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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes
Published on: November 7, 2012
Properties of spontaneous Enterobacter cloacae mutants with temperature-conditional derepression of type I
Abstract:
Spontaneous mutants, with temperature-conditional derepression of chromosomally-encoded Type I beta-lactamase synthesis, were derived from two independent clinical isolates of Enterobacter cloacae. At the permissive temperature (28 degrees C) the mutants' beta-lactamase activity was equivalent to that of their respective parents but at restrictive temperatures (above 40 degrees C) the activity increased many hundred-fold. The increased beta-lactamase expression correlated with reduced beta-lactam susceptibility. In temperature shift-up experiments, the initial rate of beta-lactamase synthesis closely paralleled that of the parent strains induced with cefoxitin. Maximal beta-lactamase activity in the mutants was attained after about 3 h growth at restrictive temperatures and was significantly higher than that of the cefoxitin-induced parents. However, the level was not as high as that observed in isogenic temperature-stable derepressed mutants, under the same conditions. All temperature-conditional mutants showed hyper-induction of beta-lactamase synthesis at permissive temperatures. Our findings are discussed in relation to a positive control model for regulation of Type I beta-lactamase synthesis in Ent. cloacae.
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