Functional and structural characterization of the genetic environment of an extended-spectrum beta-lactamase blaVEB gene from a Pseudomonas aeruginosa isolate obtained in India

Abstract

A Pseudomonas aeruginosa clinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam. A bla(VEB-1)-like gene named bla(VEB-1a), which codes for the extended-spectrum beta-lactamase VEB-1a, was identified. The genetic environment of bla(VEB-1a) was peculiar: (i) no 5' conserved sequence (5'-CS) region was present upstream of the beta-lactamase gene, whereas bla(VEB-1)-like genes are usually associated with class 1 integrons; (ii) bla(VEB-1a) was inserted between two truncated 3'-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the bla(VEB-1a) gene. Expression of the bla(VEB-1a) gene was driven by a strong promoter located in one of these repeated sequences. In addition, cloning of the beta-lactamase content of this P. aeruginosa isolate followed by expression in Escherichia coli identified the naturally occurring AmpC beta-lactamase and a gene encoding an OXA-2-like beta-lactamase located in a class 1 integron, In78, in which an insertion sequence, ISpa7, was inserted within its 5'-CS region.

FIG. 1.

Nucleotide sequence of the bla_VEB-1a genetic environment. Arrows indicate the transcriptional orientation of the gene. The deduced amino acid sequences of the genes are designated in single-letter code below the nucleotide sequence. The ribosomal binding site (RBS) of bla_VEB-1a is in boldface, and the stop codons are shown by asterisks. The core and inverse core sites are indicated by boxes and dashed boxes, respectively. The Res are underlined, and the breakpoints are indicated by multiplication signs. Within Re1, the −10 and −35 promoter sequences of P_Re1 as well as the +1 transcriptional initiation site are shown by shaded boxes.

FIG. 2.

Schematic representation of the constructs used in the study and of the bla_VEB-1a and bla_OXA-2-a environments found in P. aeruginosa 10.2. (A) The breakpoints and the Res in the bla_VEB-1a environment are designated by broken lines and black triangles, respectively. (B) The ISpa7 inverted repeats are indicated by empty triangles; the terminal IRi and inverted repeat right (IRt) sequences are shown by black circles. The coding regions are shown as boxes, with arrows indicating the orientations of transcription and white circles indicating the 59-be. The restriction sites used for cloning are indicated. Boldface dashed lines indicate regions that were identified by PCR (the sequences of the primers used are available upon request). (C) Constructs pInt-Veb (2), pVeb (2), and pRe1-Veb (this study) were used for promoter strength and studies of the specific activities of the β-lactamases. The bla_VEB-1 genes were inserted in an orientation opposite that of P_lac, thus removing any contribution of this promoter to β-lactamase expression. Promoters P_lac, P_Re1, and P_ant are indicated by broken arrows. The −10 and −35 promoter sequences of P_Re1 as well as the +1 transcriptional initiation site are indicated by shaded boxes. The numbering is according to the 135-bp Re1 sequence. β-Lactamase specific activities (in units per milligram of protein) obtained with cefuroxime as the substrate are indicated to the right of the representation of each construct. Standard deviations calculated from five independent cultures are indicated in parentheses.

FIG. 3.

Comparison of the sequences of Res Re1, Re2, and Re3. Dashes indicate gaps introduced to optimize alignment, and the shaded boxes indicate different nucleotides. The promoter sequences of the P_Re1 promoter that was characterized in this study are underlined. The −10 and −35 promoter regions in the Re sequences that are identical or related to P_Re1 as well as the +1 transcriptional initiation site are boxed. Nucleotide numbering (nucleotides 1 to 135) is according to that of the Re1 sequence.