Molecular cloning and expression of the imipenem-hydrolyzing beta-lactamase gene from Pseudomonas maltophilia in Escherichia coli.
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.
Dufresne
J
J
Département de Microbiologie et Immunologie, Faculté de Médecine, Université Laval, Québec, Canada.
Vézina
G
G
Levesque
R C
RC
D000595:Amino Acid Sequence; D001345:Autoradiography; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006868:Hydrolysis; D015378:Imipenem / Q000378:metabolism*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D015378:Imipenem; D004262:DNA Restriction Enzymes; D001618:beta-Lactamases