Preclinical Class 1 Integron with a Complete Tn402-Like Transposition Module

Abstract

The presence of integrons was assessed in gut bacteria isolated from wild-caught prawns. A pseudomonad was recovered that contained a Tn402-like class 1 integron with a complete transposition module and two gene cassettes. One cassette was identical to a previously described cassette from a chromosomal class 3 integron in Delftia tsuruhatensis.

Integrons are genetic elements that contribute to lateral gene transfer. They possess a site-specific recombination system that captures and expresses genes that are found as part of mobile gene cassettes (6, 7). Integrons are comprised of an integrase gene (intI) whose product catalyzes the insertion of gene cassettes into a recombination site, attI (6, 18) (Fig. 1). Integron-associated gene cassettes exhibit great sequence diversity (1, 12, 19), but the best-known cassettes are those that contain antibiotic resistance genes.

FIG. 1.

Model for the origin of Tn402 class 1 integrons. (A and B) A class 1 integron resident in the chromosome of an environmental betaproteobacterium is captured by a Tn402 transposon (A) to generate a hybrid structure combining the ability of integrons to sample the environmental gene cassette pool with the mobility of the Tn402 transposon (B). (C) The integron described in this article, where the hybrid structure has targeted a resolvase gene resident in a Pseudomonas species and the integron has acquired environmental gene cassettes, including one known to also be present in chromosomal class 3 integrons of Delftia (21). (D) One possible form of the ancestor of the class 1 integrons found in human pathogens and commensals. It contains an antibiotic resistance gene (Ab^R), a qacE gene cassette conferring resistance to quaternary ammonium compounds, and a complete tni transposition module. For further details, see references 20 and 3.

The class 1 integrons are of great clinical importance, being responsible for the ongoing accumulation of antibiotic resistance genes (14, 15, 17, 18). There were at least two important steps leading to the generation of the basic class 1 integron backbone that now dominates clinical isolates in the antibiotic era (3, 20). The first of these was the insertion of the integron into a Tn402-like transposon (Fig. 1A). The second step was the generation of the 3′-conserved segment (3′-CS), involving acquisition of sulI and loss of part of the tni module (2, 18).

We are interested in the evolutionary history of these events and have shown that class 1 integrons that predate capture by Tn402-like transposons are common in the chromosomes of the Betaproteobacteria (3). The initial capture of a chromosomal class 1 integron should result in a structure that contains a complete Tn402-like transposition module but contains no antimicrobial resistance cassettes, other than perhaps qacE (5). While some class 1 integrons with complete transposition machinery have been observed in clinical contexts, they possess antibiotic resistance genes that were probably acquired some time after the initial Tn402 capture event (10, 16).

The aim of this study was to survey natural environments for bacteria containing examples of the early transposon capture event and to assess their potential for transmitting integrons and novel gene cassettes into human pathogens. We were also interested in the potential exchange of gene cassettes among different classes of integrons. Here we describe a novel Tn402-like class 1 integron in a Pseudomonas strain recovered from a prawn, and we show that it carries unusual gene cassettes, including one previously found in a chromosomal class 3 integron.

Uncooked, wild-caught prawns (Eastern King Prawn, Penaeus plebejus) were collected from retail outlets in Sydney, Australia. Bacteria were cultured from individual digestive tracts of these prawns, and the resulting mixed cultures were screened for the presence of class 1, 2, and 3 integrons (4). Cultures from most prawns tested positive for class 1 integrons, but none was positive for class 2 or 3 integrons. Two hundred single colonies isolated from one mixed culture were screened for the presence of class 1 integrons. Five intI1-positive isolates were detected, and cassette arrays were recovered from each isolate using PCR with the primers MRG284 and MRG285 (4). Sequencing of cassette array PCR amplicons revealed an unusual structure for one of these isolates, designated 11BF10. Molecular typing of this strain was carried out using 16S rRNA gene sequencing, which identified it as a species of Pseudomonas.

To further characterize the Pseudomonas strain, a fosmid library was constructed (20). Five hundred fosmid clones were screened for intI1, resulting in three positives, which were purified and DNA sequenced by primer walking. A physical map of the class 1 integron and flanking region was then constructed (Fig. 2). The class 1 integron possessed features typical of a Tn402-like class 1 integron, carrying the complete tni transposition module and having 100% nucleotide homology to the transposition genes tniR, tniQ, tniB, and tniA from Tn402. The boundaries of the integron were defined by the inverted repeats IRi and IRt, identical to those in Tn402 (8, 14, 20). We concluded that this integron represented a Tn402-like class 1 integron before the formation of the 3′-CS and before the loss of transposition activity (2, 18). The left-hand boundary of the Tn402-like integron was inserted into a partial resolvase gene, consistent with the res-hunting activity of this transposon (13). The other portion of the resolvase gene was not present at the right-hand boundary beyond IRt, suggesting further rearrangements after insertion, including deletions around the insertion point. One further feature of note was the presence of a toxin/antitoxin gene pair. Such genes are a common feature of chromosomal cassette arrays (1, 11), but in this case they lie outside the integron.

FIG. 2.

Schematic diagram of 16.6 kb of the class 1 integron and flanking regions in Pseudomonas 11BF10. Landmarks from left to right include the following: five genes for conserved hypothetical proteins; a partially deleted integrase gene (Δint); a partially deleted resolvase gene (Δres); a 25-bp inverted repeat (IRi) that marks the left boundary of the Tn402-like integron; the integron integrase gene (intI1) and its attendant recombination site (attI1); two gene cassettes, one previously found in Delftia (orfB [21]) and a novel cassette (orf132), separated by their respective recombination sites (attC); a complete tni transposition module composed of tniR, tniQ, tniB, and tniA; a 25-bp inverted repeat (IRt) that marks the right boundary of the integron; a toxin-antitoxin gene pair; and a set of three conserved hypothetical genes, including a TnpA repressor and two genes whose nearest homologues were found in Delftia acidivorans.

The class 1 integron in 11BF10 contained two gene cassettes, both with identifiable open reading frames and attendant attC sites. The first gene cassette exhibited 100% nucleotide identity over its entire length (756 bp) to a gene cassette (GC2, containing orfB) previously found in a chromosomal class 3 integron from Delftia tsuruhatensis A90 (21). The second cassette contained an open reading frame for which no homology could be found in existing databases. Gene cassettes for known antibiotic resistance determinants were not present, and neither was qacE. In previous work, we presented evidence that the ancestor of the class 1 integrons currently circulating in human pathogens arose when a chromosomal class 1 integron was captured by a Tn402-like transposon (3, 20). The integron described here appears to be a descendant of that original event but one that has either lost or never acquired a qacE cassette (5) and has been circulating in the general environment since that time. The presence of a cassette that is identical to one previously found in an environmental class 3 integron (21) demonstrates a conduit of gene transfer between class 1 and class 3 integrons in the environment. Class 1 and class 3 integron-integrases exhibit only 61% amino acid identity, but this observation suggests that both recombinases recognize the attC site of this cassette.

There are several points to be made from these data. The first is that this integron represents an example of a key intermediate in the evolution of the clinical class 1 integron, which was predicted but not observed until now (3). The second point is that such class 1 integrons are still circulating in the environment, where they are free to acquire gene cassettes from the enormous and diverse pool of these elements known to exist (9, 12). Third, this integron, and potentially others like it, has active transposition machinery, enabling it to autonomously target the res sites of diverse plasmids and potentially the Tn21-like transposons, thus assuming greater mobility. Finally, it was found in food that is consumed after only light cooking, giving it a ready pathway into human commensal flora, along with any novel gene cassettes it might carry.

Nucleotide sequence accession numbers.

The 16S rRNA genes and fosmid sequences described in this article have been submitted to GenBank and have the accession numbers HQ423157 and HQ423158, respectively. The Tn402-like integron has been assigned transposon number Tn6112 (http://www.ucl.ac.uk/eastman/tn/).