Cloning and expression of the imipenem-hydrolyzing beta-lactamase operon from Pseudomonas maltophilia in Escherichia coli.
The L-1 penicillinase structural gene, blaS, from Pseudomonas maltophilia has been cloned into the vector pACYC184. The pMON01 recombinant plasmid selected by ampicillin resistance carried a 2.6-kilobase Sau3A fragment of P. maltophilia DNA and was confirmed to express L-1 beta-lactamase by comparative isoelectric focusing. A detailed physical map was constructed, and the blaS structural gene was localized with a 17-mer oligonucleotide mixed probe encoding the L-1 N-terminal amino acid sequence. Induction studies confirmed constitutive expression. Isolation of a complete beta-lactamase operon was attempted by construction of a P. maltophilia genomic library into phage lambda 2001. A recombinant phage was selected by DNA hybridization, and the 13.4-kilobase DNA insert was physically mapped and subcloned into plasmid vectors. Expression and L-1 beta-lactamase synthesis were studied in Escherichia coli.
D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D006868:Hydrolysis; D015378:Imipenem; D009876:Operon; D010957:Plasmids; D011549:Pseudomonas / Q000235:genetics*; D013845:Thienamycins / Q000378:metabolism; D001618:beta-Lactamases / Q000096:biosynthesis / Q000235:genetics*
D004269:DNA, Bacterial; D013845:Thienamycins; D015378:Imipenem; D001618:beta-Lactamases