Cloning and expression of a cloxacillin-hydrolyzing enzyme and a cephalosporinase from Aeromonas sobria AER 14M in Escherichia coli: requirement for an E. coli chromosomal mutation for efficient expression of the class D enzyme.
Two beta-lactamase genes, asbA1 and asbB1, encoding AsbA1 and AsbB1, respectively, have been cloned from Aeromonas sobria AER 14M into Escherichia coli. AsbA1 was expressed at low but detectable levels in all E. coli laboratory cloning strains tested. AsbB1 was expressed well in the E. coli cloning strain DH5 alpha. However, no enzyme activity could be detected from the same clone when placed in E. coli MC1061. Ampicillin-resistant mutants of E. coli MC1061 were obtained that expressed high levels of enzymatically active AsbB1. Four independent mutants were examined. All four mutations mapped to one locus, designated blpA (beta-lactamase permissive). The blpA locus was distinct from other known loci that play a role in beta-lactamase expression, i.e., the two loci that affect expression of the Bacteroides fragilis metallo-beta-lactamase and the ampC regulatory genes, ampD, ampE, and ampG. Sequence analysis of asbA1 and asbB1 revealed that AsbA1 was a class C beta-lactamase most closely related to the Pseudomonas aeruginosa chromosomal cephalosporinase and probably represents the common A. sobria cephalosporinase. AsbB1 was a class D enzyme most closely related to the oxacillin-hydrolyzing enzyme OXA-1, with 34% amino acid sequence identity. Purified AsbA1 was a typical cephalosporinase with a substrate profile that reflected high rates of hydrolysis of cephaloridine compared with benzylpenicillin. Purified AsbB1 showed strong penicillinase activity, with hydrolysis rates for carbenicillin and cloxacillin 2 to 2.5 times that for benzylpenicillin. Hydrolysis of imipenem was < or = 1% of that for benzylpenicillin. Both clavulanic acid and tazobactam strongly inhibited AsbB1, while sulbactam inhibited the AsbB1 enzyme less effectively. None of the inhibitors worked well against the AsbA1 enzyme. The chelators EDTA and 1,10-o-phenanthroline did not affect the activity of either enzyme. A. sobria AER 14M was found to produce both a group 1 cephalosporinase and a novel group 2d cloxacillin-hydrolyzing beta-lactamase that has been designated here OXA-12.
Rasmussen
B A
BA
Medical Research Division, American Cyanamid Company, Pearl River, New York 10965.
Keeney
D
D
Yang
Y
Y
Bush
K
K
D000333:Aeromonas / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002510:Cephalosporinase / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D003023:Cloxacillin / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation; D017386:Sequence Homology, Amino Acid; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D065093:beta-Lactamase Inhibitors; D002510:Cephalosporinase; D001618:beta-Lactamases; D003023:Cloxacillin