycosis, heat treatment, emerging infectious disease, amphibian disease, eDNA.
    • Belliella kenyensis sp. nov., isolated from an alkaline lake.

      Akhwale, Juliah Khayeli; Göker, Markus; Rohde, Manfred; Schumann, Peter; Klenk, Hans-Peter; Boga, Hamadi Iddi; Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 7 38124 Braunschweig, Germany. (2015-02)
      A red-pigmented, Gram-reaction-negative, aerobic bacterial strain, designated No.164(T), was isolated from sediment sample from the alkaline Lake Elmenteita located in the Kenyan Rift Valley. Results of 16S rRNA gene sequence analysis indicated that the isolate represented a member of the genus Belliella, with the highest sequence similarity (97 %) to Belliella pelovolcani DSM 46698(T). Optimal growth temperature was 30-35 °C, at pH 7.0-12.0 in the presence of 0-4 % (w/v) NaCl. Flexirubins were absent. The respiratory menaquinone (MK-7), predominant cellular fatty acids (iso-C15 : 0, anteiso-C15 : 0 and a mixture of C16 : 1ω7c and/or iso-C15 : 0 2-OH) and DNA G+C content (38.1 mol%) of strain No.164(T) were consistent with those of other members of the genus Belliella. The polar lipids consisted of phosphatidylethanolamine, eight unspecified lipids and one unspecified phospholipid. Several phenotypic characteristics can be used to differentiate this isolate from those of other species of the genus Belliella. The results of polyphasic analyses presented in this study indicated that this isolate should be classified as representing a novel species of the genus Belliella. The name Belliella kenyensis sp. nov. is therefore proposed; the type strain is strain No.164(T) ( = DSM 46651(T) = CECT 8551(T)).
    • Biology of archaea from a novel family Cuniculiplasmataceae (Thermoplasmata) ubiquitous in hyperacidic environments.

      Golyshina, Olga V; Kublanov, Ilya V; Tran, Hai; Korzhenkov, Alexei A; Lünsdorf, Heinrich; Nechitaylo, Taras Y; Gavrilov, Sergey N; Toshchakov, Stepan V; Golyshin, Peter N; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-12-14)
      The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed 'G-plasma'. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1-1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with 'G-plasma'. Organisms had largest genomes among Thermoplasmatales (1.87-1.94 Mbp), that shared 98.7-98.8% average nucleotide identities between themselves and 'G-plasma' and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions.
    • Bordetella bronchiseptica promotes adherence, colonization, and cytotoxicity of Streptococcus suis in a porcine precision-cut lung slice model.

      Vötsch, Désirée; Willenborg, Maren; Baumgärtner, Wolfgang; Rohde, Manfred; Valentin-Weigand, Peter; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Taylor & Francis, 2020-12-29)
      Bordetella (B.) bronchiseptica and Streptococcus (S.) suis are major pathogens in pigs, which are frequently isolated from co-infections in the respiratory tract and contribute to the porcine respiratory disease complex (PRDC). Despite the high impact of co-infections on respiratory diseases of swine (and other hosts), very little is known about pathogen-pathogen-host interactions and the mechanisms of pathogenesis. In the present study, we established a porcine precision-cut lung slice (PCLS) model to analyze the effects of B. bronchiseptica infection on adherence, colonization, and cytotoxic effects of S. suis. We hypothesized that induction of ciliostasis by a clinical isolate of B. bronchiseptica may promote subsequent infection with a virulent S. suis serotype 2 strain. To investigate this theory, we monitored the ciliary activity by light microscopy, measured the release of lactate dehydrogenase, and calculated the number of PCLS-associated bacteria. To study the role of the pore-forming toxin suilysin (SLY) in S. suis-induced cytotoxicity, we included a SLY-negative isogenic mutant and the complemented mutant strain. Furthermore, we analyzed infected PCLS by histopathology, immunofluorescence microscopy, and field emission scanning electron microscopy. Our results showed that pre-infection with B. bronchiseptica promoted adherence, colonization, and, as a consequence of the increased colonization, the cytotoxic effects of S. suis, probably by reduction of the ciliary activity. Moreover, cytotoxicity induced by S. suis is strictly dependent on the presence of SLY. Though the underlying molecular mechanisms remain to be fully clarified, our results clearly support the hypothesis that B. bronchiseptica paves the way for S. suis infection.
    • Capsule and fimbriae modulate the invasion of Haemophilus influenzae in a human blood-cerebrospinal fluid barrier model.

      Häuser, Svenja; Wegele, Christian; Stump-Guthier, Carolin; Borkowski, Julia; Weiss, Christel; Rohde, Manfred; Ishikawa, Hiroshi; Schroten, Horst; Schwerk, Christian; Adam, Rüdiger; et al. (Elsevier, 2018-07-17)
      The Gram-negative bacterium Haemophilus influenzae (H. influenzae) can commensally colonize the upper respiratory tract, but also cause life threatening disease including epiglottitis, sepsis and meningitis. The H. influenzae capsule protects the bacteria against both phagocytosis and opsonization. Encapsulated H. influenzae strains are classified into serotypes ranging from a to f dependent on their distinct polysaccharide capsule. Due to the implementation of vaccination the incidence of invasive H. influenzae type b (Hib) infections has strongly decreased and infections with other capsulated types, including H. influenzae type f (Hif), are emerging. The pathogenesis of H. influenzae meningitis is not clarified. To enter the central nervous system (CNS) the bacteria generally have to cross either the blood-brain barrier (BBB) or the blood-cerebrospinal fluid barrier (BSCFB). Using a cell culture model of the BCSFB based on human choroid plexus papilloma (HIBCPP) cells and different H. influenzae strains we investigated whether Hib and Hif invade the cells, and if invasion differs between encapsulated vs. capsular-deficient and fimbriated vs. non-fimbriated variants. We find that Hib can adhere to and invade into HIBCPP cells. Invasion occurs in a strongly polar fashion, since the bacteria enter the cells preferentially from the basolateral "blood "side. Fimbriae and capsule attenuate invasion into choroid plexus (CP) epithelial cells, and capsulation can influence the bacterial distribution pattern. Finally, analysis of clinical Hib and Hif isolates confirms the detected invasive properties of H. influenzae. Our data point to roles of capsule and fimbriae during invasion of CP epithelial cells.
    • Caulifigura coniformis gen. nov., sp. nov., a novel member of the family Planctomycetaceae isolated from a red biofilm sampled in a hydrothermal area.

      Kallscheuer, Nicolai; Wiegand, Sandra; Boedeker, Christian; Peeters, Stijn H; Jogler, Mareike; Heuer, Anja; Jetten, Mike S M; Rohde, Manfred; Jogler, Christian; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-06-24)
      Pan44T, a novel strain belonging to the phylum Planctomycetes, was isolated from a red biofilm in a hydrothermal area close to the island Panarea in the Tyrrhenian Sea north of Sicily, Italy. The strain forms white colonies on solid medium and displays the following characteristics: cell division by budding, formation of rosettes, presence of matrix or fimbriae and long stalks. The cell surface has an interesting and characteristic texture made up of triangles and rectangles, which leads to a pine cone-like morphology of the strain. Strain Pan44T is mesophilic (temperature optimum 26 °C), slightly alkaliphilic (pH optimum 8.0), aerobic and heterotrophic. The strain has a genome size of 6.76 Mb with a G + C content of 63.2%. Phylogenetically, the strain is a member of the family Planctomycetaceae, order Planctomycetales, class Planctomycetia. Our analysis supports delineation of strain Pan44T from all known genera in this family, hence, we propose to assign it to a novel species within a novel genus, for which we propose the name Caulifigura coniformis gen. nov., sp. nov., represented by Pan44T (DSM 29405T = LMG 29788T) as the type strain.
    • Cell sheet technology: Influence of culture conditions on in vitro-cultivated corneal stromal tissue for regenerative therapies of the ocular surface.

      Hasenzahl, Meike; Müsken, Mathias; Mertsch, Sonja; Schrader, Stefan; Reichl, Stephan; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley, 2021-02-03)
      The in vitro reconstruction of stromal tissue by long-term cultivation of corneal fibroblasts is a smart approach for regenerative therapies of ocular surface diseases. However, systematic investigations evaluating optimized cultivation protocols for the realization of a biomaterial are lacking. This study investigated the influence of supplements to the culture media of human corneal fibroblasts on the formation of a cell sheet consisting of cells and extracellular matrix. Among the supplements studied are vitamin C, fetal bovine serum, L-glutamine, components of collagen such as L-proline, L-4-hydroxyproline and glycine, and TGF-β1, bFGF, IGF-2, PDGF-BB and insulin. After long-term cultivation, the proliferation, collagen and glycosaminoglycan content and light transmission of the cell sheets were examined. Biomechanical properties were investigated by tensile tests and the ultrastructure was characterized by electron microscopy, small-angle X-ray scattering, antibody staining and ELISA. The synthesis of extracellular matrix was significantly increased by cultivation with insulin or TGF-β1, each with vitamin C. The sheets exhibited a high transparency and suitable material properties. The production of a transparent, scaffold-free, potentially autologous, in vitro-generated construct by culturing fibroblasts with extracellular matrix synthesis-stimulating supplements represents a promising approach for a biomaterial that can be used for ocular surface reconstruction in slowly progressing diseases.
    • Characterization of Five Zoonotic Streptococcus suis Strains from Germany, Including One Isolate from a Recent Fatal Case of Streptococcal Toxic Shock-Like Syndrome in a Hunter.

      Eisenberg, Tobias; Hudemann, Christoph; Hossain, Hamid M; Hewer, Angela; Tello, Khodr; Bandorski, Dirk; Rohde, M; Valentin-Weigand, Peter; Baums, Christoph Georg; Infectious Diseases, College of Veterinary Medicine, University Leipzig, Leipzig. (2015-12)
      A Streptococcus suis isolate from a German hunter with streptococcal toxic shock-like syndrome (STSLS) and four additional zoonotic isolates were genotyped as mrp(+) epf* (variant 1890) sly(+) cps2(+). All five zoonotic German strains were characterized by high multiplication in human blood samples ex vivo, but induction of only low levels of proinflammatory cytokines compared to a Chinese STSLS strain.
    • Characterization of the first cultured representative of Verrucomicrobia subdivision 5 indicates the proposal of a novel phylum.

      Spring, Stefan; Bunk, Boyke; Spröer, Cathrin; Schumann, Peter; Rohde, M; Tindall, Brian J; Klenk, Hans-Peter; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016)
      The recently isolated strain L21-Fru-ABTrepresents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-ABTrepresents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-ABTand related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.
    • Comparative genomic analysis of eight novel haloalkaliphilic bacteriophages from Lake Elmenteita, Kenya.

      Akhwale, Juliah Khayeli; Rohde, M; Rohde, Christine; Bunk, Boyke; Spröer, Cathrin; Klenk, Hans-Peter; Boga, Hamadi Iddi; Wittmann, Johannes; HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig Germany. (PLOS, 2019-01-01)
      We report complete genome sequences of eight bacteriophages isolated from Haloalkaline Lake Elmenteita found on the floor of Kenyan Rift Valley. The bacteriophages were sequenced, annotated and a comparative genomic analysis using various Bioinformatics tools carried out to determine relatedness of the bacteriophages to each other, and to those in public databases. Basic genome properties like genome size, percentage coding density, number of open reading frames, percentage GC content and gene organizations revealed the bacteriophages had no relationship to each other. Comparison to other nucleotide sequences in GenBank database showed no significant similarities hence novel. At the amino acid level, phages of our study revealed mosaicism to genes with conserved domains to already described phages. Phylogenetic analyses of large terminase gene responsible for DNA packaging and DNA polymerase gene for replication further showed diversity among the bacteriophages. Our results give insight into diversity of bacteriophages in Lake Elmenteita and provide information on their evolution. By providing primary sequence information, this study not only provides novel sequences for biotechnological exploitation, but also sets stage for future studies aimed at better understanding of virus diversity and genomes from haloalkaline lakes in the Rift Valley.
    • Comparing polysaccharide decomposition between the type strains Gramella echinicola KMM 6050(T) (DSM 19838(T)) and Gramella portivictoriae UST040801-001(T) (DSM 23547(T)), and emended description of Gramella echinicola Nedashkovskaya et al. 2005 emend. Shahina et al. 2014 and Gramella portivictoriae Lau et al. 2005.

      Panschin, Irina; Huang, Sixing; Meier-Kolthoff, Jan P; Tindall, Brian J; Rohde, Manfred; Verbarg, Susanne; Lapidus, Alla; Han, James; Trong, Stephan; Haynes, Matthew; et al. (2016)
      Strains of the genus Gramella (family Flavobacteriacae, phylum Bacteroidetes) were isolated from marine habitats such as tidal flat sediments, coastal surface seawater and sea urchins. Flavobacteriaceae have been shown to be involved in the decomposition of plant and algal polysaccharides. However, the potential to decompose polysaccharides may differ tremendously even between species of the same genus. Gramella echinicola KMM 6050(T) (DSM 19838(T)) and Gramella portivictoriae UST040801-001(T) (DSM 23547(T)) have genomes of similar lengths, similar numbers of protein coding genes and RNA genes. Both genomes encode for a greater number of peptidases compared to 'G. forsetii'. In contrast to the genome of 'G. forsetii', both genomes comprised a smaller set of CAZymes. Seven polysaccharide utilization loci were identified in the genomes of DSM 19838(T) and DSM 23547(T). Both Gramella strains hydrolyzed starch, galactomannan, arabinoxylan and hydroxyethyl-cellulose, but not pectin, chitosan and cellulose (Avicel). Galactan and xylan were hydrolyzed by strain DSM 19838(T), whereas strain DSM 23547(T) hydrolyzed pachyman and carboxy-methyl cellulose. Conclusively, both Gramella type strains exhibit characteristic physiological, morphological and genomic differences that might be linked to their habitat. Furthermore, the identified enzymes mediating polysaccharide decomposition, are of biotechnological interest.
    • Comparison of mcl-Poly(3-hydroxyalkanoates) synthesis by different Pseudomonas putida strains from crude glycerol: citrate accumulates at high titer under PHA-producing conditions.

      Poblete-Castro, Ignacio; Binger, Danielle; Oehlert, Rene; Rohde, Manfred; Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014)
      Achieving a sustainable society requires, among other things, the use of renewable feedstocks to replace chemicals obtained from petroleum-derived compounds. Crude glycerol synthesized inexpensively as a byproduct of biodiesel production is currently considered a waste product, which can potentially be converted into value-added compounds by bacterial fermentation. This study aimed at evaluating several characterized P. putida strains to produce medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHA) using raw glycerol as the only carbon/energy source.
    • Complete genome sequence and description of Salinispira pacifica gen. nov., sp. nov., a novel spirochaete isolated form a hypersaline microbial mat.

      Ben Hania, Wajdi; Joseph, Manon; Schumann, Peter; Bunk, Boyke; Fiebig, Anne; Spröer, Cathrin; Klenk, Hans-Peter; Fardeau, Marie-Laure; Spring, Stefan; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2015)
      During a study of the anaerobic microbial community of a lithifying hypersaline microbial mat of Lake 21 on the Kiritimati atoll (Kiribati Republic, Central Pacific) strain L21-RPul-D2(T) was isolated. The closest phylogenetic neighbor was Spirochaeta africana Z-7692(T) that shared a 16S rRNA gene sequence identity value of 90% with the novel strain and thus was only distantly related. A comprehensive polyphasic study including determination of the complete genome sequence was initiated to characterize the novel isolate. Cells of strain L21-RPul-D2(T) had a size of 0.2 - 0.25 × 8-9 μm, were helical, motile, stained Gram-negative and produced an orange carotenoid-like pigment. Optimal conditions for growth were 35°C, a salinity of 50 g/l NaCl and a pH around 7.0. Preferred substrates for growth were carbohydrates and a few carboxylic acids. The novel strain had an obligate fermentative metabolism and produced ethanol, acetate, lactate, hydrogen and carbon dioxide during growth on glucose. Strain L21-RPul-D2(T) was aerotolerant, but oxygen did not stimulate growth. Major cellular fatty acids were C14:0, iso-C15:0, C16:0 and C18:0. The major polar lipids were an unidentified aminolipid, phosphatidylglycerol, an unidentified phospholipid and two unidentified glycolipids. Whole-cell hydrolysates contained L-ornithine as diagnostic diamino acid of the cell wall peptidoglycan. The complete genome sequence was determined and annotated. The genome comprised one circular chromosome with a size of 3.78 Mbp that contained 3450 protein-coding genes and 50 RNA genes, including 2 operons of ribosomal RNA genes. The DNA G + C content was determined from the genome sequence as 51.9 mol%. There were no predicted genes encoding cytochromes or enzymes responsible for the biosynthesis of respiratory lipoquinones. Based on significant differences to the uncultured type species of the genus Spirochaeta, S. plicatilis, as well as to any other phylogenetically related cultured species it is suggested to place strain L21-RPul-D2(T) (=DSM 27196(T) = JCM 18663(T)) in a novel species and genus, for which the name Salinispira pacifica gen. nov., sp. nov. is proposed.