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Geochemical Rate-RNA Integration Study: Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Gene Transcription and Photosynthetic Capacity of Planktonic Photoautotrophs. Jorge E. Corredor, 2004.A pilot field experiment to assess the relationship between traditional biogeochemical rate measurements and transcriptional activity of microbial populations was carried out at the LEO 15 site off Tuckerton, N.J . Here, we report the relationship between photosynthetic capacity of autotrophic plankton and transcriptional activity of the large subunit gene (rbcL) for ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the enzyme responsible for primary carbon fixation during photosynthesis . Similar diel patterns of carbon fixation and rbcL gene expression were observed in three of four time series, with maxima for photosynthetic capacity (Pmax) and rbcL mRNA occurring between 10 a.m . and 1 p.m. . The lowest Pmax and rbcL levels were detected between 6 p.m . and 10:30 p.m. . A significant correlation was found between Pmax and form ID rbcL mRNA (R2 = 0.56) and forms IA and IB (R2 = 0.41 and 0.47, respectively) . The correlation between the abundance of "diatom" rbcL and Pmax mRNA was modest (R2 = 0.49; n = 12) but improved dramatically (R2 = 0.97; n = 10) upon removal of two outliers which represented afternoon samples with high Pmax but lower mRNA levels . Clone libraries from reverse transcription-PCR-amplified rbcL mRNA indicated the presence of several chromophytic algae (diatoms, prymnesiophytes, and chrysophytes) and some eukaryotic green flagellates . Analogous results were obtained from amplified small rRNA sequences and secondary pigment analysis . These results suggest that diatoms were a major contributor to carbon fixation at LEO 15 at the time of sampling and that photosynthetic carbon fixation was partially controlled by transcriptional regulation of the RubisCO gene . Synergistic Effects on Crystalline Cellulose Degradation between Cellulosomal Cellulases from Clostridium cellulovorans. Koichiro Murashima, 2002.Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome . In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA . EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively . The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed . These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose . The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation . Reactions were also performed by adding different cellulosomes in a sequential manner . When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed . On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed . These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS . Transposition of Tn5367 in Mycobacterium marinum, Using a Conditionally Recombinant Mycobacteriophage. Jan Rybniker, 2003.Mycobacterium marinum is a close relative of the obligate human pathogen Mycobacterium tuberculosis . As with M . tuberculosis, M . marinum causes intracellular infection of poikilothermic vertebrates and skin infection in humans . It is considered a valid model organism for the study of intracellular pathogenesis of mycobacteria . Low transformation efficiencies for this species have precluded approaches using mutant libraries in pathogenesis studies . We have adapted the conditionally replicating mycobacteriophage phAE94, originally developed as a transposon mutagenesis tool for M . tuberculosis, to meet the specific requirements of M . marinum . Conditions permissive for phage replication in M . tuberculosis facilitated highly efficient transposon delivery in M . marinum . Using this technique we succeeded in generating a representative mutant library of this species, and we conclude that TM4-derived mycobacteriophages are temperature-independent suicide vectors for M . marinum . In Situ Production of Exopolysaccharides during Sourdough Fermentation by Cereal and Intestinal Isolates of Lactic Acid Bacteria. Markus Tieking, 2003.EPS formed by lactobacilli in situ during sourdough fermentation may replace hydrocolloids currently used as texturizing, antistaling, or prebiotic additives in bread production . In this study, a screening of >100 strains of cereal-associated and intestinal lactic acid bacteria was performed for the production of exopolysaccharides (EPS) from sucrose . Fifteen strains produced fructan, and four strains produced glucan . It was remarkable that formation of glucan and fructan was most frequently found in intestinal isolates and strains of the species Lactobacillus reuteri, Lactobacillus pontis, and Lactobacillus frumenti from type II sourdoughs . By the use of PCR primers derived from conserved amino acid sequences of bacterial levansucrase genes, it was shown that 6 of the 15 fructan-producing lactobacilli and none of 20 glucan producers or EPS-negative strains carried a levansucrase gene . In sourdough fermentations, it was determined whether those strains producing EPS in MRS medium modified as described by Stolz et al . () and containing 100 g of sucrose liter-1 as the sole source of carbon also produce the same EPS from sucrose during sourdough fermentation in the presence of 12% sucrose . For all six EPS-producing strains evaluated in sourdough fermentations, in situ production of EPS at levels ranging from 0.5 to 2 g/kg of flour was demonstrated . Production of EPS from sucrose is a metabolic activity that is widespread among sourdough lactic acid bacteria . Thus, the use of these organisms in bread production may allow the replacement of additives .
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