L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product.
Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments. Therefore, the genes encoding asparaginases I and II are highly sequence related.
D001215:Asparaginase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D001216:Asparagine / Q000031:analogs & derivatives / Q000494:pharmacology; D003429:Cross Reactions; D004269:DNA, Bacterial / Q000032:analysis; D004274:DNA, Recombinant / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D012689:Sequence Homology, Nucleic Acid
D004269:DNA, Bacterial; D004274:DNA, Recombinant; C024848:beta-aspartylhydroxamic acid; D001216:Asparagine; D001215:Asparaginase