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Most surveys for class 1 integrons are at least partly predicated on PCR screening that targets integron conserved regions. However, class 1 integrons are structurally diverse, so dependence on conserved regions may lead to missing clinically relevant examples of class 1 integrons. Here, we surveyed a commensal population of bacteria from patients in an intensive care unit to identify class 1 integrons irrespective of their structure or genetic context. We identified several examples of class 1 integrons linked to complete Tn 402 -like or Tn 402 hybrid transposition modules and diverse insertion points with respect to the inverted repeat IRi boundary. The diversity and abundance of class 1 integrons identified are such that many novel elements seen here would not have been identified by commonly used methods, and they revealed an additional level of complexity.

Integron cassette arrays in a dozen cultivars of the most prevalent group of Vibrio isolates obtained from mucus expelled by a scleractinian coral (Pocillopora damicornis) colony living on the Great Barrier Reef were sequenced and compared. Although all cultivars showed >99% identity across recA, pyrH and rpoB genes, no two had more than 10% of their integron-associated gene cassettes in common, and some individuals shared cassettes exclusively with distantly-related members of the genus. Of cassettes shared within the population, a number appear to have been transferred between Vibrio isolates, as assessed by phylogenetic analysis. Prominent among the mucus Vibrio cassettes with potentially inferable functions are acetyltransferases, some with close similarity to known antibiotic-resistance determinants. A subset of these potential resistance cassettes were shared exclusively between the mucus Vibrio cultivars, Vibrio coral pathogens and human pathogens, thus illustrating a direct link between these microbial niches through exchange of integron-associated gene cassettes.

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds., National Health and Medical Research Council (Australia) (NHMRC grant 488502), National Institutes of Health (U.S.) (Grant GM62414-0 ), Ontario. Ministry of Revenue (Challenge Fund)

The integron/gene cassette system contributes to lateral gene transfer of genetic information in bacterial communities, with gene cassette-encoded proteins potentially playing an important role in adaptation to stress. Class 1 integrons are a particularly important class as they themselves seem to be broadly disseminated among the Proteobacteria and have an established role in the spread of antibiotic resistance genes. The abundance and structure of class 1 integrons in freshwater sediment bacterial communities was assessed through sampling of 30 spatially distinct sites encompassing different substrate and catchment types from the Greater Melbourne Area of Victoria, Australia. Real-time PCR was used to demonstrate that the abundance of intI1 was increased as a result of ecosystem perturbation, indicated by classification of sample locations based on the catchment type and a strong positive correlation with the first principal component factor score, comprised primarily of the heavy metals zinc, mercury, lead and copper. Additionally, the abundance of intI1 at sites located downstream from treated sewage outputs was associated with the percentage contribution of the discharge to the basal flow rate. Characterization of class 1 integrons in bacteria cultured from selected sediment samples identified an association with complete Tn402-like transposition modules, and the potential for coselection of heavy-metal and antibiotic resistance mechanisms in benthic environments. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.

In a survey of class 1 integrons from human stools, an unusual class 1 integron from a strain of Enterobacter cloacae was isolated and characterized in detail. Sequence analysis of a fosmid containing the class 1 integron revealed a complex set of transposons which included two Tn 402 -like transposons. One of these transposons, Tn 6007 , included a class 1 integron with two non-antibiotic-resistance-type gene cassettes and a complete transposition module. This tni module is a hybrid with a boundary within the res site compared to Tn 402 , implying that a site-specific recombination event generated either Tn 6007 or Tn 402 . The second Tn 402 -like transposon, Tn 6008 , possesses neither a mer operon nor an integron, and most of its tni module has been deleted. Tn 6007 , Tn 6008 , and the 2,478 bases between them, collectively designated Tn 6006 , have transposed into a Tn 5036 /Tn 3926 -like transposon as a single unit. Tn 6006 , Tn 6007 , and Tn 6008 could all transpose as discrete entities. Database analysis also revealed that a version of Tn 6008 was present in the genome of Xanthomonas campestris pv. vesicatoria. Overall, the E. cloacae isolate further demonstrated that functional class 1 integrons/transposons are probably common in bacterial communities and have the potential to add substantially to the problem of multidrug-resistant nosocomial infections.

The class 1 integron In28, found in the multidrug resistance transposon Tn 1403 , was found to be located in the res site of the backbone transposon and is flanked by a 5-bp direct duplication, indicating that it reached this position by transposition. In28 has a backbone structure related to that of In4, but has lost internal sequences, including the sul1 gene, due to an IS 6100 -mediated deletion. In28 also lacks the partial copy of IS 6100 found in In4 and contains different gene cassettes, blaP1, cmlA1, and aadA1 . In1, the class 1 integron found in the multidrug resistance plasmid R46, is also located in a putative res site and belongs to the In4 group. In1 has a shorter internal deletion than In28 and has also lost one end. Additional integrons with structures related to In4 were also found in databases, and most of them had also lost either one end or internal regions or both. Tn 610 belongs to this group.

The class 1 integron integrase, IntI1, recognizes two distinct types of recombination sites, attI sites, found in integrons, and members of the 59-be family, found in gene cassettes. The efficiencies of the integrative version of the three possible reactions, i.e., between two 59-be, between attI1 and a 59-be, or between two attI1 sites, were compared. Recombination events involving two attI1 sites were significantly less efficient than the reactions in which a 59-be participated, and the attI1 × 59-be reaction was generally preferred over the 59-be × 59-be reaction. Recombination of attI1 with secondary sites was less efficient than the 59-be × secondary site reaction.

The multidrug resistance plasmid pBWH301 was shown to contain a sull-associated integron with five inserted gene cassettes, aacA7-catB3-aadB-oxa2-orfD, all of which can be mobilized by the integron-encoded DNA integrase. The aadB, oxa2, and orfD cassettes are identical to known cassettes. The aacA7 gene encodes a protein that is a member of one of the three known families of aminoglycoside acetyltransferases classified as AAC(6')-I. The chloramphenicol acetyltransferase encoded by the catB3 gene is closely related to members of a recently identified family of chloramphenicol acetyltransferases. The catB3 gene displays a relatively high degree of sequence identity to a chromosomally located open reading frame in Pseudomonas aeruginosa, and this may represent evidence for the acquisition by a cassette of a chromosomal gene.

Integrons are genetic elements which are capable of acquiring genes by site-specific recombination. The most common integron structure consists of two conserved segments flanking a variable region where many different antibiotic resistance genes have been found. The integrons In6 and In7, present in the plasmids pSa and pDGO100, respectively, are unusual in that they include a duplication of the sulI gene which is located within the integron 3′-conserved segment. To further investigate the structure of these integrons, the DNA sequence of the segment located between the two sulI genes was determined. In In7 this segment is 2822 bases long and includes a trimethoprim resistance gene, dhfrX , at one end. The corresponding region in In6 is 4.5 kb and is nearly identical to the In7 segment over the first 2105 bases. In the region unique to In6, a cat gene, conferring chloramphenicol resistance, has replaced the dhfrX gene of In7. This location thus represents a second variable region where different antibiotic resistance genes are found, but the way in which genes become associated with this second variable region is not known. The overall similarity of the structures of In6 and In7 suggests that the additional DNA segments found in these integrons have a common origin, and a possible mechanism for the origin of integrons with partial 3′-conserved segment duplications is presented.

A new trimethoprim resistance determinant, designated dhfrX, was identified in the In7 integron of pDGO100. The sequence of the dhfrX dihydrofolate reductase is up to 28% identical to the sequences of several known dihydrofolate reductase proteins. The dhfrX gene is adjacent to the second 3'-conserved segment of the In7 integron, but the first 77 bases of this segment are not present.