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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

1999; American Society for Microbiology; Volume: 43; Issue: 9 Linguagem: Inglês

10.1128/aac.43.9.2193

1098-6596

Gian María Rossolini, Nicola Franceschini, Laura Lauretti, Berardo Caravelli, Maria Letizia Riccio, Moreno Galleni, Jean‐Marie Frère, Gianfranco Amicosante,

Bacterial biofilms and quorum sensing

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.