Integron-associated gene cassettes in Halifax Harbour: assessment of a mobile gene pool in marine sediments

The integron/gene cassette systems identified in bacteria comprise a class of genetic elements that allow adaptation by acquisition of gene cassettes. Integron gene cassettes have been shown to facilitate the spread of drug resistance in human pathogens but their role outside a clinical setting has not been explored extensively. We sequenced 2145 integron gene cassettes from four marine sediment samples taken from the vicinity of Halifax Nova Scotia, Canada, increasing the number of gene cassettes obtained from environmental microbial communities by 10-fold. Sequence analyses reveals that the majority of these cassettes encode novel proteins and that this study is consistent with previous claims of high cassette diversity as we estimate a Chao1 diversity index of ∼3000 cassettes from these samples. The functional distribution of environmental cassettes recovered in this study, when compared with that of cassettes from the only other source with significant sampling ( Vibrio genomes) suggests that alternate selection regimes might be acting on these two gene pools. The majority of cassettes recovered in this study encode novel, unknown proteins. In instances where we obtained multiple alleles of a novel protein we demonstrate that non-synonymous versus synonymous substitution rates ratios suggest relaxed selection. Cassette-encoded proteins with known homologues represent a variety of functions and prevalent among these are isochorismatases; proteins involved in iron scavenging. Phylogenetic analysis of these isochorismatases as well as of cassette-encoded acetyltransferases reveals a patchy distribution, suggesting multiple sources for the origin of these cassettes. Finally, the two most environmentally similar sample sites considered in this study display the greatest overlap of cassette types, consistent with the hypothesis that cassette genes encode adaptive proteins.     

Marine integrons containing novel integrase genes,attachment sites,attI, and associated gene cassettes in polluted sediments from Suez and Tokyo Bays

In order to understand the structure and biological significance of integrons and associated gene cassettes in marine polluted sediments, metagenomic DNAs were extracted from sites at Suez and Tokyo Bays. PCR amplicons containing new integrase genes, intI, linked with novel gene cassettes, were recovered and had sizes from 1.8 to 2.5 kb. This approach uncovered, for the first time, the structure and diversity of both marine integron attachment site, attI, and the first gene cassette, the most efficiently expressed integron-associated gene cassette. The recovered 13 and 20 intI phylotypes, from Suez and Tokyo Bay samples, respectively, showed a highly divergence, suggesting a difference in integron composition between the sampling sites. Some intI phylotypes showed similarity with that from Geobacter metallireducens, belonging to Deltaproteobacteria, the dominant class in both sampling sites, as determined by 16S rRNA gene analysis. Thirty distinct families of putative attI site, as determined by the presence of an attI-like simple site, were recovered. A total of 146 and 68 gene cassettes represented Suez and Tokyo Bay unsaturated cassette pools, respectively. Gene cassettes, including a first cassette, from both sampling sites encoded two novel families of glyoxalase/bleomycin antibiotic-resistance protein. Gene cassettes from Suez Bay encoded proteins similar to haloacid dehalogenases, protein disulfide isomerases and death-on-curing and plasmid maintenance system killer proteins. First gene cassettes from Tokyo Bay encoded a xenobiotic-degrading protein, cardiolipin synthetase, esterase and WD40-like β propeller protein. Many of the first gene cassettes encoded proteins with no ascribable function but some of them were duplicated and possessed signal functional sites, suggesting efficient adaptive functions to their bacterial sources. Thus, each sampling site had a specific profile of integrons and cassette types consistent with the hypothesis that the environment shapes the genome.     

The gene cassette metagenome is a basic resource for bacterial genome evolution

Lateral gene transfer has been proposed as a fundamental process underlying bacterial diversity. Transposons, plasmids and phage are widespread and have been shown to significantly contribute to lateral gene transfer. However, the processes by which disparate genes are assembled and integrated into the host regulatory network to yield new phenotypes are poorly known. Recent discoveries about the integron/gene cassette system indicate it has the potential to play a role in this process. Gene cassettes are small mobile elements typically consisting of a promoterless orf and a recombination site. Integrons are capable of acquisition and re-arrangement of gene cassettes and of the expression of their associated genes. The potential of the integron/gene cassette system is thus largely determined by the diversity contained within the cassette pool and the rate at which integrons sample this pool. We show here using a polymerase chain reaction (PCR) approach by which the environmental gene cassette (EGC) metagenome can be directly sampled that this metagenome contains both protein-coding and non-protein coding genes. Environmental gene cassette-associated recombination sites showed greater diversity than previously seen in integron arrays. Class 1 integrons were shown to be capable of accessing this gene pool through tests of recombinational activity with a representative range of EGCs. We propose that gene cassettes represent a vast, prepackaged genetic resource that could be thought of as a metagenomic template for bacterial evolution.     

New enzymes from environmental cassette arrays: functional attributes of a phosphotransferase and an RNA-methyltransferase

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7") from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg(2+)-binding residues. Unlike related APH(4) or APH(7") enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.     

High diversity and rapid spatial turnover of integron gene cassettes in soil

Integrons are genetic elements that promote rapid adaptation in bacteria by capturing exogenous, mobile gene cassettes. Recently, a subset of gene cassettes has facilitated the global spread of antibiotic resistance. However, outside clinical settings, very little is known about their diversity and spatial ecology. To address this question, we sequenced integron gene cassettes from soils sampled across Australia and Antarctica. We recovered 44 970 open reading frames that encoded 27 215 unique proteins, representing an order of magnitude more cassettes than previous sequencing efforts. We found that cassettes have extremely high local richness, significantly greater than previously predicted, with estimates ranging from 4000 to 18 000 unique cassettes per 0.3 g of soil. We show that cassettes have a heterogeneous distribution across space, and that they exhibit rapid turnover with distance. Similarity between samples drops to between 0.1% and 10% at distances of as little as 100 m. Together, these data provide key insights into the ecology and size of the gene cassette metagenome.     

Mobile gene cassettes: a fundamental resource for bacterial evolution

Horizontal gene transfer increases genetic diversity in prokaryotes to a degree not allowed by the limitations of reproduction by binary fission. The integron/gene cassette system is one of the most recently characterized examples of a system that facilitates horizontal gene transfer. This system, discovered in the context of multidrug resistance, is recognized in a clinical context for its role in allowing pathogens to adapt to the widespread use of antibiotics. Recent studies suggest that gene cassettes are common and encode functions relevant to many adaptive traits. To estimate the diversity of mobile cassettes in a natural environment, a molecular technique was developed to provide representative distributions of cassette populations at points within a sampling area. Subsequently, statistical methods analogous to those used for calculating species diversity were employed to assess the diversity of gene cassettes within the sample area in addition to gaining an estimate of cassette pool size. Results indicated that the number of cassettes within a 5x10-m sample area was large and that the overall mobile cassette metagenome was likely to be orders of magnitude larger again. Accordingly, gene cassettes appear to be capable of mobilizing a significant genetic resource and consequently have a substantial impact on bacterial adaptability.     

IntI2 integron integrase in Tn7

Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination. Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21. A second class of integron is found on transposon Tn7 and its relatives. We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon. This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes. The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1. In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid. The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons. We also observed a fourth excisable cassette downstream of those described previously in Tn7. The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids. IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies. The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7. However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E.     

Integrons and gene cassettes: hotspots of diversity in bacterial genomes

Integrons are genetic units found in many bacterial species that are defined by their ability to capture small mobile elements called gene cassettes. Cassettes usually contain only one gene, potentially any gene, and an attC recombination site, and thousands of cassettes have been sequenced. A specialized IntI site-specific recombinase encoded by the integron recognizes attC and incorporates cassettes into an attI site located adjacent to the intI gene. Over 100 types of integrons have been found, most in bacterial chromosomes. They can all potentially share the same cassettes and, as recombination between attC in a cassette and an attI can occur repeatedly, an integron can contain from zero to hundreds of cassettes. Cassette arrays that are not located next to an intI gene, or solo cassettes at apparently random sites, are also seen. Hence, integrons contribute to generation of diversity in bacterial, plasmid, and transposon genomes and facilitate extensive sharing of information among bacteria.     

Novel and diverse integron integrase genes and integron-like gene cassettes are prevalent in deep-sea hydrothermal vents

The lack of information about mobile DNA in deep-sea hydrothermal vents limits our understanding of the phylogenetic diversity of the mobile genome of bacteria in these environments. We used culture-independent techniques to explore the diversity of the integron/mobile gene cassette system in a variety of hydrothermal vent communities. Three samples, which included two different hydrothermal vent fluids and a mussel species that contained essentially monophyletic sulfur-oxidizing bacterial endosymbionts, were collected from Suiyo Seamount, Izu-Bonin, Japan, and Pika site, Mariana arc. First, using degenerate polymerase chain reaction (PCR) primers, we amplified integron integrase genes from metagenomic DNA from each sample. From vent fluids, we discovered 74 new integrase genes that were classified into 11 previously undescribed integron classes. One integrase gene was recorded in the mussel symbiont and was phylogenetically distant from those recovered from vent fluids. Second, using PCR primers targeting the gene cassette recombination site (59-be), we amplified and subsequently identified 60 diverse gene cassettes. In multicassette amplicons, a total of 13 59-be sites were identified. Most of these sites displayed features that were atypical of the features previously well conserved in this family. The Suiyo vent fluid was characterized by gene cassette open reading frames (ORFs) that had significant homologies with transferases, DNA-binding proteins and metal transporter proteins, while the majority of Pika vent fluid gene cassettes contained novel ORFs with no identifiable homologues in databases. The symbiont gene cassette ORFs were found to be matched with DNA repair proteins, methionine aminopeptidase, aminopeptidase N, O-sialoglycoprotein endopeptidase and glutamate synthase, which are proteins expected to play a role in animal/symbiont metabolism. The success of this study indicates that the integron/gene cassette system is common in deep-sea hydrothermal vents, an environment type well removed from anthropogenic disturbance.     

Quantitative and qualitative impact of hospital effluent on dissemination of the integron pool

There is increasing evidence that human activity, and especially the resulting effluent, has a major role in the dissemination of bacterial antibiotic-resistance determinants in the environment. Hospitals are the major antibiotic consumers and thus facilitate the spread of antibiotic resistance. Questions are increasingly being raised about the management of hospital effluents, but their involvement in antibiotic-resistance dissemination has never been assessed. Integrons are a paradigm of genetic transfer between the environmental resistome and both commensal and pathogenic bacteria. In order to assess the impact of hospital activities on antibiotic-resistance dissemination in the environment, we monitored integrons and their gene cassettes in hospital effluents, and their release in the environment. We found that bacterial communities present in a hospital effluent contained a high proportion of integrons. In terms of both their gene cassette diversity and gene cassette arrays, the urban effluent and municipal wastewater treatment plant (WWTP) influent were most similar, whereas the hospital effluent and recirculation sludge exhibited very specific patterns. We found that anthropogenic activities led to the release of abundant integrons and antibiotic-resistance gene cassettes, but we observed no specific impact of hospital activities on the receiving environment. Furthermore, although the WWTP did not reduce the normalized integron copy number, it reduced the diversity of gene cassette arrays contained in the raw wastewater, underlining the effect of the biological treatment on the anthropogenic integron pool arriving at the WWTP.     

Deletion of Integron-Associated Gene Cassettes Impact on the Surface Properties of Vibrio rotiferianus DAT722

Background

The integron is a genetic recombination system that catalyses the acquisition of genes on mobilisable elements called gene cassettes. In Vibrio species, multiple acquired gene cassettes form a cassette array that can comprise 1–3% of the bacterial genome. Since 75% of these gene cassettes contain genes encoding proteins of uncharacterised function, how the integron has driven adaptation and evolution in Vibrio species remains largely unknown. A feature of cassette arrays is the presence of large indels. Using Vibrio rotiferianus DAT722 as a model organism, the aim of this study was to determine how large cassette deletions affect vibrio physiology with a view to improving understanding into how cassette arrays influence bacterial host adaptation and evolution.

Methodology/Principal Findings

Biological assays and proteomic techniques were utilised to determine how artificially engineered deletions in the cassette array of V. rotiferianus DAT722 affected cell physiology. Multiple phenotypes were identified including changes to growth and expression of outer membrane porins/proteins and metabolic proteins. Furthermore, the deletions altered cell surface polysaccharide with Proton Nuclear Magnetic Resonance on whole cell polysaccharide identifying changes in the carbohydrate ring proton region indicating that gene cassette products may decorate host cell polysaccharide via the addition or removal of functional groups.

Conclusions/Significance

From this study, it was concluded that deletion of gene cassettes had a subtle effect on bacterial metabolism but altered host surface polysaccharide. Deletion (and most likely rearrangement and acquisition) of gene cassettes may provide the bacterium with a mechanism to alter its surface properties, thus impacting on phenotypes such as biofilm formation. Biofilm formation was shown to be altered in one of the deletion mutants used in this study. Reworking surface properties may provide an advantage to the bacterium’s interactions with organisms such as bacteriophage, protozoan grazers or crustaceans.     

Genetic diversity analyses of class 1 integrons and their associated antimicrobial resistance genes in Enterobacteriaceae strains recovered from aquatic habitats in China

Aims: To characterize the molecular diversity of class 1 integrons and antibiotic resistance (AR) genes of Enterobacteriaceae strains recovered from aquatic habitats in Jinan, Shandong Province, China. Methods and Results: Six hundred and thirty‐eight antimicrobial‐resistant Enterobacteriaceae isolated from wastewater were examined for class 1 integron. Of these, 293 were positive for the class 1 integrase gene intI1; among these, 34 gene cassettes and 29 AR genes were detected. Twenty‐nine distinct gene cassette arrays were identified by restriction fragment length polymorphism (RFLP). Seven strains harboring novel gene cassette arrays were subjected to further study, in which antimicrobial susceptibility profiles were determined, and the presence of other AR genes outside of the integrons was assayed. Several of the resistance determinants were found to be transferable by conjugation or transformation. Conclusions: This study established the assessment of class 1 integron and antimicrobial resistance gene patterns among environmental Enterobacteriaceae. Also, a restriction enzyme EcoRII was employed to develop a rapid and simple method for characterizing gene cassette arrays by RFLP analysis, which facilitated further study of novel gene cassette arrays. Significance and Impact of Study: These data not only illustrated the diversity of class 1 integron gene cassettes but also provided direct evidence that integrons mobilized gene cassettes, generating new linkages of resistance genes, and they could be integrated in gene transfer units such as conjugative plasmids to contribute to the dissemination of AR genes by horizontal gene transfer (HGT) in aquatic environments.     

Dimeric dUTPases, HisE, and MazG belong to a new superfamily of all-alpha NTP pyrophosphohydrolases with potential "house-cleaning" functions

Structure-guided analysis of the new dimeric dUTPase family revealed its sequence relationship to the phage T4 dCTPase, phosphoribosyl-ATP pyrophosphatase HisE, NTP pyrophosphatase MazG, and several uncharacterized protein families, including the human protein XTP3TPA (RS21-C6), which is overexpressed in embryonic and cancer cells. Comparison with the recently determined structure of a MazG-like protein from Sulfolobus solfataricus supported the unification of these enzymes in one superfamily of all-alpha NTP pyrophosphatases, suggesting that dimeric dUTPases evolved from a tetrameric MazG-like ancestor by gene duplication. Analysis of the structure of the Sulfolobus MazG points to 2-hydroxyadenosine (isoguanosine) triphosphate, a product of oxidative damage of ATP, as the most likely substrate. We predict that uncharacterized members of this superfamily perform "house-cleaning" functions by hydrolyzing abnormal NTPs and are functionally analogous to the structurally unrelated hydrolases of the Nudix superfamily. We outline probable tertiary and quaternary structures of the all-alpha NTP pyrophosphatase superfamily members.     

Oral spirochetes implicated in dental diseases are widespread in normal human subjects and carry extremely diverse integron gene cassettes

The NIH Human Microbiome Project (HMP) has produced several hundred metagenomic data sets, allowing studies of the many functional elements in human-associated microbial communities. Here, we survey the distribution of oral spirochetes implicated in dental diseases in normal human individuals, using recombination sites associated with the chromosomal integron in Treponema genomes, taking advantage of the multiple copies of the integron recombination sites (repeats) in the genomes, and using a targeted assembly approach that we have developed. We find that integron-containing Treponema species are present in ～80% of the normal human subjects included in the HMP. Further, we are able to de novo assemble the integron gene cassettes using our constrained assembly approach, which employs a unique application of the de Bruijn graph assembly information; most of these cassette genes were not assembled in whole-metagenome assemblies and could not be identified by mapping sequencing reads onto the known reference Treponema genomes due to the dynamic nature of integron gene cassettes. Our study significantly enriches the gene pool known to be carried by Treponema chromosomal integrons, totaling 826 (598 97% nonredundant) genes. We characterize the functions of these gene cassettes: many of these genes have unknown functions. The integron gene cassette arrays found in the human microbiome are extraordinarily dynamic, with different microbial communities sharing only a small number of common genes.     

Recovery and evolutionary analysis of complete integron gene cassette arrays from Vibrio

Background  

Integrons are genetic elements capable of the acquisition, rearrangement and expression of genes contained in gene cassettes. Gene cassettes generally consist of a promoterless gene associated with a recombination site known as a 59-base element (59-be). Multiple insertion events can lead to the assembly of large integron-associated cassette arrays. The most striking examples are found in Vibrio, where such cassette arrays are widespread and can range from 30 kb to 150 kb. Besides those found in completely sequenced genomes, no such array has yet been recovered in its entirety. We describe an approach to systematically isolate, sequence and annotate large integron gene cassette arrays from bacterial strains.     

Integrase-directed recovery of functional genes from genomic libraries

Large population sizes, rapid growth and 3.8 billion years of evolution firmly establish microorganisms as a major source of the planet''s biological and genetic diversity. However, up to 99% of the microorganisms in a given environment cannot be cultured. Culture-independent methods that directly access the genetic potential of an environmental sample can unveil new proteins with diverse functions, but the sequencing of random DNA can generate enormous amounts of extraneous data. Integrons are recombination systems that accumulate open reading frames (gene cassettes), many of which code for functional proteins with enormous adaptive potential. Some integrons harbor hundreds of gene cassettes and evidence suggests that the gene cassette pool may be limitless in size. Accessing this genetic pool has been hampered since sequence-based techniques, such as hybridization or PCR, often recover only partial genes or a small subset of those present in the sample. Here, a three-plasmid genetic strategy for the sequence-independent recovery of gene cassettes from genomic libraries is described and its use by retrieving functional gene cassettes from the chromosomal integron of Vibrio vulnificus ATCC 27562 is demonstrated. By manipulating the natural activity of integrons, we can gain access to the caches of functional genes amassed by these structures.     

Cassette recruitment in the chromosomal Integron of Vibrio cholerae

Integrons confer a rapid adaptation capability to bacteria. Integron integrases are able to capture and shuffle novel functions embedded in cassettes. Here, we investigated cassette recruitment in the Vibrio cholerae chromosomal integron during horizontal transfer. We demonstrated that the endogenous integrase expression is sufficiently triggered, after SOS response induction mediated by the entry of cassettes during conjugation and natural transformation, to mediate significant cassette insertions. These insertions preferentially occur at the attIA site, despite the presence of about 180 attC sites in the integron array. Thanks to the presence of a promoter in the attIA site vicinity, all these newly inserted cassettes are expressed and prone to selection. We also showed that the RecA protein is critical for cassette recruitment in the V. cholerae chromosomal integron but not in mobile integrons. Moreover, unlike the mobile integron integrases, that of V. cholerae is not active in other bacteria. Mobile integrons might have evolved from the chromosomal ones by overcoming host factors, explaining their large dissemination in bacteria and their role in antibioresistance expansion.     

Diversity of gene cassettes and the abundance of the class 1 integron-integrase gene in sediment polluted by metals

The integron-gene cassette system has typically been associated with antibiotic-resistant pathogens. However, the diversity of gene cassettes and the abundance of class 1 integrons outside of the clinical context are not fully explored. Primers targeting the conserved segments of attC recombination sites were used to amplify gene cassettes from the sediment of the Mina stream, which exhibited a higher degree of stress to metal pollution in the dry season than the rainy season. Of the 143 total analyzed sequences, 101 had no matches to proteins in the database, where cassette open reading frames could be identified by homology with database entries. There was a predominance of sequences encoding essential cellular functions. Each season that was sampled yielded a specific pool of gene cassettes. Real-time PCR revealed that 8.5 and 41.6 % of bacterial cells potentially harbored a class 1 integron in the rainy and dry seasons, respectively. In summary, our findings demonstrate that most of the gene cassettes have no ascribable function and, apparently, historically metal-contaminated sediment favors the maintenance of bacteria containing the intI1 gene. Thus, the diversity of gene cassettes is far from being fully explored deserving further attention.