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

Abstract

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

FIG. 1

Results of zymogram analysis performed after renaturing SDS-PAGE with the chromogenic cephalosporin nitrocefin as the substrate for detection of β-lactamase activity. Lanes: 1, crude extract from CCUG 4310, not induced; 2, crude extract from CCUG 4310 induced with ampicillin; 3, purified CME-1 enzyme; 4, crude extract from E. coli DH5α(pBlaA-4c); 5, crude extract from E. coli DH5α(pACYC184). The crude extracts were prepared as described in Table 1. Protein size standards are indicated in kilodaltons on the left.

FIG. 2

Restriction map of the DNA insert of plasmid pBlaA-4c and subcloning strategy. Thick lines represent insert sequences, while thin lines represent vector sequences. The location and orientation of the blaA_CME ORF is indicated. Crude extracts prepared from early-stationary-phase cultures of E. coli clones carrying each recombinant plasmid were assayed for production of β-lactamase activity (β-lact.) as described in the Materials and Methods section. Abbreviations: Ac, AccI; Av, AvaII; Av/Sm, AvaII-SmaI junction; B, BamHI; C, ClaI; C/N, ClaI-NspV junction; N, NspV; P, PstI; RI, EcoRI; S/B, Sau3AI-BamHI junction; Sa, SalI; Sm, SmaI; X, XhoI.

FIG. 3

Nucleotide sequence of the blaA_CME gene and flanking regions. Nucleotide 1 corresponds to the first base of the AccI restriction site located upstream of the gene. The deduced amino acid sequence of the CME-1 protein is reported below the nucleotide sequence. The underlined region corresponds to the experimentally determined signal peptide for secretion.

FIG. 4

Sequence alignment of the CME-1 protein (in boldface) with its closest class A neighbors. Identical residues are indicated by an asterisk; conservative substitutions are indicated by a colon. The enzyme names and corresponding sequence references are the same as those in Table 2. The conserved residues of the ABL consensus sequence (ABL cons.) (26) are reported above the alignment, and some relevant amino acid positions, according to the ABL numbering scheme (3), are also indicated. The Ω-loop region is indicated by a horizontal bar below the sequences.

FIG. 5

Unrooted tree showing the phyletic relationships among 19 different class A β-lactamases, including the CME-1 enzyme and its closest neighbors. Sequence names are the same as those in Table 2. Numbers at each branching point indicate the number/1,000 bootstrap trials returned for that point.

FIG. 6

SDS-PAGE analysis of the purification steps of the CME-1 protein. Lanes: A, clarified extract of E. coli BL21(DE3)(pBlaA-CNB); B, pooled fractions with β-lactamase activity eluted from the S-Sepharose column; C, pooled fractions with β-lactamase activity eluted from the Superdex-75 column. Protein size standards are indicated (in kilodaltons) on the right.