Cloning of a Chryseobacterium (Flavobacterium) meningosepticum chromosomal gene (blaACME) encoding an extended-spectrum class A β-lactamase related to the Bacteroides cephalosporinases and the VEB-1 and PER β-lactamases

In addition to the BlaB metallo-beta-lactamase, Chryseobacterium (Flavobacterium) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine P-lactamase, named CME-1, with an alkaline isoelectric pH. The blaA(CME), gene that encodes the latter enzyme was isolated from a genomic library constructed in the Escherichia coli plasmid vector pACYC184 by screening for cefuroxime-resistant clones. Sequence analysis revealed that the CME-1 enzyme is a new class A p-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-I) and PER beta-lactamases and the Bacteroides chromosomal cephalosporinases. The blaA(CME), determinant is located on the chromosome and exhibits features typical of those of C. meningosepticum resident genes. The CME-1 protein was purified from an E. coli strain that overexpresses the cloned gene via a T7-based expression system by means of an anion-exchange chromatography step followed by a gel permeation chromatography step. Kinetic parameters for several substrates were determined. CME-1 is a clavulanic acid-susceptible extended-spectrum p-lactamase that hydrolyzes most cephalosporins, penicillins, and monobactams but that does not hydrolyze cephamycins and carbapenems. The enzyme exhibits strikingly different kinetic parameters for different classes of p-lactams, with both ii, and k(cat),,, values much higher for cephalosporins than for penicillins and monobactams. However, the variability of both kinetic parameters resulted in overall similar acylation rates (k(cat)/K-m ratios) for all types of beta-lactam substrates.