CARB-9, a carbenicillinase encoded in the VCR region of Vibrio cholerae non-O1, non-O139 belongs to a family of cassette-encoded beta-lactamases

Abstract

The gene bla(CARB-9) was located in the Vibrio cholerae super-integron, but in a different location relative to bla(CARB-7). CARB-9 (pI 5.2) conferred beta-lactam MICs four to eight times lower than those conferred by CARB-7, differing at Ambler's positions V97I, L124F, and T228K. Comparison of the genetic environments of all reported bla(CARB) genes indicated that the CARB enzymes constitute a family of cassette-encoded beta-lactamases.

FIG. 1.

Phylogenetic neighbor-joining tree of the bla_CARB genes. Relevant features of compared sequences are in Table 1. The RTG cluster of presumptive ancestors of the carbenicillinases (5, 16) is indicated. The tree was rooted with bla_RTG-1. Bootstrap percentages (based on 1,000 replicates) of 70% (or higher) of key nodes are shown.

FIG. 2.

Alignment of CARB-9 (288 amino acids) with the CARB enzymes. CARB-1 and CARB-2 were formerly named PSE-4 and PSE-1, respectively. Identical residues are indicated by dots. Amino acid motifs conserved in all penicillin-recognizing enzymes (12) are signaled by shaded boxes (I to VII). Residues highly conserved among class A β-lactamases are indicated by asterisks. The unique change, G144D, between CARB-7 and CARB-9 and the remainder of class A β-lactamases reported to date is signaled by the arrow; this change would be located far from the active site, as inferred from the crystallographic structure reported for CARB-1 (15). The three residues that discriminate between CARB-7 and CARB-9 are underlined. Amino acids that differentiate CARB-6 from CARB-1, -2, and -3 and which are identical to residues in the CARB-7 sequence are shown in boldface. Sequences are numbered as described by Ambler et al. Gaps (dashes) at positions 58, 239, and 253 are indicated (1).

FIG. 3.

Flanking regions of bla_CARB genes. Relevant features of compared sequences are in Table 1. Numbers on the left correspond to nucleotide positions in the GenBank reports. Gaps (dashes) were introduced in order to maximize the alignment (Clustal X). Codifying regions are signaled by shaded boxes, and the names of corresponding genes are indicated. The 5′ and/or 3′ ends not included in the alignment are represented by < and >, respectively. cmp, complementary sequence. The truncated codifying sequence (aadA?) at the end of N29 shared 97% identity with the integron-encoded gene aadA10 from the P. aeruginosa plasmid R388-R151 (GenBank accession no. U37105). Promoter (Pr) regions (−35 and −10) and the RBS reported previously are shown in boldface, and those identified in this work (the BPROM program, http://www.softberry.com/berry.phtml?topic=promoter) are additionally underlined. For clarity, VCRs and reported attC elements (underlined sequences) were defined from the third nucleotide upstream to the motif TAAC in the ICS, up to the G in the GTT of the CS. Consensus sequences for both ICS and CS are shown below the alignment. The identities among all available sequences (*) and positions highly conserved among Vibrio repeated sequences (VXRs; #) (24) are indicated. The regions of highest identity are boxed (boxes 1 to 5). Changes between the VCRs associated with bla_CARB-7 and bla_CARB-9 are signaled (▴). Horizontal arrows indicate the boundaries of the bla_CARB cassettes (5′ to 3′), and vertical arrows indicate the putative recombination points (between G and T in the GTT of the CS).