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Role of Feedback Regulation of Pantothenate Kinase (CoaA) in Control of Coenzyme A Levels in Escherichia coli. Charles O. Rock, 2003.Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters . The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex . CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA . CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active . The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2] . Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein . These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels . Genetic and Culture-Based Approaches for Detecting Macrolide Resistance in Chlamydia pneumoniae. Paul F. Riska, 2004.Three clinical Chlamydia pneumoniae isolates for which the MIC of azithromycin increased after treatment were investigated for genetic evidence of macrolide resistance . Attempts to induce antibiotic resistance in vitro were made . No genetic mechanism was identified for the phenotypic change in these C . pneumoniae isolates . No macrolide resistance was obtained in vitro . Evidence for Temporal Regulation of the Two Pseudomonas cellulosa Xylanases Belonging to Glycoside Hydrolase Family 11. Kaveh Emami, 2002.Pseudomonas cellulosa is a highly efficient xylan-degrading bacterium . Genes encoding five xylanases, and several accessory enzymes, which remove the various side chains that decorate the xylan backbone, have been isolated from the pseudomonad and characterized . The xylanase genes consist of xyn10A, xyn10B, xyn10C, xyn10D, and xyn11A, which encode Xyn10A, Xyn10B, Xyn10C, Xyn10D, and Xyn11A, respectively . In this study a sixth xylanase gene, xyn11B, was isolated which encodes a 357-residue modular enzyme, designated Xyn11B, comprising a glycoside hydrolase family 11 catalytic domain appended to a C-terminal X-14 module, a homologue of which binds to xylan . Localization studies showed that the two xylanases with glycoside hydrolase family (GH) 11 catalytic modules, Xyn11A and Xyn11B, are secreted into the culture medium, whereas Xyn10C is membrane bound . xyn10C, xyn10D, xyn11A, and xyn11B were all abundantly expressed when the bacterium was cultured on xylan or ß-glucan but not on medium containing mannan, whereas glucose repressed transcription of these genes . Although all of the xylanase genes were induced by the same polysaccharides, temporal regulation of xyn11A and xyn11B was apparent on xylan-containing media . Transcription of xyn11A occurred earlier than transcription of xyn11B, which is consistent with the predicted mode of action of the encoded enzymes . Xyn11A, but not Xyn11B, exhibits xylan esterase activity, and the removal of acetate side chains is required for xylanases to hydrolyze the xylan backbone . A transposon mutant of P . cellulosa in which xyn11A and xyn11B were inactive displayed greatly reduced extracellular but normal cell-associated xylanase activity, and its growth rate on medium containing xylan was indistinguishable from wild-type P . cellulosa . Based on the data presented here, we propose a model for xylan degradation by P . cellulosa in which the GH11 enzymes convert decorated xylans into substituted xylooligosaccharides, which are then hydrolyzed to their constituent sugars by the combined action of cell-associated GH10 xylanases and side chain-cleaving enzymes . A Stable Bioluminescent Construct of Escherichia coli O157:H7 for Hazard Assessments of Long-Term Survival in the Environment. Jennifer M. Ritchie, 2003.A chromosomally lux-marked (Tn5 luxCDABE) strain of nontoxigenic Escherichia coli O157:H7 was constructed by transposon mutagenesis and shown to have retained the O157, H7, and intimin phenotypes. The survival characteristics of this strain in the experiments performed (soil at -5, -100, and -1,500 kPa matric potential and artificial groundwater) were indistinguishable from the wild-type strain . Evaluation of potential luminescence was found to be a rapid, cheap, and quantitative measure of viable E . coli O157:H7 Tn5 luxCDABE populations in environmental samples . In the survival studies, bioluminescence of the starved populations of E . coli O157:H7 Tn5 luxCDABE could be reactivated to the original levels of light emission, suggesting that these populations remain viable and potentially infective to humans . The attributes of the construct offer a cheap and low-risk substitute to the use of verocytotoxin-producing E . coli O157:H7 in long-term survival studies . Cloning and Characterization of the xyl1 Gene, Encoding an NADH-Preferring Xylose Reductase from Candida parapsilosis, and Its Functional Expression in Candida tropicalis. Jung-Kul Lee, 2003.Xylose reductase (XR) is a key enzyme in D-xylose metabolism, catalyzing the reduction of D-xylose to xylitol . An NADH-preferring XR was purified to homogeneity from Candida parapsilosis KFCC-10875, and the xyl1 gene encoding a 324-amino-acid polypeptide with a molecular mass of 36,629 Da was subsequently isolated using internal amino acid sequences and 5' and 3' rapid amplification of cDNA ends . The C . parapsilosis XR showed high catalytic efficiency (kcat/Km = 1.46 s-1 mM-1) for D-xylose and showed unusual coenzyme specificity, with greater catalytic efficiency with NADH (kcat/Km = 1.39 x 104 s-1 mM-1) than with NADPH (kcat/Km = 1.27 x 102 s-1 mM-1), unlike all other aldose reductases characterized . Studies of initial velocity and product inhibition suggest that the reaction proceeds via a sequentially ordered Bi Bi mechanism, which is typical of XRs . Candida tropicalis KFCC-10960 has been reported to have the highest xylitol production yield and rate . It has been suggested, however, that NADPH-dependent XRs, including the XR of C . tropicalis, are limited by the coenzyme availability and thus limit the production of xylitol . The C . parapsilosis xyl1 gene was placed under the control of an alcohol dehydrogenase promoter and integrated into the genome of C . tropicalis . The resulting recombinant yeast, C . tropicalis BN-1, showed higher yield and productivity (by 5 and 25%, respectively) than the wild strain and lower production of by-products, thus facilitating the purification process . The XRs partially purified from C . tropicalis BN-1 exhibited dual coenzyme specificity for both NADH and NADPH, indicating the functional expression of the C . parapsilosis xyl1 gene in C . tropicalis BN-1 . This is the first report of the cloning of an xyl1 gene encoding an NADH-preferring XR and its functional expression in C . tropicalis, a yeast currently used for industrial production of xylitol .
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