CAU-1, a subclass B3 metallo-beta-lactamase of low substrate affinity encoded by an ortholog present in the Caulobacter crescentus chromosome

Abstract

The sequenced chromosome of Caulobacter crescentus CB15 encodes a hypothetical protein that exhibits significant similarity (30 to 35% identical residues) to metallo-beta-lactamases of subclass B3. An allelic variant of this gene (divergent by 3% of its nucleotides) was cloned in Escherichia coli from C. crescentus type strain DSM4727. Expression studies confirmed the metallo-beta-lactamase activity of its product, CAU-1. The enzyme produced in E. coli was purified by two ion-exchange chromatography steps. CAU-1 contains a 29-kDa polypeptide with an alkaline isoelectric pH (> 9), and unlike the L1 enzyme of Stenotrophomonas maltophilia, the native form is monomeric. Kinetic analysis revealed a preferential activity toward penicillins, carbapenems, and narrow-spectrum cephalosporins, while oxyimino cephalosporins were poorly or not hydrolyzed. Affinities for the various beta-lactams were poor overall (K(m) values were always > 100 microM and often > 400 microM). The interaction with divalent ion chelators appeared to occur by a mechanism similar to that prevailing in other members of subclass B3. In C. crescentus, the CAU-1 enzyme is produced independently of beta-lactam exposure and, interestingly, the bla(CAU) determinant is bracketed by three other genes, including two genes encoding enzymes involved in methionine biosynthesis and a gene encoding a putative transcriptional regulator, in an operon-like structure. The CAU-1 enzyme is the first example of a metallo-beta-lactamase in a member of the alpha subdivision of the class Proteobacteria:

FIG. 1.

Genetic organization of the C. crescentus CB15 chromosomal region containing ORF CC2139, corresponding to the bla_CAU gene. ORFs are indicated by arrows. The position and orientation of the primers used for PCR cloning and mapping procedures as described in Materials and Methods are indicated above and below the map. The amplicons obtained from the genome of C. crescentus DSM4727^T with primers CAU-OP/F and CAU-MET/R or primers BLA-CAU/F and CAU-OP/R are indicated by thick lines below the map, and the restriction sites used to confirm their identities are also shown. The 2.2-kb amplicon obtained with primers CAU-OP/F and CAU-MET/R corresponds to the insert of plasmid pFG2002 (thin lines represent vector sequences). Plasmid pFG2003, used for expression experiments, was generated from pFG2002 following partial removal of the CC2138 ORF as described in Materials and Methods.

FIG. 2.

Sequence alignment of subclass B3 metallo-β-lactamases. Positions of zinc ligand residues are numbered in accordance with the BBL scheme (15). Secondary structure elements (S, strands; 3₁₀ and H, helices; L, loops) of the L1 enzyme (36) are indicated above the sequences. References for the sequences are as follows: L1, reference ; FEZ-1, reference ; GOB-1, reference ; THIN-B, reference . The four amino acid residues in the protein encoded by ORF CC2139 of C. crescentus CB15 that differ from CAU-1 are indicated below the sequence.

FIG. 3.

SDS-PAGE analysis of the purified CAU-1 protein (10 μg, lane 3), of the DEAE-Sepharose eluate (lane 2), and of the crude extract of DH5α(pFG2003) (lane 1). Protein molecular size standards (lane 4) are reported in kilodaltons on the right.

FIG. 4.

Distribution of resident metallo-β-lactamases in bacterial lineages. Only phyla in which such enzymes have been detected are shown, along with the corresponding species. Bacterial classification is according to Bergey's Manual of Systematic Bacteriology (16).