RESUMO
Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression. They were originally identified as a mechanism used by Gram-negative bacteria to collect antibiotic resistance genes and express multiple resistance phenotypes in synergy with transposons. More recently, their role has been broadened with the discovery of chromosomal integron (CI) structures in the genomes of hundreds of bacterial species. This review focuses on the resources carried in these elements, on their unique recombination mechanisms, and on the different mechanisms controlling the cassette dynamics. We discuss the role of the toxin/antitoxin (TA) cassettes for the stabilization of the large cassette arrays carried in the larger CIs, known as superintegrons. Finally, we explore the central role played by single-stranded DNA in the integron cassette dynamics in light of the recent discovery that the integron integrase expression is controlled by the SOS response.
Assuntos
Bactérias/genética , Integrons , Antitoxinas/genética , Toxinas Bacterianas/genéticaRESUMO
UNLABELLED: The role of chromosomal toxin-antitoxin (TA) systems, which are ubiquitous within the genomes of free-living bacteria, is still debated. We have scanned the Vibrio cholerae N16961 genome for class 2 TA genes and identified 18 gene pair candidates. Interestingly, all but one are located in the chromosome 2 superintegron (SI). The single TA found outside the SI is located on chromosome 1 and is related to the well-characterized HipAB family, which is known to play a role in antibiotic persistence. We investigated this clustering within the SI and its possible biological consequences by performing a comprehensive functional analysis on all of the putative TA systems. We demonstrate that the 18 TAs identified encode functional toxins and that their cognate antitoxins are able to neutralize their deleterious effects when expressed in Escherichia coli. In addition, we reveal that the 17 predicted TA systems of the SI are transcribed and expressed in their native context from their own promoters, a situation rarely found in integron cassettes. We tested the possibility of interactions between noncognate pairs of all toxins and antitoxins and found no cross-interaction between any of the different TAs. Although these observations do not exclude other roles, they clearly strengthen the role of TA systems in stabilizing the massive SI cassette array of V. cholerae. IMPORTANCE: The chromosomal toxin-antitoxin systems have been shown to play various, sometimes contradictory roles, ranging from genomic stabilization to bacterial survival via persistence. Determining the interactions between TA systems hosted within the same bacteria is essential to understand the hierarchy between these different roles. We identify here the full set of class 2 TAs carried in the Vibrio cholerae N16961 genome and found they are all, with a single exception, located in the chromosome 2 superintegron. Their characterization, in terms of functionality, expression, and possible cross-interactions, supports their main role as being the stabilization of the 176-cassette-long array of the superintegron but does not exclude dual roles, such as stress response elements, persistence, and bacteriophage defense through abortive infection mechanisms.
Assuntos
Antitoxinas/metabolismo , Toxinas Bacterianas/metabolismo , Integrons/fisiologia , Vibrio cholerae/metabolismo , Antitoxinas/genética , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Regiões Promotoras Genéticas , Vibrio cholerae/genéticaRESUMO
Toxin-antitoxin (TA) systems have been reported in the genomes of most bacterial species, and their role when located on the chromosome is still debated. TA systems are particularly abundant in the massive cassette arrays associated with chromosomal superintegrons (SI). Here, we describe the characterization of two superintegron cassettes encoding putative TA systems. The first is the phd-doc(SI) system identified in Vibrio cholerae N16961. We determined its distribution in 36 V. cholerae strains and among five V. metschnikovii strains. We show that this cassette, which is in position 72 of the V. cholerae N16961 cassette array, is functional, carries its own promoter, and is expressed from this location. Interestingly, the phd-doc(SI) system is unable to control its own expression, most likely due to the absence of any DNA-binding domain on the antitoxin. In addition, this SI system is able to cross talk with the canonical P1 phage system. The second cassette that we characterized is the ccd(Vfi) cassette found in the V. fischeri superintegron. We demonstrate that CcdB(Vfi) targets DNA-gyrase, as the canonical CcB(F) toxin, and that ccd(Vfi) regulates its expression in a fashion similar to the ccd(F) operon of the conjugative plasmid F. We also establish that this cassette is functional and expressed in its chromosomal context in V. fischeri CIP 103206T. We tested its functional interactions with the ccdAB(F) system and found that CcdA(Vfi) is specific for its associated CcdB(Vfi) and cannot prevent CcdB(F) toxicity. Based on these results, we discuss the possible biological functions of these TA systems in superintegrons.
Assuntos
Integrons/genética , Vibrio/genética , Antitoxinas/genética , Escherichia coli/genética , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Toxinas Biológicas/genética , Vibrio cholerae/genéticaRESUMO
Candida albicans is a major fungal pathogen of humans. Although its genome has been sequenced more than two decades ago, there are still over 4300 uncharacterized C. albicans genes. We previously generated an ORFeome as well as a collection of destination vectors to facilitate overexpression of C. albicans ORFs. Here, we report the construction of â¼2500 overexpression mutants and their evaluation by in vitro spotting on rich medium and in a liquid pool experiment in rich medium, allowing the identification of genes whose overexpression has a fitness cost. The candidates were further validated at the individual strain level. This new resource allows large-scale screens in different growth conditions to be performed routinely. Altogether, based on the concept of identifying functionally related genes by cluster analysis, the availability of this overexpression mutant collection will facilitate the characterization of gene functions in C. albicans.
Assuntos
Candida albicans , Genoma Fúngico , Candida albicans/genética , Proteínas Fúngicas/genéticaRESUMO
Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.
Assuntos
Bacteriófago mu/genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Mutagênese Insercional/métodos , Plasmídeos/genética , Bacteriófago mu/metabolismo , Mapeamento Cromossômico , Cromossomos Bacterianos , Conjugação Genética , DNA Bacteriano/genética , Escherichia coli/metabolismoRESUMO
In many bacteria, gene replacement can only be achieved using RP4-based conjugative systems where exogenous DNA is delivered to the recipient on conditionally replicating plasmids carrying an oriT(RP4). In the commonly used strains SM10 and S17-1 conjugative functions are provided by an RP4 plasmid inserted in the chromosome. Those strains, besides mobilizing oriT(RP4)-carrying plasmids, also transfer their own chromosomal genes, which can be inconvenient for several reasons. We describe here a new version of an RP4-based conjugative system that allows for efficient transfer of mobilizable plasmids without the transfer of chromosomal genes, providing fully controlled and efficient exogeneous DNA delivery to non-transformable bacteria.
Assuntos
Bactérias/genética , Conjugação Genética , Plasmídeos/genética , Sequência de Bases , Cromossomos Bacterianos/genética , Clonagem Molecular , DNA Bacteriano/genética , Vetores Genéticos/genética , Dados de Sequência Molecular , Origem de ReplicaçãoRESUMO
We describe the construction of the pSW family of conditionally replicating plasmids which are based on the IncX oriV origin (oriV(R6Kgamma)) of replication that is dependent on the pir-encoded protein. We constructed several Escherichia coli derivatives expressing pir from different chromosomal loci, and the pir gene could be transduced by phage P1 to any E. coli strain. These chromosomal constructions generate dapA and thyA knockouts, which lead to diaminopimelate or thymidine auxotrophies, respectively, and they serve to provide absolute counterselection even in rich media. These strains can be easily counterselected if used in plasmid transfer experiments into markerless recipients, and they have been demonstrated to work efficiently in E. coli xVibrio or E. coli xBartonella matings. We constructed different pSW plasmids carrying either the oriT(RP4) or the oriT(R388), and we demonstrated that these derivatives can be efficiently transferred using RP4 and R388 conjugation machineries, respectively. We also constructed two plasmids expressing the R388 conjugation machinery, but lacking the oriT(R388). We demonstrated that these plasmids enabled efficient and exclusive transfer of a pSW-oriT(R388) derivative from E. coli to V. cholerae, and we offer an alternative to the popular RP4-based delivery system.
Assuntos
Conjugação Genética , Escherichia coli/genética , Plasmídeos/genética , Replicação do DNA , Proteínas de Escherichia coli/genética , Dados de Sequência Molecular , Recombinação Genética , Análise de Sequência de DNARESUMO
We constructed a family of conditionally replicating plasmids, the pTX1 family, which are based on the IncPalpha oriV origin of replication that is dependent on the trfA-encoded protein. We constructed several Escherichia coli derivatives expressing trfA from different chromosomal loci, which can be transduced by phage P1 to any E. coli strain. The pTX1 plasmids also carry the oriTRP4 origin of transfer, and can be conjugated to E. coli, Vibrio cholerae and likely to a broad range of bacteria from the commonly used donor strains SM10 and S17-1, which sustain replication of the plasmids through the trfA gene carried by their integrated RP4. If TrfA is not provided in trans, these plasmids behave as suicide vectors. As such they can be used as a platform for a variety of applications such as those developed on the popular conditionally replicating plasmids carrying the oriVR6Kgamma origin of replication that is controlled by the Pi protein. Their ability to be used as efficient suicide vectors for gene disruption in V. cholerae has been demonstrated.
Assuntos
Replicação do DNA , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/crescimento & desenvolvimento , Vetores Genéticos , Vibrio cholerae/genética , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd(F)) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd(O157)). The ccd(F) and the ccd(O157) systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd(F) system. We have shown that the chromosomal ccd(O157) system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB(O157) toxin targets the DNA gyrase, and the CcdA(O157) antitoxin is degraded by the Lon protease. The ccd(O157) chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd(F). ccd(O157) is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd(F) system is able to mediate postsegregational killing in an E. coli strain harboring the ccd(O157) system in its chromosome. This shows that the plasmidic ccd(F) system is functional in the presence of its chromosomal counterpart.
Assuntos
Antitoxinas/metabolismo , Escherichia coli O157/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Sequência de Bases , Cromossomos Bacterianos/genética , Primers do DNA , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/metabolismo , Cinética , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.
Assuntos
Integrases/metabolismo , Integrons/fisiologia , Vibrio cholerae/enzimologia , Vibrio cholerae/genética , Sequência de Bases , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutagênese Insercional , Recombinação Genética , Alinhamento de Sequência , Homologia de Sequência do Ácido NucleicoRESUMO
The capture and spread of antibiotic resistance determinants by integrons underlies the rapid evolution of multiple antibiotic resistance among diverse Gram-negative clinical isolates. The association of multiple resistance integrons (MRIs) with mobile DNA elements facilitates their transit across phylogenetic boundaries and augments the potential impact of integrons on bacterial evolution. Recently, ancestral chromosomal versions, the super-integrons (SIs), were found to be genuine components of the genomes of diverse bacterial species. SIs possess evolutionary characteristics and stockpiles of adaptive functions, including cassettes related to antibiotic resistance determinants previously characterized in clinical isolates, which suggest that MRIs and their resistance genes were originally recruited from SIs and their pool of amassed genes. However, the recombination activity of integrons has never been demonstrated in a bacterium other than Escherichia coli. We introduced a naturally occurring MRI (TpR, SulR) on a conjugative plasmid into Vibrio cholerae, a species known to harbour a SI. We show that MRIs can randomly recruit genes directly from the cache of SI cassettes. By applying a selective constraint for the development of antibiotic resistance, we demonstrate bacterial resistance evolution through the recruitment a novel, but phenotypically silent, chloramphenicol acetyltransferase gene from the V. cholerae SI and its precise insertion into the MRI. The resulting resistance profile (CmR, TpR, SulR) could then be disseminated by conjugation to other clinically relevant pathogens at high frequency. These results demonstrate that otherwise phenotypically sensitive strains may still be a genetic source for the evolution of resistance to clinically relevant antibiotics through integron-mediated recombination events.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Evolução Molecular , Bactérias Gram-Negativas/efeitos dos fármacos , Integrases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/química , Cloranfenicol O-Acetiltransferase/genética , Conjugação Genética , Elementos de DNA Transponíveis/genética , Escherichia coli/genética , Bactérias Gram-Negativas/genética , Dados de Sequência Molecular , Plasmídeos/genética , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genéticaRESUMO
Integrons are natural tools for bacterial evolution and innovation. Their involvement in the capture and dissemination of antibiotic-resistance genes among Gram-negative bacteria is well documented. Recently, massive ancestral versions, the superintegrons (SIs), were discovered in the genomes of diverse proteobacterial species. SI gene cassettes with an identifiable activity encode proteins related to simple adaptive functions, including resistance, virulence, and metabolic activities, and their recruitment was interpreted as providing the host with an adaptive advantage. Here, we present extensive comparative analysis of SIs identified among the Vibrionaceae. Each was at least 100 kb in size, reaffirming the participation of SIs in the genome plasticity and heterogeneity of these species. Phylogenetic and localization data supported the sedentary nature of the functional integron platform and its coevolution with the host genome. Conversely, comparative analysis of the SI cassettes was indicative of both a wide range of origin for the entrapped genes and of an active cassette assembly process in these bacterial species. The signature attC sites of each species displayed conserved structural characteristics indicating that symmetry rather than sequence was important in the recognition of such a varied collection of target recombination sequences by a single site-specific recombinase. Our discovery of various addiction module cassettes within each of the different SIs indicates a possible role for them in the overall stability of large integron cassette arrays.