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Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation. Lingyun Rui, 2004.Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase  -subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity) . In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation . Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (Vmax of 9.3 versus 4.5 nmol · min–1 · mg of protein–1 and unchanged Km), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography . The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol · min–1 · mg of protein–1 [mean ± standard deviation]) at 91 µM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM . The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (Vmax of 2.61 versus 0.95 nmol · min–1 · mg of protein–1 and unchanged Km) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol · min–1 · mg of protein–1) . The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol · min–1 · mg of protein–1) . A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed . Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis .
Direct Quantitation of the Numbers of Individual Penicillin-Binding Proteins per Cell in Staphylococcus aureus. Michael J. Pucci, 2002.The penicillin-binding proteins (PBPs) are a set of enzymes that participate in bacterial peptidoglycan assembly . The absolute numbers of each PBP were determined by direct measurement and have been reported for two Staphylococcus aureus strains, RN4220 (methicillin-sensitive S . aureus) and RN450M (methicillin-resistant S . aureus) . From the specific activity of the labeled penicillin and the absolute number of disintegrations per minute, and from the number of CFU per milliliter calculated from proteins and optical density, a determination of the number of PBPs per cell was made . These numbers ranged from approximately 150 to 825 PBPs/cell and represent the first direct determination of absolute numbers of PBPs in S . aureus . Mutations in the 16S rRNA Genes of Helicobacter pylori Mediate Resistance to Tetracycline. Catharine A. Trieber, 2002.Low-cost and rescue treatments for Helicobacter pylori infections involve combinations of several drugs including tetracycline . Resistance to tetracycline has recently emerged in H . pylori . The 16S rRNA gene sequences of two tetracycline-resistant clinical isolates (MIC = 64 µg/ml) were determined and compared to the consensus H . pylori 16S rRNA sequence . One isolate had four nucleotide substitutions, and the other had four substitutions and two deletions . Natural transformation with the 16S rRNA genes from the resistant organisms conferred tetracycline resistance on susceptible strains . 16S rRNA genes containing the individual mutations were constructed and tested for the ability to confer resistance . Only the 16S rRNA gene containing the triple mutation, AGA965-967TTC, was able to confer tetracycline resistance on H . pylori 26695 . The MICs of tetracycline for the transformed strains were equivalent to those for the original clinical isolates . The two original isolates were also metronidazole resistant, but this trait was not linked to the tetracycline resistance phenotype . Serial passage of several H . pylori strains on increasing concentrations of tetracycline yielded mutants with only a very modest increase in tetracycline resistance to a MIC of 4 to 8 µg/ml . These mutants all had a deletion of G942 in the 16S rRNA genes . The mutations in the 16S rRNA are clearly responsible for tetracycline resistance in H . pylori . Expression of Heteropolymeric Ferritin Improves Iron Storage in Saccharomyces cerevisiae. Hye-Jin Kim, 2003.Saccharomyces cerevisiae was engineered to express different amount of heavy (H)- and light (L)-chain subunits of human ferritin by using a low-copy integrative vector (YIp) and a high-copy episomal vector (YEp) . In addition to pep4::HIS3 allele, the expression host strain was bred to have the selection markers leu2- and ura3- for YIplac128 and YEp352, respectively . The heterologous expression of phytase was used to determine the expression capability of the host strain . Expression in the new host strain (2805-a7) was as high as that in the parental strain (2805), which expresses high levels of several foreign genes . Following transformation, Northern and Western blot analyses demonstrated the expression of H- and L-chain genes . The recombinant yeast was more iron tolerant, in that transformed cells formed colonies on plates containing more than 25 mM ferric citrate, whereas none of the recipient strain cells did . Prussian blue staining indicated that the expressed isoferritins were assembled in vivo into a complex that bound iron . The expressed subunits showed a clear preference for the formation of heteropolymers over homopolymers . The molar ratio of H to L chains was estimated to be 1:6.8 . The gel-purified heteropolymer took up iron faster than the L homopolymer, and it took up more iron than the H homopolymer did . The iron concentrations in transformants expressing the heteropolymer, L homopolymer, and H homopolymer were 1,004, 760, and 500 µg per g (dry weight) of recombinant yeast cells, respectively . The results indicate that heterologously expressed H and L subunits coassemble into a heteropolymer in vivo and that the iron-carrying capacity of yeast is further enhanced by the expression of heteropolymeric isoferritin .
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