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Lasch P.,Robert Koch Institute | Jacob D.,Robert Koch Institute | Grunow R.,Robert Koch Institute | Schwecke T.,Robert Koch Institute | And 2 more authors.
TrAC - Trends in Analytical Chemistry | Year: 2016

MALDI-TOF MS is a relatively new technology which has revolutionized the way microorganisms are identified. The technique provides specific biomarker profiles which can be employed for rapid, accurate and cost-effective microbial identification.The focus of this mini-review is on the current status and new advances in the application of MALDI-TOF MS for identification of highly pathogenic bacteria (HPB). Data from selected studies with HPB of the genera Bacillus, Burkholderia, Brucella, Francisella and Yersinia are reviewed and molecular assignments of HPB-specific high abundance biomarkers are presented. Furthermore, experiences from an European inter-laboratory ring trial on HPB are outlined. © 2016 Elsevier B.V.


Tillich U.M.,Wildau University of Applied Sciences | Tillich U.M.,Humboldt University of Berlin | Wolter N.,Wildau University of Applied Sciences | Franke P.,Wildau University of Applied Sciences | And 2 more authors.
BMC Biotechnology | Year: 2014

Background: Temperature tolerance is an important aspect for commercial scale outdoor cultivation of microalgae and cyanobacteria. While various genes are known to be related to Synechocystis sp. PCC6803's heat shock response, there is very limited published data concerning the specific genes involved in long term thermal tolerance. We have previously used random mutagenesis and adaptive evolution to generate a mixture of strains of Synechocystis sp. PCC6803 with significantly increased thermal tolerance. The genetic modifications leading to the phenotypes of the newly generated strains are the focus of this work.Results: We used a custom screening platform, based on 96-deepwell microplate culturing in an in house designed cultivation chamber integrated in a liquid handling robot for screening and selection; in addition we also used a more conventional system. The increased thermal tolerances of the isolated monoclonal strains were validated in larger bioreactors and their whole genomes sequenced. Comparison of the sequence information to the parental wild type identified various mutations responsible for the enhanced phenotypes. Among the affected genes identified are clpC, pnp, pyk2, sigF, nlpD, pyrR, pilJ and cya1.Conclusions: The applied methods (random mutagenesis, in vivo selection, screening, validation, whole genome sequencing) were successfully applied to identify various mutations, some of which are very unlikely to have been identified by other approaches. Several of the identified mutations are found in various strains and (due to their distribution) are likely to have occurred independently. This, coupled with the relatively low number of affected genes underscores the significance of these specific mutations to convey thermal tolerance in Synechocystis. © 2014 Tillich et al.; licensee BioMed Central Ltd.


Schulze K.,Wildau University of Applied Sciences | Lang I.,Algenol Biofuels Germany GmbH | Enke H.,Algenol Biofuels Germany GmbH | Grohme D.,Wildau University of Applied Sciences | Frohme M.,Wildau University of Applied Sciences
BMC Research Notes | Year: 2015

Abstract Background: Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype. A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein. Methods: Based on these findings we developed a method for fluorescence microscopy in order to distinguish producing and non-producing cells with respect to their pigmentation phenotype. By applying a specific filter set the emitted fluorescence of a producer cell with a reduced PC content appeared orange. The emitted fluorescence of a non-producing cell with a wt pigmentation phenotype was detected in red, and dead cells in green. In an automated process multiple images of each sample were taken and analyzed with a plugin for the image analysis software ImageJ to identify dead (green), non-producing (red) and producing (orange) cells. Results: The results of the presented validation experiments revealed a good identification with 98 % red cells in the wt sample and 90 % orange cells in the producer sample. The detected wt pigmentation phenotype (red cells) in the producer sample were either not fully induced yet (in 48 h induced cultures) or already reverted to a non-producing cells (in long-term photobioreactor cultivations), emphasizing the sensitivity and resolution of the method. Conclusions: The fluorescence microscopy method displays a useful technique for a rapid detection of non-producing single cells in an ethanol producing cell population. © 2015 Schulze et al.; licensee BioMed Central.


Dienst D.,Algenol Biofuels Germany GmbH | Dienst D.,Humboldt University of Berlin | Georg J.,Albert Ludwigs University of Freiburg | Abts T.,Algenol Biofuels Germany GmbH | And 8 more authors.
Biotechnology for Biofuels | Year: 2014

Background: The production of biofuels in photosynthetic microalgae and cyanobacteria is a promising alternative to the generation of fuels from fossil resources. To be economically competitive, producer strains need to be established that synthesize the targeted product at high yield and over a long time. Engineering cyanobacteria into forced fuel producers should considerably interfere with overall cell homeostasis, which in turn might counteract productivity and sustainability of the process. Therefore, in-depth characterization of the cellular response upon long-term production is of high interest for the targeted improvement of a desired strain. Results: The transcriptome-wide response to continuous ethanol production was examined in Synechocystis sp. PCC6803 using high resolution microarrays. In two independent experiments, ethanol production rates of 0.0338% (v/v) ethanol d§ssup§-1§esup§ and 0.0303% (v/v) ethanol d§ssup§-1§esup§ were obtained over 18 consecutive days, measuring two sets of biological triplicates in fully automated photobioreactors. Ethanol production caused a significant (∼40%) delay in biomass accumulation, the development of a bleaching phenotype and a down-regulation of light harvesting capacity. However, microarray analyses performed at day 4, 7, 11 and 18 of the experiment revealed only three mRNAs with a strongly modified accumulation level throughout the course of the experiment. In addition to the overexpressed adhA (slr1192) gene, this was an approximately 4 fold reduction in cpcB (sll1577) and 3 to 6 fold increase in rps8 (sll1809) mRNA levels. Much weaker modifications of expression level or modifications restricted to day 18 of the experiment were observed for genes involved in carbon assimilation (Ribulose bisphosphate carboxylase and Glutamate decarboxylase). Molecular analysis of the reduced cpcB levels revealed a post-transcriptional processing of the cpcBA operon mRNA leaving a truncated mRNA cpcA* likely not competent for translation. Moreover, western blots and zinc-enhanced bilin fluorescence blots confirmed a severe reduction in the amounts of both phycocyanin subunits, explaining the cause of the bleaching phenotype. Conclusions: Changes in gene expression upon induction of long-term ethanol production in Synechocystis sp. PCC6803 are highly specific. In particular, we did not observe a comprehensive stress response as might have been expected. © 2014 Dienst et al.; licensee BioMed Central Ltd.


Reddy G.K.,Bielefeld University | Lindner S.N.,Bielefeld University | Lindner S.N.,Algenol Biofuels Germany GmbH | Wendisch V.F.,Bielefeld University
Applied and Environmental Microbiology | Year: 2015

Corynebacterium glutamicum uses the Embden-Meyerhof-Parnas pathway of glycolysis and gains 2 mol of ATP per mol of glucose by substrate-level phosphorylation (SLP). To engineer glycolysis without net ATP formation by SLP, endogenous phosphorylating NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was replaced by nonphosphorylating NADPdependent glyceraldehyde-3-phosphate dehydrogenase (GapN) from Clostridium acetobutylicum, which irreversibly converts glyceraldehyde-3-phosphate (GAP) to 3-phosphoglycerate (3-PG) without generating ATP. As shown recently (S. Takeno, R. Murata, R. Kobayashi, S. Mitsuhashi, and M. Ikeda, Appl Environ Microbiol 76:7154-7160, 2010, http://dx.doi.org/10.1128/AEM.01464-10), this ATP-neutral, NADPH-generating glycolytic pathway did not allow for the growth of Corynebacterium glutamicum with glucose as the sole carbon source unless hitherto unknown suppressor mutations occurred; however, these mutations were not disclosed. In the present study, a suppressor mutation was identified, and it was shown that heterologous expression of udhA encoding soluble transhydrogenase from Escherichia coli partly restored growth, suggesting that growth was inhibited by NADPH accumulation. Moreover, genome sequence analysis of second-site suppressor mutants that were able to grow faster with glucose revealed a single point mutation in the gene of non-proton-pumping NADH:ubiquinone oxidoreductase (NDH-II) leading to the amino acid change D213G, which was shared by these suppressor mutants. Since related NDH-II enzymes accepting NADPH as the substrate possess asparagine or glutamine residues at this position, D213G, D213N, and D213Q variants of C. glutamicum NDH-II were constructed and were shown to oxidize NADPH in addition to NADH. Taking these findings together, ATP-neutral glycolysis by the replacement of endogenous NAD-dependent GAPDH with NADP-dependent GapN became possible via oxidation of NADPH formed in this pathway by mutant NADPH-accepting NDH-IID213G and thus by coupling to electron transport phosphorylation (ETP). © 2015, American Society for Microbiology.

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