Molecular cloning and expression of the imipenem-hydrolyzing beta-lactamase gene from Pseudomonas maltophilia in Escherichia coli

Abstract

The L-1 penicillinase structural gene, blaS, from Pseudomonas maltophilia was cloned into the vector pACYC184. The pMON01 recombinant plasmid selected by ampicillin resistance carried a 2.6-kilobase (kb) 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 NH2-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; the 13.4-kb DNA insert was physically mapped and subcloned into the high-copy-number plasmid pACYC184 and into the low-copy-number vector pLG338. The expression of the cloned blaS L-1 structural gene and levels of beta-lactamase synthesis were studied in Escherichia coli. The protein synthesized was found to be similar to the L-1 beta-lactamase of the prototype P. maltophilia, although expression levels were gene dosage dependent for beta-lactamase synthesis.