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Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol. Gönül Vardar, 2004.Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol . To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants . The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography . Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein) . From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively) . The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein) . Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol . Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol) . Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol . Isolation of a Negative Control Mutant of the Nitrogen Assimilation Control Protein, NAC, in Klebsiella aerogenes. Brian K. Janes, 2003.A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated . Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression . This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant . It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter . Identification of a Third Sulfate Activation System in Sinorhizobium sp . Strain BR816: the CysDN Sulfate Activation Complex. Carla Snoeck, 2003.Sinorhizobium sp . strain BR816 possesses two nodPQ copies, providing activated sulfate (3'-phosphoadenosine-5'-phosphosulfate [PAPS]) needed for the biosynthesis of sulfated Nod factors . It was previously shown that the Nod factors synthesized by a nodPQ double mutant are not structurally different from those of the wild-type strain . In this study, we describe the characterization of a third sulfate activation locus . Two open reading frames were fully characterized and displayed the highest similarity with the Sinorhizobium meliloti housekeeping ATP sulfurylase subunits, encoded by the cysDN genes . The growth characteristics as well as the levels of Nod factor sulfation of a cysD mutant (FAJ1600) and a nodP1 nodQ2 cysD triple mutant (FAJ1604) were determined . FAJ1600 shows a prolonged lag phase only with inorganic sulfate as the sole sulfur source, compared to the wild-type parent . On the other hand, FAJ1604 requires cysteine for growth and produces sulfate-free Nod factors . Apigenin-induced nod gene expression for Nod factor synthesis does not influence the growth characteristics of any of the strains studied in the presence of different sulfur sources . In this way, it could be demonstrated that the "household" CysDN sulfate activation complex of Sinorhizobium sp . strain BR816 can additionally ensure Nod factor sulfation, whereas the symbiotic PAPS pool, generated by the nodPQ sulfate activation loci, can be engaged for sulfation of amino acids . Finally, our results show that rhizobial growth defects are likely the reason for a decreased nitrogen fixation capacity of bean plants inoculated with cysD mutant strains, which can be restored by adding methionine to the plant nutrient solution . Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR. Palmer A. Orlandi, 2003.Cyclospora cayetanensis is a coccidian parasite that causes protracted diarrheal illness in humans . C . cayetanensis is the only species of this genus thus far associated with human illness, although Cyclospora species from other primates have been named . The current method to detect the parasite uses a nested PCR assay to amplify a 294-bp region of the small subunit rRNA gene, followed by restriction fragment length polymorphism (RFLP) or DNA sequence analysis . Since the amplicons generated from C . cayetanensis and Eimeria species are the same size, the latter step is required to distinguish between these different species . The current PCR-RFLP protocol, however, cannot distinguish between C . cayetanensis and these new isolates . The differential identification of such pathogenic and nonpathogenic parasites is essential in assessing the risks to human health from microorganisms that may be potential contaminants in food and water sources . Therefore, to expand the utility of PCR to detect and identify these parasites in a multiplex assay, a series of genus- and species-specific forward primers were designed that are able to distinguish sites of limited sequence heterogeneity in the target gene . The most effective of these unique primers were those that identified single-nucleotide polymorphisms (SNPs) at the 3' end of the primer . Under more stringent annealing and elongation conditions, these SNP primers were able to differentiate between C . cayetanensis, nonhuman primate species of Cyclospora, and Eimeria species . As a diagnostic tool, the SNP PCR protocol described here presents a more rapid and sensitive alternative to the currently available PCR-RFLP detection method . In addition, the specificity of these diagnostic primers removes the uncertainty that can be associated with analyses of foods or environmental sources suspected of harboring potential human parasitic pathogens .
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