Comparison of the Chromosomal and Extrachromosomal Genetic Determinants Controlling Staphylococcal Penicillinase Production

Sawai, Tetsuo; Mitsuhashi, Susumu; Yamagishi, Saburo

doi:10.1111/j.1348-0421.1968.tb00425.x

Cited by 3 publications

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“…Judging from these data and the known amino acid composition, there is no obvious phylogenetic relationship between the penicillinases from different organisms or episomes in the Enterobacteriaceae. Evidence has recently been reported which indicates that the situation could be the reverse in the case of the staphylococcal penicillinases (36). Evolutionary aspects of the structure of purified penicillinases were recently discussed by Ambler and Meadway (1) and by Pollock (29,30).…”

Section: Discussionmentioning

confidence: 99%

Resistance of Escherichia coli to Penicillins VI. Purification and Characterization of the Chromosomally Mediated Penicillinase Present in ampA -Containing Strains

1970

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The chromosomally mediated penicillinase present in three strains of Escherichia coli K-12 has been purified and characterized. Two of the strains carried the ampA gene and the third the wild-type allele. The purification involves release of the enzyme by spheroplast formation, dialysis, chromatography on sulfoethyl cellulose, and chromatography on hydroxylapatite. Enzyme from the two mutants appeared homogeneous in polyacrylamide gel electrophoresis. Enzyme from the wild-type strain gave two bands. Immunologically, the enzymes from all three strains were identical. Ultracentrifugation gave a homogeneous peak with a sedimentation coefficient of 3.45. Gel filtration gave an estimated molecular weight of29,000. The Nterminal amino acid residue was found to be alanine. Complete amino acid analysis showed a lack of cysteine. Ultraviolet spectra were recorded at three different pH values. The extinction coefficient at 280 nm is 21.0 for a 1% solution at pH 6.8. The optimal pH is 7.3. With enzyme from one of the resistant mutants, the following Km and turnover number values were obtained: for penicillin G, 12 ;uM and 2,080; for D-MATERIALS AND METHODS Bacterial strains. All strains used were E. coli K-12. Strain Gllal which carries the amp Al allele is a spontaneous mutant obtained from the wild-type strain Gll (10). The ampAl allele was transduced into strain D2, which afterwards was mutated to the highly ampicillin-resistant strain D3 (5). A second mutation step induced streptomycin resistance in D3, thus producing strain D31 (5). Strains Gllal and D2 both form single cell colonies on plates containing D-ampicillin concentrations of 15 to 20,g/ml, whereas strain D31 can form single-cell colonies on plates with D-ampicillin concentrations of 75 to 100 ,Ag/ml (for resistance determinations, see references 6 and 24). Media and growth conditions. The growth medium 218

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