Cloning of a Chryseobacterium ( Flavobacterium ) meningosepticum Chromosomal Gene ( blaA CME ) Encoding an Extended-Spectrum Class A β-Lactamase Related to the Bacteroides Cephalosporinases and the VEB-1 and PER β-Lactamases

Abstract

ABSTRACT In addition to the BlaB metallo-β-lactamase, Chryseobacterium ( Flavobacterium ) meningosepticum CCUG 4310 (NCTC 10585) constitutively produces a 31-kDa active-site serine β-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 β-lactamase structurally divergent from the other members of this class, being most closely related to the VEB-1 (also named CEF-1) and PER β-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 β-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 β-lactams, with both K m 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 β-lactam substrates.