New insertion sequence elements in the upstream region of cfiA in imipenem-resistant Bacteroides fragilis strains

Abstract

The 747-bp cfiA gene, which encodes a metallo-beta-lactamase, and the regions flanking cfiA in six imipenem-resistant and four imipenem-susceptible Bacteroides fragilis strains isolated in Japan were analyzed by PCR and DNA sequencing. The nucleotide sequences of the cfiA genes (designated cfiA(1) to cfiA(10)) of all 10 strains tested varied from that of the standard cfiA gene from B. fragilis TAL2480. However, putative proteins encoded by the cfiA variants contained conserved amino acid residues important for zinc binding and hairpin loop formation, suggesting that cfiA variants have the capability of producing metallo-beta-lactamases with full catalytic activities. PCR assay indicated that six metallo-beta-lactamase-producing, imipenem-resistant strains had an insertion mutation in the region immediately upstream of cfiA. Nucleotide sequencing of the PCR-amplified fragments along with the upstream region of cfiA revealed that there were five new kinds of insertion sequence (IS) elements (designated IS612, IS613, IS614, IS615, and IS616, with a size range of 1,594 to 1,691 bp), of which only IS616 was found to be almost identical to IS1188, one of the IS elements previously identified in the upstream region of cfiA. These elements had target site duplications of 4 or 5 bp in length, terminal inverted repeats (14, 15, or 17 bp in size), and a large open reading frame encoding a putative transposase which is required for the transcription of IS elements. Each element was inserted such that the transcriptional direction of the transposase was opposite to that of cfiA. A computer-aided homology search revealed that, based on the homology of their putative transposases, the sizes of their terminal inverted repeat sequences, and their target site duplications, IS612, IS613, IS614, and IS615 belong to the IS4 family, which includes IS942, previously found in some drug-resistant B. fragilis strains, but that IS616 belongs to the IS1380 family. All the IS elements appear to have putative promoter motif sequences (the -7 region's TAnnTTTG motif and the -33 region's TTG or TG) in their end regions, suggesting that the IS elements provide a promoter for the transcription of cfiA upon insertion. These data provide additional proof that various IS elements may exist to provide a promoter to express the cfiA gene.

FIG. 1.

(A) Locations of six primers used for PCR and sequencing analyses of the cfiA gene; (B) deduced amino acid sequences of the products from the cfiA genes (cfiA₁ to cfiA₁₀) from 10 B. fragilis strains used in this study. These are compared with the sequence of the product of the standard cfiA gene in B. fragilis TAL 2480 (GenBank accession no. M34831) (32). Amino acid residues (His99, His101, Asp103, His162, Cys181, and His223) thought to be involved in the binding of the two Zn²⁺ ions are shown in boldface. Fully conserved amino acid residues are denoted by asterisks.

FIG. 2.

Features of new IS elements and related IS elements. (A) Nucleotide sequences of terminal regions of IS elements and flanking regions. The sequences of IS elements, including IS942 (25), IS1170 (32), IS1380 (29), IS1247 (33), and IS1412 (9), and flanking sequences are shown in uppercase and lowercase letters, respectively. IRL and IRR indicate imperfect terminal inverted repeat sequences at the left and right end regions of each IS element, respectively. IRL and IRR sequences, which are underlined, are located in the upstream and downstream regions of an open reading frame, respectively. The target site sequences possibly duplicated upon the transposition of each element are shown in boldface. (B) Locations of the target sites for the new IS elements IS1186 (21) and IS942 in the upstream region of cfiA. The target sites are shown by bars above or below the nucleotide sequence. (C) Putative promoter sequences in new IS elements. Sequences homologous to consensus sequences of B. fragilis promoters, TAnnTTTG in the −7 region and TnTG, TTG, or TG in the −33 region (2), are underlined. Putative ribosome-binding sites, cfiA start codons, and target site sequences are indicated in boldface, double underlining, and italics, respectively.