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Analysis of the Effects of Chlorhexidine on Oral Biofilm Vitality and Structure Based on Viability Profiling and an Indicator of Membrane Integrity. C. K. Hope, 2004.Multispecies biofilms modeling interproximal plaque were grown on a hydroxyapatite substratum in a constant-depth film fermentor and then immersed in a viewing solution containing fluorescent indicators of membrane integrity . Confocal laser scanning microscopy (CLSM) revealed the structure and spatial distribution of cell vitality within the biofilms . Chlorhexidine gluconate (CHX) was added to the viewing solution to achieve concentrations of 0.05 and 0.2% (wt/vol) before further CLSM time-lapse series were captured . Image analysis showed that exposure to 0.2% CHX caused the biofilm to contract at a rate of 1.176 µm min–1 along the z axis and also effected changes in total fluorescence measurements and viability profiles through the biofilms after a delay of 3 to 5 min . At a concentration of 0.05% CHX, total fluorescence measurements for the biofilm exhibited barely detectable changes after 5 min . Fluorescence profiles (fluorescence versus time versus depth), however, clearly showed that a time-dependent effect was present, but the clearest indicator of the effect of dilute CHX over time was viability profiling . These findings suggest the possibility of using fluorescent indicators of membrane integrity in conjunction with viability profiling to evaluate the penetration of the bactericidal effects of membrane-active antimicrobial compounds into biofilm . Potential Role of a Novel Psychrotolerant Member of the Family Geobacteraceae, Geopsychrobacter electrodiphilus gen . nov., sp . nov., in Electricity Production by a Marine Sediment Fuel Cell. Dawn E. Holmes, 2004.Previous studies have shown that members of the family Geobacteraceae that attach to the anodes of sediment fuel cells are directly involved in harvesting electricity by oxidizing organic compounds to carbon dioxide and transferring the electrons to the anode . In order to learn more about this process, microorganisms from the anode surface of a marine sediment fuel cell were enriched and isolated with Fe(III) oxide . Two unique marine isolates were recovered, strains A1T and A2 . They are gram-negative, nonmotile rods, with abundant c-type cytochromes . Phylogenetic analysis of the 16S rRNA, recA, gyrB, fusA, rpoB, and nifD genes indicated that strains A1T and A2 represent a unique phylogenetic cluster within the Geobacteraceae . Both strains were able to grow with an electrode serving as the sole electron acceptor and transferred ca . 90% of the electrons available in their organic electron donors to the electrodes . These organisms are the first psychrotolerant members of the Geobacteraceae reported thus far and can grow at temperatures between 4 and 30°C, with an optimum temperature of 22°C . Strains A1T and A2 can utilize a wide range of traditional electron acceptors, including all forms of soluble and insoluble Fe(III) tested, anthraquinone 2,6-disulfonate, and S0 . In addition to acetate, both strains can utilize a number of other organic acids, amino acids, long-chain fatty acids, and aromatic compounds to support growth with Fe(III) nitrilotriacetic acid as an electron acceptor . The metabolism of these organisms differs in that only strain A1T can use acetoin, ethanol, and hydrogen as electron donors, whereas only strain A2 can use lactate, propionate, and butyrate . The name Geopsychrobacter electrodiphilus gen . nov., sp . nov., is proposed for strains A1T and A2, with strain A1T (ATCC BAA-880T; DSM 16401T; JCM 12469) as the type strain . Strains A1T and A2 (ATCC BAA-770; JCM 12470) represent the first organisms recovered from anodes that can effectively couple the oxidation of organic compounds to an electrode . Thus, they may serve as important model organisms for further elucidation of the mechanisms of microbe-electrode electron transfer in sediment fuel cells . Explorative Multivariate Analyses of 16S rRNA Gene Data from Microbial Communities in Modified-Atmosphere-Packed Salmon and Coalfish. Knut Rudi, 2004.Modified-atmosphere packaging (MAP) of foods in combination with low-temperature storage extends product shelf life by limiting microbial growth . We investigated the microbial biodiversity of MAP salmon and coalfish by using an explorative approach and analyzing both the total amounts of bacteria and the microbial group composition (both aerobic and anaerobic bacteria) . Real-time PCR analyses revealed a surprisingly large difference in the microbial loads for the different fish samples . The microbial composition was determined by examining partial 16S rRNA gene sequences from 180 bacterial isolates, as well as by performing terminal restriction fragment length polymorphism analysis and cloning 92 sequences from PCR products of DNA directly retrieved from the fish matrix . Twenty different bacterial groups were identified . Partial least-squares (PLS) regression was used to relate the major groups of bacteria identified to the fish matrix and storage time . A strong association of coalfish with Photobacterium phosphoreum was observed . Brochothrix spp . and Carnobacterium spp., on the other hand, were associated with salmon . These bacteria dominated the fish matrixes after a storage period . Twelve Carnobacterium isolates were identified as either Carnobacterium piscicola (five isolates) or Carnobacterium divergens (seven isolates), while the eight Brochothrix isolates were identified as Brochothrix thermosphacta by full-length 16S rRNA gene sequencing . Principal-component analyses and PLS analysis of the growth characteristics (with 49 different substrates) showed that C . piscicola had distinct substrate requirements, while the requirements of B . thermosphacta and C . piscicola were quite divergent . In conclusion, our explorative multivariate approach gave a picture of the total microbial biodiversity in MAP fish that was more comprehensive than the picture that could be obtained previously . Such information is crucial in controlled food production when, for example, the hazard analysis of critical control points principle is used . A Single Mutation in the Activation Site of Bovine Trypsinogen Enhances Its Accumulation in the Fermentation Broth of the Yeast Pichia pastoris. José Hanquier, 2003.We produced bovine trypsinogen in the yeast Pichia pastoris . Little or no trypsinogen was detected when the gene with its native leader sequence was expressed under the control of the strong aox1 promoter, suggesting that expression of the wild-type bovine trypsinogen was toxic to the cells . We altered the trypsinogen native propeptide sequence by replacing the lysine at position 6 with an aspartic acid, thus destroying the site in the propeptide cleaved by enterokinase and by trypsin . This mutant accumulated up to 10 mg of trypsinogen per liter in shake flask cultures and about 40 mg/liter in 6-liter fermentors . Trypsinogen could be activated in vitro with a dipeptidyl-aminopeptidase, which selectively removed the modified trypsinogen propeptide; the resulting trypsin was fully active and showed evidence of glycosylation . Thus, we have developed a novel protein production scheme that can be used for the expression of proteins, such as proteases, that are deleterious to the producing organism . This system relies on the expression of a zymogen that cannot be activated in vivo coupled with its in vitro purification and activation . Metabolic Engineering of the Carotenoid Biosynthetic Pathway in the Yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma). Jan C. Verdoes, 2003.The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X . dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants . In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a ß-carotene-accumulating mutant of X . dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain . This increase was mainly due to an increase of the ß-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower . Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids . Furthermore, we showed that in metabolic engineered X . dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via ß-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-ß-  -caroten-4-one (HDCO) . The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains .
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