Rasmussen B A, Keeney D, Yang Y, Bush K
Medical Research Division, American Cyanamid Company, Pearl River, New York 10965.
Antimicrob Agents Chemother. 1994 Sep;38(9):2078-85. doi: 10.1128/AAC.38.9.2078.
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.
已从温和气单胞菌AER 14M中克隆出分别编码AsbA1和AsbB1的两个β-内酰胺酶基因asbA1和asbB1，并将其导入大肠杆菌。在所有测试的大肠杆菌实验室克隆菌株中，AsbA1均以低水平但可检测到的水平表达。AsbB1在大肠杆菌克隆菌株DH5α中表达良好。然而，当将同一克隆置于大肠杆菌MC1061中时，未检测到酶活性。获得了大肠杆菌MC1061的氨苄青霉素抗性突变体，其表达高水平的具有酶活性的AsbB1。对四个独立的突变体进行了检测。所有四个突变均定位于一个位点，命名为blpA（β-内酰胺酶允许位点）。blpA位点与其他在β-内酰胺酶表达中起作用的已知位点不同，即影响脆弱拟杆菌金属β-内酰胺酶和ampC调节基因ampD、ampE和ampG表达的两个位点。asbA1和asbB1的序列分析表明，AsbA1是一种C类β-内酰胺酶，与铜绿假单胞菌染色体头孢菌素酶关系最为密切，可能代表了温和气单胞菌常见的头孢菌素酶。AsbB1是一种D类酶，与苯唑西林水解酶OXA-1关系最为密切，氨基酸序列同一性为34%。纯化的AsbA1是一种典型的头孢菌素酶，其底物谱显示与苄青霉素相比，头孢菌素的水解速率较高。纯化的AsbB1显示出很强的青霉素酶活性，羧苄青霉素和氯唑西林的水解速率是苄青霉素的2至2.5倍。亚胺培南的水解率小于或等于苄青霉素的1%。克拉维酸和他唑巴坦均强烈抑制AsbB1，而舒巴坦对AsbB1酶的抑制效果较差。这些抑制剂对AsbA1酶均无良好效果。螯合剂乙二胺四乙酸（EDTA）和1,10-邻菲啰啉对两种酶的活性均无影响。发现温和气单胞菌AER 14M可产生一种1类头孢菌素酶和一种新型的2d类氯唑西林水解β-内酰胺酶，在此命名为OXA-12。
