IS1999 increases expression of the extended-spectrum beta-lactamase VEB-1 in Pseudomonas aeruginosa

Abstract

The integron-borne bla(VEB-1) gene encodes an extended-spectrum beta-lactamase. This gene was associated mostly with IS1999 and rarely with an additional IS2000 element in Pseudomonas aeruginosa isolates from Thailand, whereas IS1999 was only very rarely associated with bla(VEB-1) in Enterobacteriaceae. Expression experiments and promoter study identified promoter sequences in IS1999 that increased the expression of VEB-1 in P. aeruginosa.

FIG.1.

Schematic map of the constructs used in this study. Constructs 1 (pDA-1, pInt-Veb, and pVeb), 2 (pDA-2, pInt-1999-Veb, pInt-1999*-Veb, p1999R-Veb, p1999-Veb, and p1999L-Veb), and 3 (pDA-3, pInt-1999-2000-Veb, and p1999R-2000R-Veb) were cloned from genomic DNAs of P. aeruginosa clinical isolates 14, 1, and JES, respectively. The bla_VEB-1 gene was inserted in opposite orientation to P_lac, thus removing any contribution of promoter P_lac in β-lactamase expression. The stop codon resulting from a site-directed mutagenesis experiment is shown by an asterisk (construct pInt-1999*-Veb). Restriction sites that were used at each cloning step are underlined. The coding regions are shown as boxes, with an arrow indicating the orientation of their transcription. The IR of IS1999 and IS2000 are shown by filled and empty triangles, respectively. IRL and IRR of IS1999 are indicated for pDA-2. The broken arrows indicate the promoter P_lac. Thin dashed lines indicate ligation in the multiple-cloning site of the shuttle vector pBBR1MCS.3.

FIG. 2.

Comparative study (using cefepime as the substrate) of the specific activity of β-lactamase VEB-1 from cultures of E. coli DH10B (left panel) and P. aeruginosa KG2505 (right panel) strains harboring recombinant plasmids. Error bars represent standard deviations calculated from five independent cultures. No measurable activity was detected for E. coli DH10B(pVEB) and for E. coli DH10B(p1999L-VEB).

FIG. 3.

(A) Structure of IS1999. The IS1999 IR are shown by filled triangles. The arrows indicate the orientation of transcription. The outward-directed promoter, P_out, and the promoter of the transposase gene, P_in, are indicated by broken arrows. The −10 and −35 regions for P_in and P_out are underlined. Nucleotide position 115 (according to the sequence GenBank AF133697) in IS1999 corresponds to the transcription start of P_out. The IRL sequence is boxed. (B) Mapping of transcription initiation. Primer Vebprom was extended using RNAs from cultures of E. coli DH10B(pInt-1999-Veb) (lane 1) or P. aeruginosa KG2505(pInt-1999-Veb) (lane 2) as the templates. Equal volumes (2 μl) of the extension product obtained from P. aeruginosa and E. coli were loaded onto the gel. Size markers were from sequencing reactions generated from pInt-1999-Veb DNA primed with Vebprom. G-, A-, T-, and C-specific lanes are indicated. The nucleotide sequences on the left side correspond to that of the complementary strand, which was deduced from the sequencing reaction. The −10 and −35 promoter sequences of P_out regions are shown, and the +1 transcriptional initiation site is indicated by an arrowhead. Similar results were obtained for RNA extracted from E. coli DH10B and P. aeruginosa KG2505 harboring pInt-1999-2000-Veb recombinant plasmid (data not shown).

FIG. 3.

(A) Structure of IS1999. The IS1999 IR are shown by filled triangles. The arrows indicate the orientation of transcription. The outward-directed promoter, P_out, and the promoter of the transposase gene, P_in, are indicated by broken arrows. The −10 and −35 regions for P_in and P_out are underlined. Nucleotide position 115 (according to the sequence GenBank AF133697) in IS1999 corresponds to the transcription start of P_out. The IRL sequence is boxed. (B) Mapping of transcription initiation. Primer Vebprom was extended using RNAs from cultures of E. coli DH10B(pInt-1999-Veb) (lane 1) or P. aeruginosa KG2505(pInt-1999-Veb) (lane 2) as the templates. Equal volumes (2 μl) of the extension product obtained from P. aeruginosa and E. coli were loaded onto the gel. Size markers were from sequencing reactions generated from pInt-1999-Veb DNA primed with Vebprom. G-, A-, T-, and C-specific lanes are indicated. The nucleotide sequences on the left side correspond to that of the complementary strand, which was deduced from the sequencing reaction. The −10 and −35 promoter sequences of P_out regions are shown, and the +1 transcriptional initiation site is indicated by an arrowhead. Similar results were obtained for RNA extracted from E. coli DH10B and P. aeruginosa KG2505 harboring pInt-1999-2000-Veb recombinant plasmid (data not shown).