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Degradation of Aromatics and Chloroaromatics by Pseudomonas sp . Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase. Stefan R. Kaschabek, 2002.The degradation of 3-oxoadipate in Pseudomonas sp . strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA . 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels . Estimation of the native molecular mass gave a value of 115,000 ± 5,000 Da with a Superose 12 column . Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities . The subunit A and B values were 32,900 and 27,000 Da . Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da . The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV . The pH optimum was 8.4 . Km values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively . Reversibility of the reaction with succinate was shown . The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate . Some activity was observed with 4-methyl-3-oxoadipate . Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase . 3-Oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose . Estimation of the native molecular mass gave 162,000 ± 5,000 Da with a Superose 6 column . The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa . On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4 . The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG . The pH optimum was 7.8 . Km values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively . Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions . The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found . Profiling Early Osmostress-Dependent Gene Expression in Escherichia coli Using DNA Macroarrays. Arnim Weber, 2002.DNA macroarray technology was used to monitor early transcriptional alterations of Escherichia coli in response to an osmotic upshift imposed by the addition of 0.4 M NaCl . Altered mRNA levels of 152 genes were detected; 45 genes showed increased expression while the expression of the remaining 107 genes was reduced . Northern blot analysis of several selected genes differing in their relative expression values confirmed the results obtained by the array technology . Plants in the Pink: Cytokinin Production by Methylobacterium. Mary E. Lidstrom, 2002. The Earthworm Gut: an Ideal Habitat for Ingested N2O-Producing Microorganisms. Marcus A. Horn, 2003.The in vivo production of nitrous oxide (N2O) by earthworms is due to their gut microbiota, and it is hypothesized that the microenvironment of the gut activates ingested N2O-producing soil bacteria . In situ measurement of N2O and O2 with microsensors demonstrated that the earthworm gut is anoxic and the site of N2O production . The gut had a pH of 6.9 and an average water content of approximately 50% . The water content within the gut decreased from the anterior end to the posterior end . In contrast, the concentration of N2O increased from the anterior end to the mid-gut region and then decreased along the posterior part of the gut . Compared to the soil in which worms lived and fed, the gut of the earthworm was highly enriched in total carbon, organic carbon, and total nitrogen and had a C/N ratio of 7 (compared to a C/N ratio of 12 in soil) . The aqueous phase of gut contents contained up to 80 mM glucose and numerous compounds that were indicative of anaerobic metabolism, including up to 9 mM formate, 8 mM acetate, 3 mM lactate, and 2 mM succinate . Compared to the soil contents, nitrite and ammonium were enriched in the gut up to 10- and 100-fold, respectively . The production of N2O by soil was induced when the gut environment was simulated in anoxic microcosms for 24 h (the approximate time for passage of soil through the earthworm) . Anoxia, high osmolarity, nitrite, and nitrate were the dominant factors that stimulated the production of N2O . Supplemental organic carbon had a very minimal stimulatory effect on the production of N2O, and addition of buffer or ammonium had essentially no effect on the initial N2O production rates . However, a combination of supplements yielded rates greater than that obtained mathematically for single supplements, suggesting that the maximum rates observed were due to synergistic effects of supplements . Collectively, these results indicate that the special microenvironment of the earthworm gut is ideally suited for N2O-producing bacteria and support the hypothesis that the in situ conditions of the earthworm gut activate ingested N2O-producing soil bacteria during gut passage .
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