Integrating and conjugative elements (ICEs) are one of the three principal types of self-transmissible mobile genetic elements in bacteria. ICEs, like plasmids, transfer via conjugation; but unlike plasmids and similar to many phages, these elements integrate into and replicate along with the host chromosome. Members of the SXT/R391 family of ICEs have been isolated from several species of gram-negative bacteria, including Vibrio cholerae, the cause of cholera, where they have been important vectors for disseminating genes conferring resistance to antibiotics. Here we developed a plasmid-based system to capture and isolate SXT/R391 ICEs for sequencing. Comparative analyses of the genomes of 13 SXT/R391 ICEs derived from diverse hosts and locations revealed that they contain 52 perfectly syntenic and nearly identical core genes that serve as a scaffold capable of mobilizing an array of variable DNA. Furthermore, selection pressure to maintain ICE mobility appears to have restricted insertions of variable DNA into intergenic sites that do not interrupt core functions. The variable genes confer diverse element-specific phenotypes, such as resistance to antibiotics. Functional analysis of a set of deletion mutants revealed that less than half of the conserved core genes are required for ICE mobility; the functions of most of the dispensable core genes are unknown. Several lines of evidence suggest that there has been extensive recombination between SXT/R391 ICEs, resulting in re-assortment of their respective variable gene content. Furthermore, our analyses suggest that there may be a network of phylogenetic relationships among sequences found in all types of mobile genetic elements.
SummaryElements that excise and integrate, such as prophages, and transfer by conjugation, such as plasmids, have been found in various bacteria. These elements appear to have a diversified set of characteristics including cell-to-cell contact using pili or cell aggregation, transfer of single-stranded or double-stranded DNA, low or high specificity of integration and serine or tyrosine recombinases. This has led to a highly heterogeneous nomenclature, including conjugative transposons, integrative 'plasmids', genomic islands and numerous unclassified elements. However, all these elements excise by site-specific recombination, transfer the resulting circular form by conjugation and integrate by recombination between a specific site of this circular form and a site in the genome of their host. Whereas replication of the circular form probably occurs during conjugation, this replication is not involved in the maintenance of the element. In this review, we show that these elements share very similar characteristics and, therefore, we propose to classify them as integrative and conjugative elements (ICEs). These elements evolve by acquisition or exchanges of modules with various transferable elements including at least ICEs and plasmids. The ICEs are probably widespread among the bacteria.
Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94DX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNAseq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.
SummaryIn vibrios and enterobacteria lateral gene transfer is often facilitated by integrating conjugative elements (ICEs) of the SXT/R391 family. SXT/R391 ICEs integrate by site-specific recombination into prfC and transfer by conjugation, a process that is initiated at a specific locus called the origin of transfer (oriTSXT). We identified genomic islands (GIs) harbouring a sequence that shares > 63% identity with oriTSXT in three species of Vibrio. Unlike SXT/R391 ICEs, these GIs are integrated into a gene coding for a putative stress-induced protein and do not appear to carry any gene coding for a conjugative machinery or for mobilization proteins. Our results show that SXT/R391 ICEs trigger the excision and mediate the conjugative transfer in trans of the three Vibrio GIs at high frequency. GIs' excision is independent of the ICEencoded recombinase and is controlled by the ICEencoded transcriptional activator SetCD, which is expressed during the host SOS response. Both mobI and traI, two ICE-borne genes involved in oriT recognition, are essential for GIs' transfer. We also found that SXT/R391 ICEs mobilize in trans over 1 Mb of chromosomal DNA located 5Ј of the GIs' integration site. Together these results support a novel mechanism of mobilization of GIs by ICEs of the SXT/R391 family.
The Vibrio cholerae SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics. SXT integrates in a site-specific fashion at prfC and excises from the chromosome to form a circular but nonreplicative extrachromosomal form. Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int. Here we found that Int is necessary and sufficient for SXT integration and that int expression in recipient cells requires the SXT activators SetC and SetD. Although no xis-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision. We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration. Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs. Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors. In the absence of prfC, SXT integrated into several secondary attachment sites but preferentially into the 5 end of pntB. SXT excision and transfer from a donor containing pntB::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility.Integrative and conjugative elements (ICEs) are now recognized as a large and diverse class of mobile genetic elements in both gram-negative and gram-positive organisms (9). ICEs excise from the chromosomes of their hosts, transfer to a new host via conjugation, and then integrate into the chromosome again. These elements encode diverse excision, recombination, and conjugation systems, as well as many other properties, such as resistance to antibiotics (25, 30), nitrogen fixation (27), and degradation of aromatic compounds (24). ICE integration can be more or less site specific, and a large variety of sequences are used as targets for integration.The SXT element is a Vibrio cholerae-derived ICE that has also been referred to as a conjugative transposon (29) and a constin (17). SXT was originally isolated in 1993 from MO10, a V. cholerae serogroup O139 clinical isolate. The MO10-derived SXT, SXT MO10 , encodes resistance to sulfamethoxazole, trimethoprim, chloramphenicol, and streptomycin (29). After the extensive cholera outbreaks on the Indian subcontinent caused by V. cholerae O139 in late 1992 and 1993, V. cholerae O1 reemerged and was found to harbor an ICE, designated SXT ET , that is very similar to SXT MO10 but contains different antibiotic resistance genes (15). Other SXT variants that lack antibiotic resistance genes have also been recognized in recent V. cholerae O139 isolates. SXT-related ICEs are cu...
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