Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.

Integrons constitute a novel family of DNA elements which evolved by site-specific integration of discrete units between two conserved segments. On the In4 integron of Tn1696, a precisely inserted gene cassette of 1,549 bp conferring nonenzymatic chloramphenicol resistance (cmlA) is present between the streptomycin-spectinomycin resistance (aadA2) gene cassette and the 3'-conserved segment of the integron. In this study, we present the nucleotide sequence of the cmlA gene cassette of Tn1696, show its similarity to bacterial efflux systems and other transport proteins, and present evidence for alterations that its expression exerts on bacterial membranes. The cmlA gene cassette apparently carries its own promoter(s), a situation that has not heretofore been observed in the integrons of multiresistance plasmids and transposons of gram-negative bacteria. One or more of these promoters were shown to be functionally active in expressing a cat marker gene from promoter-probe vectors. The putative CmlA polypeptide appears to provoke a reduction of the content of the major porins OmpA and OmpC.     

产超广谱β-内酰胺酶细菌中I类整合子的研究进展

产超广谱β-内酰胺酶(Extended-spectrum beta-lactamase, ESBLs)细菌的多重耐药性是临床用药的一大难题, 近年研究发现其耐药性的产生与整合子密切相关, 其中临床最常见、研究最深入的是I类整合子。整合子是一种可移动基因元件, 在整合酶的作用下捕捉外源基因盒并使之表达, 是具有基因整合和切除功能的天然克隆和表达系统。研究表明I类整合子可连续捕捉和整合多种耐药基因, 以质粒或转座子为载体在细菌之间传播耐药性, 使ESBLs细菌多重耐药趋势十分严峻。本文就I类整合子的结构特征、I类整合子对耐药基因盒的整合作用及其与ESBLs细菌耐药性的关系等方面进行综述。     

Gene cassettes and cassette arrays in mobile resistance integrons

Gene cassettes are small mobile elements, consisting of little more than a single gene and recombination site, which are captured by larger elements called integrons. Several cassettes may be inserted into the same integron forming a tandem array. The discovery of integrons in the chromosome of many species has led to the identification of thousands of gene cassettes, mostly of unknown function, while integrons associated with transposons and plasmids carry mainly antibiotic resistance genes and constitute an important means of spreading resistance. An updated compilation of gene cassettes found in sequences of such 'mobile resistance integrons' in GenBank was facilitated by a specially developed automated annotation system. At least 130 different (<98% identical) cassettes that carry known or predicted antibiotic resistance genes were identified, along with many cassettes of unknown function. We list exemplar GenBank accession numbers for each and address some nomenclature issues. Various modifications to cassettes, some of which may be useful in tracking cassette epidemiology, are also described. Despite potential biases in the GenBank dataset, preliminary analysis of cassette distribution suggests interesting differences between cassettes and may provide useful information to direct more systematic studies.     

Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination

An integron is a genetic unit that includes the determinants of the components of a site-specific recombination system capable of capturing and mobilizing genes that are contained in mobile elements called gene cassettes. An integron also provides a promoter for expression of the cassette genes, and integrons thus act both as natural cloning systems and as expression vectors. The essential components of an integron are an int gene encoding a site-specific recombinase belonging to the integrase family, an adjacent site, attl, that is recognized by the integrase and is the receptor site for the cassettes, and a promoter suitably oriented for expression of the cassette-encoded genes. The cassettes are mobile elements that include a gene (most commonly an antibiotic-resistance gene) and an integrase-specific recombination site that is a member of a family of sites known as 59-base elements. Cassettes can exist either free in a circularized form or integrated at the attl site, and only when integrated is a cassette formally part of an integron. A single site-specific recombination event involving the integron-associated attl site and a cassette-associated 59-base element leads to insertion of a free circular cassette into a recipient integron. Multiple cassette insertions can occur, and integrons containing several cassettes have been found in the wild. The integrase also catalyses excisive recombination events that can lead to loss of cassettes from an integron and generate free circular cassettes. Due to their ability to acquire new genes, integrons have a clear role in the evolution of the genomes of the plasmids and transposons that contain them. However, a more general role in evolution is also likely. Events involving recombination between a specific 59-base-element site and a nonspecific secondary site have recently been shown to occur. Such events should lead either to the insertion of cassettes at non-specific sites or to the formation of stable cointegrates between different plasmid molecules, and a cassette situated outside the integron context has recently been identified.     

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.     

A novel family of potentially mobile DNA elements encoding site-specific gene-integration functions: integrons

A family of novel mobile DNA elements is described, examples of which are found at several independent locations and encode a variety of antibiotic resistance genes. The complete elements consist of two conserved segments separated by a segment of variable length and sequence which includes inserted antibiotic resistance genes. The conserved segment located 3' to the inserted resistance genes was sequenced from Tn21 and R46, and the sequences are identical over a region of 2026 bases, which includes the sulphonamide resistance gene sull, and two further open reading frames of unknown function. The complete sequences of both the 3' and 5' conserved regions of the DNA element have been determined. A 59-base sequence element, found at the junctions of inserted DNA sequences and the conserved 3' segment, is also present at this location in the R46 sequence. A copy of one half of this 59-base element is found at the end of the sull gene, suggesting that sull, though part of the conserved region, was also originally inserted into an ancestral element by site-specific integration. Inverted or direct terminal repeats or short target site duplications, both of which are characteristics of class I and class II transposons, are not found at the outer boundaries of the elements described here. Furthermore, the conserved regions do not encode any proteins related to known transposition proteins, except the DNA integrase encoded by the 5' conserved region which is implicated in the gene insertion process. Mobilization of this element has not been observed experimentally; mobility is implied from the identification of the element in at least four independent locations, in Tn21, R46 (IncN), R388 (IncW) and Tn1696. The definitive features of these novel elements are (i) that they include site-specific integration functions (the integrase and the insertion site); (ii) that they are able to acquire various gene units and act as an expression cassette by supplying the promoter for the inserted genes. As a consequence of acquiring different inserted genes, the element exists in a variety of forms which differ in the number and nature of the inserted genes. This family of elements appears formally distinct from other known mobile DNA elements and we propose the name DNA integration elements, or integrons.     

Transposon Tn21, flagship of the floating genome.

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Transposon Tn21, Flagship of the Floating Genome

The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.     

Structural and functional characterization of tnpI, a recombinase locus in Tn21 and related beta-lactamase transposons

A novel discrete mobile DNA element from Tn21 from the plasmid R100.1 is described, and its mobilization function was confirmed experimentally. In addition, the element behaves as a recombinase-active locus (tnpI) which facilitates insertions of antibiotic resistance genes as modules or cassettes at defined hot spots or integration sites. A similar tnpI sequence was detected by DNA hybridization in a series of beta-lactamase transposons and plasmids and localized on their physical maps. The genetic function of the locus cloned from Tn21 into pACYC184 was tested for conduction and integration into the plasmids R388 and pOX38Km, and the results suggested recombinase-integrase activity and recA independence. DNA sequence analysis of the tnpI locus revealed no inverted or direct terminal repeats or transposition features of class I and class II transposons. The coding capacity revealed three putative open reading frames encoding 131, 134, and 337 amino acids. Orf3 encoded a putative polypeptide product of 337 amino acids that shared highly significant identity with the carboxyl region of integrase proteins. A comparison and an alignment of the tnpI locus from Tn21 and its flanking sequences identified similar sequences in plasmids and in transposons. The alignment revealed discrete nucleotide changes in these tnpI-like loci and a conserved 3' and 5' GTTA/G hot spot as a duplicated target site. Our data confirm the remarkable ubiquity of tnpI associated with antibiotic resistance genes. We present a model of transposon modular evolution into more complex multiresistant units via tnpI and site-specific insertions, deletions, and DNA rearrangements at this locus.     

Increased levels of sigJ mRNA in late stationary phase cultures of Mycobacterium tuberculosis detected by DNA array hybridisation

Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance. Such acquisition is mediated by an integron-encoded integrase, which captures genes that are part of gene cassettes. To test whether integrons occur in environments with no known history of antibiotic exposure, PCR primers were designed to conserved regions of the integrase gene and the gene cassette recombination site. Amplicons generated from four environmental DNA samples contained features typical of the integrons found in antibiotic-resistant and pathogenic bacteria. The sequence diversity of the integrase genes in these clones was sufficient to classify them within three new classes of integron. Since they are derived from environments not associated with antibiotic use, integrons appear to be more prevalent in bacteria than previously observed.     

Recombination activity of a distinctive integron-gene cassette system associated with Pseudomonas stutzeri populations in soil

Class 1 integrons have strongly influenced the evolution of multiple antibiotic resistance. Diverse integrons have recently been detected directly in a range of natural environments. In order to characterize the properties of these environmental integrons, we sought to isolate organisms containing integrons from soils, which resulted in the isolation of Pseudomonas stutzeri strain Q. Further isolation efforts targeted at this species resulted in recovery of two other strains (P and BAM). 16S rRNA sequences and chromosome mapping showed that these three strains are very closely related clonal variants in a single genomovar of P. stutzeri. Only strains Q and BAM were found to contain an integron and an associated gene cassette array. The intI and attI components of these strains showed 99 and 90% identity, respectively. The structure of these integrons and their associated gene cassettes was similar to that reported previously for other integron classes. The two integrons contained nonoverlapping sets of cassette-associated genes. In contrast, many of the cassette-associated recombination sites in the two integrons were similar and were considered to constitute a distinct subfamily consisting of 59-base element (59-be) recombination sites (the Pseudomonas subfamily). The recombination activity of P. stutzeri integron components was tested in cointegrate assays. IntIPstQ was shown to catalyze site-specific recombination between its cognate attI site and 59-be sites from antibiotic resistance gene cassettes. While IntIPstQ did not efficiently mediate recombination between members of the Pseudomonas 59-be subfamily and other 59-be types, the former sites were functional when they were tested with IntI1. We concluded that integrons present in P. stutzeri possess recombination activity and represent a hot spot for genomic diversity in this species.     

Class 1 integrons potentially predating the association with tn402-like transposition genes are present in a sediment microbial community

Integrons are genetic elements that contribute to lateral gene transfer in bacteria as a consequence of possessing a site-specific recombination system. This system facilitates the spread of genes when they are part of mobile cassettes. Most integrons are contained within chromosomes and are confined to specific bacterial lineages. However, this is not the case for class 1 integrons, which were the first to be identified and are one of the single biggest contributors to multidrug-resistant nosocomial infections, carrying resistance to many antibiotics in diverse pathogens on a global scale. The rapid spread of class 1 integrons in the last 60 years is partly a result of their association with a specific suite of transposition functions, which has facilitated their recruitment by plasmids and other transposons. The widespread use of antibiotics has acted as a positive selection pressure for bacteria, especially pathogens, which harbor class 1 integrons and their associated antibiotic resistance genes. Here, we have isolated bacteria from soil and sediment in the absence of antibiotic selection. Class 1 integrons were recovered from four different bacterial species not known to be human pathogens or commensals. All four integrons lacked the transposition genes previously considered to be a characteristic of this class. At least two of these integrons were located on a chromosome, and none of them possessed antibiotic resistance genes. We conclude that novel class 1 integrons are present in a sediment environment in various bacteria of the beta-proteobacterial class. These data suggest that the dispersal of this class may have begun before the "antibiotic era."     

Integrons: Novel DNA elements which capture genes by site-specific recombination

Integrons are unusual DNA elements which include a gene encoding a site-specific DNA recombinase, a DNA integrase, and an adjacent site at which a wide variety of antibiotic resistance and other genes are found as inserts. One or more genes can be found in the insert region, but each gene is part of an independent gene cassette. The inserted genes are expressed from a promoter in the conserved sequences located 5 to the genes, and integrons are thus natural expression vectors. A model for gene insertion in which circular gene cassettes are inserted individually via a single site-specific recombination event has been proposed and verified experimentally. The gene cassettes include a gene coding region and, at the 3 end of the gene an imperfect inverted repeat, a 59-base element. The 59-base elements are a diverse family of elements which function as sites recognized by the DNA integrase. Site-specific insertion of individual genes thus represents a further mechanism which contributes to the evolution of the genomes of Gram-negative bacteria and their plasmids and transposons.Members of the most studied class of integrons, which include thesulI gene in the conserved sequences, are believed to be mobile DNA elements on the basis that they are found in many independent locations, and a discrete boundary is found at the outer end of the 5-conserved segment. However, the length of the 3-conserved segment is variable in the integrons examined to date, and it is likely that this variability has arisen as the result of insertion and deletion events. Though the true extent of the 3-conserved segment remains to be determined, it seems likely that these integrons are mobile DNA elements. The second known class of integrons comprises members of the Tn7 transposon family.     

Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements

The high and sometimes inappropriate use of antibiotics has accelerated the development of antibiotic resistance, creating a major challenge for the sustainable treatment of infections world-wide. Bacterial communities often respond to antibiotic selection pressure by acquiring resistance genes, i.e. mobile genetic elements that can be shared horizontally between species. Environmental microbial communities maintain diverse collections of resistance genes, which can be mobilized into pathogenic bacteria. Recently, exceptional environmental releases of antibiotics have been documented, but the effects on the promotion of resistance genes and the potential for horizontal gene transfer have yet received limited attention. In this study, we have used culture-independent shotgun metagenomics to investigate microbial communities in river sediments exposed to waste water from the production of antibiotics in India. Our analysis identified very high levels of several classes of resistance genes as well as elements for horizontal gene transfer, including integrons, transposons and plasmids. In addition, two abundant previously uncharacterized resistance plasmids were identified. The results suggest that antibiotic contamination plays a role in the promotion of resistance genes and their mobilization from environmental microbes to other species and eventually to human pathogens. The entire life-cycle of antibiotic substances, both before, under and after usage, should therefore be considered to fully evaluate their role in the promotion of resistance.     

Origin, evolution, and migration of drug resistance genes

Current views on the mechanisms responsible for the emergence of multiple drug resistance in clinical bacterial isolates are considered. Hypotheses on the origin of resistance genes derived from determinants of actinomycetes, antibiotic producers, and chromosomal genes of bacteria involved in cellular metabolism are reviewed. The mechanisms underlying the diffusion of resistance determinants by means of bacterial mobile elements (plasmids, transposons, and integrons) are discussed. Examples of the horizontal transfer of resistance determinants between Gram-positive and Gram-negative bacteria are presented.     

The evolution of class 1 integrons and the rise of antibiotic resistance

Class 1 integrons are central players in the worldwide problem of antibiotic resistance, because they can capture and express diverse resistance genes. In addition, they are often embedded in promiscuous plasmids and transposons, facilitating their lateral transfer into a wide range of pathogens. Understanding the origin of these elements is important for the practical control of antibiotic resistance and for exploring how lateral gene transfer can seriously impact on, and be impacted by, human activities. We now show that class 1 integrons can be found on the chromosomes of nonpathogenic soil and freshwater Betaproteobacteria. Here they exhibit structural and sequence diversity, an absence of antibiotic resistance genes, and a phylogenetic signature of lateral transfer. Some examples are almost identical to the core of the class 1 integrons now found in pathogens, leading us to conclude that environmental Betaproteobacteria were the original source of these genetic elements. Because these elements appear to be readily mobilized, their lateral transfer into human commensals and pathogens was inevitable, especially given that Betaproteobacteria carrying class 1 integrons are common in natural environments that intersect with the human food chain. The strong selection pressure imposed by the human use of antimicrobial compounds then ensured their fixation and global spread into new species.     

Integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution

Integrons are horizontal gene transfer (HGT) systems containing elements necessary for site-specific recombination and expression of foreign DNA. The overall phylogenetic distribution of integrons and range of genes that can be transferred by integrons are unknown. This report contains an exploration of integrons in an environmental microbial community and an investigation of integron evolution. First, using culture-independent techniques, we explored the diversity of integrons and integron-transferred genes in heavy-metal-contaminated mine tailings. Using degenerate primers, we amplified integron integrase genes from the tailings. We discovered 14 previously undescribed integrase genes, including six novel gene lineages. In addition, we found 11 novel gene cassettes in this sample. One of the gene cassettes that we sequenced is similar to a gene that codes for a step in a pathway for nitroaromatic catabolism, a group of compounds associated with mining activity. This suggests that integrons may be important for gene transfer in response to selective pressures other than the presence of antibiotics. We also investigated the evolution of integrons by statistically comparing the phylogenies of 16S rRNA and integrase genes from the same organisms, using sequences from GenBank and various sequencing projects. We found significant differences between the organismal (16S rRNA) and integrase trees, and we suggest that these differences may be due to HGT.     

整合子-基因盒系统与细菌耐药机制的研究进展

大量研究表明整合子-基因盒系统是微生物耐药的主要机制,由其介导的耐药基因水平转移是细菌耐药机制产生的主要途径。已知的整合子被分为两大类：传统的整合子和超级整合子。前者存在于转座子、质粒和细菌染色体,其基因盒编码产物可使细菌耐受一种或多种抗菌药物及消毒剂;而后者则只存在于细菌的染色体上,它携带的基因盒更多,且其编码产物则更加复杂,目前只在特定菌株中发现超级整合子。本文就整合子的结构、分布、检测及它对细菌耐药性的影响等几个方面的研究进展进行讨论。     

Integrons,transposons and IS elements promote diversification of multidrug resistance plasmids and adaptation of their hosts to antibiotic pollutants from pharmaceutical companies

Plasmids are important vehicles for the dissemination of antibiotic resistance genes (ARGs) among bacteria by conjugation. Here, we determined the complete nucleotide sequences of nine different plasmids previously obtained by exogenous plasmid isolation from river and creek sediments and wastewater from a pharmaceutical company. We identified six IncP/P-1ε plasmids and single members of IncL, IncN and IncFII-like plasmids. Genetic structures of the accessory regions of the IncP/P-1ε plasmids obtained implied that multiple insertions and deletions had occurred, mediated by different transposons and Class 1 integrons with various ARGs. Our study provides compelling evidence that Class 1 integrons, Tn402-like transposons, Tn3-like transposons and/or IS26 played important roles in the acquisition of ARGs across all investigated plasmids. Our plasmid sequencing data provide new insights into how these mobile genetic elements could mediate the acquisition and spread of ARGs in environmental bacteria.