(A) Primers used for studying the environment of In60 (Table 1). (B) Schematic presentation of integron In60 carrying bla_CTX-M-9 (accession number AF174129). Open arrows, ORFs; dotted arrow, the unsequenced fragment of the sul1 gene; open box, the region that shows high similarity to orf3 of K. ascorbata; gray arrow, the previously reported sequence (13); •, 59-be; ○, shorter 59-be; diagonally striped box, the recombination site attI1; ♦, hypothetical recombination region; solid boxes, IR. Relevant nucleotide translational start and stop codons of genes and ORFs are numbered. (C) Comparison of the variable regions of different sul1-type complex integrons. The partial duplication of In6 (PD) is also shown (16). (D) Recombinant plasmids (containing fragments of In60) obtained in this work.

Comparison of the region between bla_CTX-M-9 and orf1005 of the In60 sequence and the region between the kluA-1 and orf3 genes of Kluyvera ascorbata (AJ272538). The relevant amino acid and translational stop codons (∗) are indicated. Dashes and dots indicate gaps and identical nucleotides, respectively. Nucleotide positions in In60 are shown. Regions sharing 78% identity between both sequences are in bold.

Comparison of regions to which the proposed DNA recombinase preferential site of In60 and of other sul1-type complex integrons and/or plasmids belong (18). The β-lactamase gene detected downstream of these regions is presented in brackets. Solid lines and dots indicate nucleotide identities, and the asterisk represents the point break where the nucleotide sequence changes. In6 contains a direct tandem duplication of the last 392 bases of the common region (16).

Sequence of the imperfect 83-bp IR (in bold and underlined) flanking orf1005. Imperfect direct duplications found at both ends of IS3000 are indicated in italics. The remnant sequence of the aadA2 59-be is boldface and boxed. The translational starting points of both orf1005 and qacEΔ1 are marked with asterisks, and the stop codon of orf1005 is overlined. Nucleotide positions in In60 are shown.