Genetic and biochemical characterization of CGB-1, an Ambler class B carbapenem-hydrolyzing beta-lactamase from Chryseobacterium gleum

Abstract

Chryseobacterium gleum (previously included in the Flavobacterium IIb species) is a gram-negative aerobe that is a source of nosocomial infections. An Ambler class B beta-lactamase gene was cloned and expressed in Escherichia coli from reference strain C. gleum CIP 103039 that had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. The purified beta-lactamase, CGB-1, with a pI value of 8.6 and a determined relative molecular mass of ca. 26 kDa, hydrolyzed penicillins; narrow- and expanded-spectrum cephalosporins; and carbapenems. CGB-1 was a novel member of the molecular subclass B1 of metallo-enzymes. It had 83 and 42% amino acid identity with IND-1 from Chryseobacterium indologenes and BlaB from C. meningosepticum, respectively. Thus, in addition to the previously characterized clavulanic acid-inhibited extended-spectrum beta-lactamase CGA-1 of Ambler class A, C. gleum produces a very likely chromosome-borne class B beta-lactamase.

FIG. 1.

Nucleotide sequence of the cloned 2,004-bp fragment of recombinant plasmid pCGB-1 containing the bla_CGB-1 coding region and a truncated ORF encoding a putative capreomycin resistance determinant. The deduced amino acid sequence is designated in the single-letter code below the nucleotide sequence. The putative terminator sequence is indicated by two inverted arrows, and stop codons are indicated by stars.

FIG. 2.

Comparison of the amino acid sequence of β-lactamase CGB-1 with those of IND-1, IND-2, IND-3, and IND-4 from C. indologenes (4) and BlaB from C. meningosepticum (23). The BBL numbering scheme is indicated above the sequences (8). Broken lines and dots indicated identical and deleted amino acid residues, respectively. The vertical arrow indicates the putative cleavage site for the leader peptide of CGB-1. Amino acids that may be involved in binding of Zn²⁺ or/and water are indicated by a star.