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Greifswald, Germany

Kabisch J.,University of Greifswald | Thurmer A.,University of Gottingen | Hubel T.,Miltenyi Biotec GmbH | Popper L.,SternEnzym GmbH and Co. KG | And 3 more authors.
Journal of Biotechnology | Year: 2013

The genome sequence of Bacillus subtilis ATCC 6051 and its suitability as an expression host for recombinant protein production was determined. The comparison of this undomesticated wild type with the widely used laboratory strain B. subtilis 168 reveals a high degree of congruency between the two strains. Differences could only be detected on the level of point mutations or small insertions. B. subtilis ATCC 6051 shows none of the auxotrophies known for B. subtilis 168 and is able to produce polyketides. It exhibits better use of complex media and higher genomic stability through reduced natural competence. Consequently, B. subtilis ATCC 6051 was genetically modified to yield an optimized strain for the production of heterologously expressed proteins under control of an acetoin-inducible promoter. © 2012 Elsevier B.V.

Kabisch A.,University of Greifswald | Otto A.,University of Greifswald | Konig S.,University of Greifswald | Konig S.,University Pierre and Marie Curie | And 7 more authors.
ISME Journal | Year: 2014

Members of the phylum Bacteroidetes are abundant in many marine ecosystems and are known to have a pivotal role in the mineralization of complex organic substrates such as polysaccharides and proteins. We studied the decomposition of the algal glycans laminarin and alginate by 'Gramella forsetii' KT0803, a bacteroidetal isolate from North Sea surface waters. A combined application of isotope labeling, subcellular protein fractionation and quantitative proteomics revealed two large polysaccharide utilization loci (PULs) that were specifically induced, one by alginate and the other by laminarin. These regulons comprised genes of surface-exposed proteins such as oligomer transporters, substrate-binding proteins, carbohydrate-active enzymes and hypothetical proteins. Besides, several glycan-specific TonB-dependent receptors and SusD-like substrate-binding proteins were expressed also in the absence of polysaccharide substrates, suggesting an anticipatory sensing function. Genes for the utilization of the beta-1,3-glucan laminarin were found to be co-regulated with genes for glucose and alpha-1,4-glucan utilization, which was not the case for the non-glucan alginate. Strong syntenies of the PULs of 'G. forsetii' with similar loci in other Bacteroidetes indicate that the specific response mechanisms of 'G. forsetii' to changes in polysaccharide availability likely apply to other Bacteroidetes. Our results can thus contribute to an improved understanding of the ecological niches of marine Bacteroidetes and their roles in the polysaccharide decomposition part of carbon cycling in marine ecosystems. © 2014 International Society for Microbial Ecology All rights reserved.

Welsch N.,University of Greifswald | Homuth G.,University of Greifswald | Schweder T.,University of Greifswald | Schweder T.,Institute of Marine Biotechnology
Applied Microbiology and Biotechnology | Year: 2015

In order to improve the overproduction of “difficult to express” proteins, a low-temperature expression system for Bacillus subtilis based on the cold-inducible promoter of the desaturase-encoding des gene was constructed. Selected regulatory DNA sequence elements from B. subtilis genes known to be cold-inducible were fused to different model genes. It could be demonstrated that these regulatory elements are able to mediate increased heterologous gene expression, either by improved translation efficiency or by higher messenger RNA (mRNA) stability. In case of a cold-adapted β-galactosidase from Pseudoalteromonas haloplanktis TAE79A serving as the model, significantly higher expression was achieved by fusing its coding sequence to the so-called “downstream box” sequence of cspB encoding the major B. subtilis cold-shock protein. The combination of this fusion with a cspB 5′-UTR stem-loop structure resulted in further enhancement of the β-galactosidase expression. In addition, integration of the transcription terminator of the B. subtilis cold-inducible bkd operon downstream of the target genes caused a higher mRNA stability and enabled thus a further significant increase in expression. Finally, the fully optimized expression system was validated by overproducing a B. subtilis xylanase as well as an α-glucosidase from Saccharomyces cerevisiae, the latter known for tending to form inclusion bodies. These analyses verified the applicability of the engineered expression system for extracellular and intracellular protein synthesis in B. subtilis, thereby confirming the suitability of this host organism for the overproduction of critical, poorly soluble proteins. © 2015, Springer-Verlag Berlin Heidelberg.

Welsch N.,University of Greifswald | Homuth G.,University of Greifswald | Schweder T.,University of Greifswald | Schweder T.,Institute of Marine Biotechnology
Applied Microbiology and Biotechnology | Year: 2012

The suitability of three β-galactosidases as reporter enzymes for promoter expression analyses was investigated in Bacillus subtilis with respect to various temperature conditions during cultivation and assay procedures. Starting from the hypothesis that proteins derived from diverse habitats have different advantages as reporters at different growth temperatures, the beta-galactosidases from the thermophilic organism Bacillus stearothermophilus, from the mesophilic bacterium Escherichia coli and from the psychrophilic organism Pseudoalteromonas haloplanktis TAE79 were analysed under control of the constitutive B. subtilis lepA promoter. Subsequent expression of the β-galactosidase genes and determination of specific activities was performed at different cultivation and assay temperatures using B. subtilis as host. Surprisingly, the obtained results demonstrated that the highest activities over a broad cultivation temperature range were obtained using the β-galactosidase from the mesophilic bacterium E. coli whereas the enzymes from the thermophilic and psychrophilic bacteria revealed a more restricted usability in terms of cultivation temperature. © 2011 Springer-Verlag.

Finke B.,Leibniz Institute for Plasma Science and Technology | Polak M.,Leibniz Institute for Plasma Science and Technology | Hempel F.,Leibniz Institute for Plasma Science and Technology | Rebl H.,University of Rostock | And 8 more authors.
Advanced Engineering Materials | Year: 2012

The application of antimicrobial surfaces to titanium alloy (Ti) implants would be beneficial to prevent implant-associated infections of joint endoprostheses and osteosyntheses. Copper (Cu) could be advantageously applied for this purpose, since it exhibits a well-known antimicrobial activity and is a trace element in the human body, i.e., it is non-toxic in small concentrations. This approach was evaluated with two plasma-based surface modification procedures: Implantation of Cu ions into Ti by means of plasma immersion ion implantation (PIII) and Coating of Ti surfaces with Cu-Ti films by means of dual high power impulse magnetron sputtering (dual HiPIMS). In this manner, the surfaces could be equipped with various amounts of Cu, as it was analyzed by X-ray photoelectron spectroscopy (XPS). The surfaces released up to 8 mmol · L -1 of Cu within 24 h, measured with atomic absorption spectroscopy (AAS). Hence, the surfaces possessed an antimicrobial potential against typical infect-associated bacteria (Staphylococcus aureus). Surfaces with a higher Cu release prepared by HiPIMS technique revealed a higher antimicrobial effect, while surfaces implanted by PIII were less cytotoxic to osteoblasts (MG-63 cells). These results show that Cu doped and coated implants could be useful for prevention and therapy of implant-associated infections. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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