Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.     

Dissemination and loss of a biofilm‐related genomic island in marine Pseudoalteromonas mediated by integrative and conjugative elements

Acquisition of genomic islands (GIs) plays a central role in the diversification and adaptation of bacteria. Some GIs can be mobilized in trans by integrative and conjugative elements (ICEs) or conjugative plasmids if the GIs carry specific transfer‐related sequences. However, the transfer mechanism of GIs lacking such elements remains largely unexplored. Here, we investigated the transmissibility of a GI found in a coral‐associated marine bacterium. This GI does not carry genes with transfer functions, but it carries four genes required for robust biofilm formation. Notably, this GI is inserted in the integration site for SXT/R391 ICEs. We demonstrated that acquisition of an SXT/R391 ICE results in either a tandem GI/ICE arrangement or the complete displacement of the GI. The GI displacement by the ICE greatly reduces biofilm formation. In contrast, the tandem integration of the ICE with the GI in cis allows the GI to hijack the transfer machinery of the ICE to excise, transfer and re‐integrate into a new host. Collectively, our findings reveal that the integration of an ICE into a GI integration site enables rapid genome dynamics and a new mechanism by which SXT/R391 ICEs can augment genome plasticity.     

The current ICE age: biology and evolution of SXT-related integrating conjugative elements

SXT is an integrating conjugative element (ICE) that was initially isolated from a 1992 Vibrio cholerae O139 clinical isolate from India. This approximately 100-kb ICE encodes resistance to multiple antibiotics. SXT or closely related ICEs are now present in most clinical and some environmental V. cholerae isolates from Asia and Africa. SXT-related ICEs are not limited to V. cholerae. It is now clear that so-called IncJ elements such as R391 are closely related to SXT. More than 25 members of the SXT/R391 family of ICEs have now been identified in environmental and clinical isolates of diverse species of gamma-proteobacteria worldwide. In this review, we discuss the diversity, evolution and biology of this family of ICEs.     

Genomic analysis of a novel integrative conjugative element in Vibrio cholerae

Integrative conjugative elements (ICEs) are a class of self-transmissible mobile elements that mediate horizontal gene transfer in bacteria, and play an important role in bacterial evolution. Since 1992, ICEs of the SXT/R391 family have been found to be widely distributed among Vibrio cholerae strains isolated in Asian countries. Here we describe ICEVchB33, an ICE found in the genomes of two V. cholerae O1 Eltor strains, one isolated in India, 1994, and the other from Mozambique, 2004. ICEVchB33 revealed a new genetic organization, different from other ICEs of the SXT/R391 family, demonstrating the genomic plasticity of these elements.     

Replication and Active Partition of Integrative and Conjugative Elements (ICEs) of the SXT/R391 Family: The Line between ICEs and Conjugative Plasmids Is Getting Thinner

Integrative and Conjugative Elements (ICEs) of the SXT/R391 family disseminate multidrug resistance among pathogenic Gammaproteobacteria such as Vibrio cholerae. SXT/R391 ICEs are mobile genetic elements that reside in the chromosome of their host and eventually self-transfer to other bacteria by conjugation. Conjugative transfer of SXT/R391 ICEs involves a transient extrachromosomal circular plasmid-like form that is thought to be the substrate for single-stranded DNA translocation to the recipient cell through the mating pore. This plasmid-like form is thought to be non-replicative and is consequently expected to be highly unstable. We report here that the ICE R391 of Providencia rettgeri is impervious to loss upon cell division. We have investigated the genetic determinants contributing to R391 stability. First, we found that a hipAB-like toxin/antitoxin system improves R391 stability as its deletion resulted in a tenfold increase of R391 loss. Because hipAB is not a conserved feature of SXT/R391 ICEs, we sought for alternative and conserved stabilization mechanisms. We found that conjugation itself does not stabilize R391 as deletion of traG, which abolishes conjugative transfer, did not influence the frequency of loss. However, deletion of either the relaxase-encoding gene traI or the origin of transfer (oriT) led to a dramatic increase of R391 loss correlated with a copy number decrease of its plasmid-like form. This observation suggests that replication initiated at oriT by TraI is essential not only for conjugative transfer but also for stabilization of SXT/R391 ICEs. Finally, we uncovered srpMRC, a conserved locus coding for two proteins distantly related to the type II (actin-type ATPase) parMRC partitioning system of plasmid R1. R391 and plasmid stabilization assays demonstrate that srpMRC is active and contributes to reducing R391 loss. While partitioning systems usually stabilizes low-copy plasmids, srpMRC is the first to be reported that stabilizes a family of ICEs.     

Environmental Vibrio spp., isolated in Mozambique, contain a polymorphic group of integrative conjugative elements and class 1 integrons

Circulation of mobile genetic elements linked to drug resistance spread was studied in Vibrio strains isolated from surface urban water (river and sea) and shellfish samples in 2002-2003 in Maputo, Mozambique. Class 1 integrons and integrating conjugative elements (ICE) were investigated by PCR and mating experiments in strains of major health interest: 10 Vibrio cholerae, six Vibrio parahaemolyticus, two Vibrio alginolyticus and one Vibrio fluvialis. Resistance to at least two antibiotics (predominantly beta-lactams) was detected in all the strains, with additional resistances to sulfamethoxazole, spectinomycin, streptomycin and/or trimethoprim. Class 1 integrons contributed partially to the expression of drug resistance and were found in five isolates: four V. cholerae (blaP1 cassette, one strain also contained the dfrA15 cassette) and one V. alginolyticus (aadA2 cassette). ICEs, apparently devoid of resistance genes, were found in eight V. cholerae, three V. parahaemolyticus and one V. fluvialis isolates. A wide variability was observed by molecular characterization of ICEs. Five ICEs were included in the SXT/R391 family and seven ICEs were not classified. Our results indicate that the SXT/R391 family and related ICEs comprise a large class of polymorphic genetic elements widely circulating in environmental Vibrio strains in Africa, beside those evidently linked to drug resistance in clinical isolates.     

Interactions between inner membrane proteins in donor and recipient cells limit conjugal DNA transfer

Conjugation enables horizontal transmission of DNA among bacteria, thereby facilitating the rapid spread of genes such as those conferring resistance to antibiotics. Cell-cell contact is required for conjugative DNA transfer but does not ensure its success. The presence of certain plasmids in potential recipient cells inhibits redundant transfer of these plasmids from competent donors despite contact between donor and recipient cells. Here, we used two closely related integrating conjugative elements (ICEs), SXT and R391, to identify genes that inhibit redundant conjugative transfer. Cells containing SXT exclude transfer of a second copy of SXT but not R391 and vice versa. The specific exclusion of SXT and R391 is dependent upon variants of TraG and Eex, ICE-encoded inner membrane proteins in donor and recipient cells, respectively. We identified short sequences within each variant that determine the exquisite specificity of self-recognition; these data suggest that direct interactions between TraG and Eex mediate exclusion.     

Identification of the origin of transfer (oriT) and a new gene required for mobilization of the SXT/R391 family of integrating conjugative elements

Integrating conjugative elements (ICEs) are self-transmissible, mobile elements that are widespread among bacteria. Following their excision from the chromosome, ICEs transfer by conjugation, a process initiated by a single-stranded DNA break at a specific locus called the origin of transfer (oriT). The SXT/R391 family of ICEs includes SXT(MO10), R391, and more than 25 related ICEs found in gammaproteobacteria. A previous study mapped the oriT locus of SXT(MO10) to a 550-bp intergenic region between traD and s043. We suspected that this was not the correct oriT locus, because the identical traD-s043 region in R391 and other SXT/R391 family ICEs was annotated as a gene of an unknown function. Here, we investigated the location and structure of the oriT locus in the ICEs of the SXT/R391 family and demonstrated that oriT(SXT) corresponds to a 299-bp sequence that contains multiple imperfect direct and inverted repeats and is located in the intergenic region between s003 and rumB'. The oriT(SXT) locus is well conserved among SXT/R391 ICEs, like R391, R997, and pMERPH, and cross-recognition of oriT(SXT) and oriT(R391) by R391 and SXT(MO10) was demonstrated. Furthermore, we identified a previously unannotated gene, mobI, located immediately downstream from oriT(SXT), which proved to be essential for SXT(MO10) transfer and SXT(MO10)-mediated chromosomal DNA mobilization. Deletion of mobI did not impair the SXT(MO10)-dependent transfer of the mobilizable plasmid CloDF13, suggesting that mobI has no role in the assembly of the SXT(MO10) mating pair apparatus. Instead, mobI appears to be involved in the recognition of oriT(SXT).     

Genomic analysis of ICEVchBan8: An atypical genetic element in Vibrio cholerae

Genomic islands (GIs) and integrative conjugative elements (ICEs) are major players in bacterial evolution since they encode genes involved in adaptive functions of medical or environmental importance. Here we performed the genomic analysis of ICEVchBan8, an unusual ICE found in the genome of a clinical non-toxigenic Vibrio cholerae O37 isolate. ICEVchBan8 shares most of its genetic structure with SXT/R391 ICEs. However, this ICE codes for a different integration/excision module is located at a different insertion site, and part of its genetic cargo shows homology to other pathogenicity islands of V. cholerae.     

Formation of SXT tandem arrays and SXT-R391 hybrids

SXT is an integrative and conjugative element (ICE) isolated from Vibrio cholerae. This approximately 100-kb ICE encodes resistance to multiple antibiotics and integrates site specifically into the chromosome. SXT excises from the chromosome to form a circular but nonreplicative extrachromosomal molecule that is required for its transfer. Here we found that a significant fraction of freshly isolated SXT exconjugants contained tandem SXT arrays. There was heterogeneity in the size of the SXT arrays detected in single exconjugant colonies. Some arrays consisted of more than five SXTs arranged in tandem. These extended arrays were unstable and did not persist during serial passages. The mechanism accounting for the generation of SXT arrays is unknown; however, array formation was not dependent upon recA and appeared to depend on conjugative transfer. While such arrays did not alter the transfer frequency of wild-type SXT, they partially complemented the transfer deficiency of a Deltaxis SXT mutant, which is ordinarily unable to generate the extrachromosomal intermediate required for SXT transfer. Exconjugants derived from donor strains that harbored tandem arrays of SXT and R391, an SXT-related element, contained functional hybrid elements that arose from recA-independent recombination between the two ICEs. Thus, arrays of SXT-related elements promote the creation of novel ICEs.     

Beyond antibiotic resistance: integrating conjugative elements of the SXT/R391 family that encode novel diguanylate cyclases participate to c‐di‐GMP signalling in Vibrio cholerae

In Vibrio cholerae, the second messenger bis‐(3′?5′)‐cyclic dimeric guanosine monophosphate (c‐di‐GMP) increases exopolysaccharides production and biofilm formation and decreases virulence and motility. As such, c‐di‐GMP is considered an important player in the transition from the host to persistence in the environment. c‐di‐GMP level is regulated through a complex network of more than 60 chromosomal genes encoding predicted diguanylate cyclases (DGCs) and phosphodiesterases. Herein we report the characterization of two additional DGCs, DgcK and DgcL, encoded by integrating conjugative elements (ICEs) belonging to the SXT/R391 family. SXT/R391 ICEs are self‐transmissible mobile elements that are widespread among vibrios and several species of enterobacteria. We found that deletion of dgcL increases the motility of V. cholerae, that overexpression of DgcK or DgcL modulates gene expression, biofilm formation and bacterial motility, and that a single amino acid change in the active site of either enzyme abolishes these phenotypes. We also show that DgcK and DgcL are able to synthesize c‐di‐GMP in vitro from GTP. DgcK was found to co‐purify with non‐covalently bound flavin mononucleotide (FMN). DgcL's enzymatic activity was augmented upon phosphorylation of its phosphorylatable response‐regulator domain suggesting that DgcL is part of a two‐component signal transduction system. Interestingly, we found orthologues of dgcK and dgcL in several SXT/R391 ICEs from two species of Vibrio originating from Asia, Africa and Central America. We propose that besides conferring usual antibiotic resistances, dgcKL‐bearing SXT/R391 ICEs could enhance the survival of vibrios in aquatic environments by increasing c‐di‐GMP level.     

Genomic and functional analysis of ICEPdaSpa1, a fish-pathogen-derived SXT-related integrating conjugative element that can mobilize a virulence plasmid

Integrating conjugative elements (ICEs) are self-transmissible mobile elements that transfer between bacteria via conjugation and integrate into the host chromosome. SXT and related ICEs became prevalent in Asian Vibrio cholerae populations in the 1990s and play an important role in the dissemination of antibiotic resistance genes in V. cholerae. Here, we carried out genomic and functional analyses of ICEPdaSpa1, an SXT-related ICE derived from a Spanish isolate of Photobacterium damselae subsp. piscicida, the causative agent of fish pasteurellosis. The approximately 102-kb DNA sequence of ICEPdaSpa1 shows nearly 97% DNA sequence identity to SXT in genes that encode essential ICE functions, including integration and excision, conjugal transfer, and regulation. However, approximately 25 kb of ICEPdaSpa1 DNA, including a tetracycline resistance locus, is not present in SXT. Most ICEPdaSpa1-specific DNA is inserted at loci where other SXT-related ICEs harbor element-specific DNA. ICEPdaSpa1 excises itself from the chromosome and is transmissible to other Photobacterium strains, as well as to Escherichia coli, in which it integrates into prfC. Interestingly, the P. damselae virulence plasmid pPHDP10 could be mobilized from E. coli in an ICEPdaSpa1-dependent fashion via the formation of a cointegrate between pPHDP10 and ICEPdaSpa1. pPHDP10-Cm integrated into ICEPdaSpa1 in a non-site-specific fashion independently of RecA. The ICEPdaSpa1::pPHDP10 cointegrates were stable, and markers from both elements became transmissible at frequencies similar to those observed for the transfer of ICEPdaSpa1 alone. Our findings reveal the plasticity of ICE genomes and demonstrate that ICEs can enable virulence gene transfer.     

Structural comparison of the integrative and conjugative elements R391, pMERPH, R997, and SXT

R391 and SXT are members of a group of eleven chromosome-borne conjugative elements found in the gamma-proteobacteria, whose members carry different antibiotic resistance traits. Recent genomic analysis of R391 and SXT revealed a highly conserved 'backbone' encoding integration/excision, conjugative transfer, and regulation functions, augmented by an array of phenotypic traits and transposable elements. In this study, PCR amplification and sequence analysis were employed to investigate the genomic structure of two further MGE of the R391 family, pMERPH (HgR) and R997 (ApR, SmR, SuR). R997 and pMERPH were found to be structurally related to R391 and SXT and share a number of virtually identical regions with them-including putative integration, conjugative transfer, and regulatory determinants-interrupted by variable DNA segments and transposable elements. The presence of a highly conserved backbone in the four elements strongly suggests their origin in a common ancestral element, which itself was a mosaic of sequences related to phages and plasmids. Subsequent genetic recombination and the acquisition of transposable elements resulted in the possession of variable phenotypic traits among the four MGE, and diversification into two distinct lineages, the first one including R391 and pMERPH, the second one containing SXT and R997.     

Structure, diversity, and mobility of the Salmonella pathogenicity island 7 family of integrative and conjugative elements within Enterobacteriaceae

Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry. Salmonella pathogenicity island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus Salmonella and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella enterica serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus, and Yersinia species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae.     

R391: a conjugative integrating mosaic comprised of phage,plasmid, and transposon elements

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.     

细菌中整合性接合元件的研究进展

整合性接合元件是近年来在细菌中发现的一种可移动的基因元件,它位于染色体上,可通过接合转移的方式介导细菌间基因的水平转移。这种基因的水平转移有助于细菌适应特定的环境条件,但许多整合性接合元件包含耐药基因,这些遗传元件的水平转移极大地加速了耐药基因在同种及不同种属之间的传播,造成细菌的耐药以至多重耐药问题日益严重,耐药机制日趋复杂;同时整合性接合元件与基因岛有着密切的联系,因此对其特征及转移机制进行研究很有必要。     

Uncovering the prevalence and diversity of integrating conjugative elements in actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.