No abstract

Mobile genetic elements have a crucial role in spreading antibiotic resistance genes among bacterial populations. Environmental and genetic factors that regulate conjugative transfer of antibiotic resistance genes in bacterial populations are largely unknown. Integrating conjugative elements (ICEs) are a diverse group of mobile elements that are transferred by means of cell-cell contact and integrate into the chromosome of the new host. SXT is a approximately 100-kilobase ICE derived from Vibrio cholerae that encodes genes that confer resistance to chloramphenicol, sulphamethoxazole, trimethoprim and streptomycin. SXT-related elements were not detected in V. cholerae before 1993 but are now present in almost all clinical V. cholerae isolates from Asia. ICEs related to SXT are also present in several other bacterial species and encode a variety of antibiotic and heavy metal resistance genes. Here we show that SetR, an SXT encoded repressor, represses the expression of activators of SXT transfer. The 'SOS response' to DNA damage alleviates this repression, increasing the expression of genes necessary for SXT transfer and hence the frequency of transfer. SOS is induced by a variety of environmental factors and antibiotics, for example ciprofloxacin, and we show that ciprofloxacin induces SXT transfer as well. Thus, we present a mechanism by which therapeutic agents can promote the spread of antibiotic resistance genes.

Many recent Asian clinical Vibrio cholerae E1 Tor O1 and O139 isolates are resistant to the antibiotics sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm), and streptomycin (Sm). The corresponding resistance genes are located on large conjugative elements (SXT constins) that are integrated into prfC on the V. cholerae chromosome. We determined the DNA sequences of the antibiotic resistance genes in the SXT constin in MO10, an O139 isolate. In SXT MO10 , these genes are clustered within a composite transposon-like structure found near the element's 5 end. The genes conferring resistance to Cm (floR), Su (sulII), and Sm (strA and strB) correspond to previously described genes, whereas the gene conferring resistance to Tm, designated dfr18, is novel. In some other O139 isolates the antibiotic resistance gene cluster was found to be deleted from the SXT-related constin. The El Tor O1 SXT constin, SXT ET , does not contain the same resistance genes as SXT MO10 . In this constin, the Tm resistance determinant was located nearly 70 kbp away from the other resistance genes and found in a novel type of integron that constitutes a fourth class of resistance integrons. These studies indicate that there is considerable flux in the antibiotic resistance genes found in the SXT family of constins and point to a model for the evolution of these related mobile elements.The intercellular spread of the genetic determinants of resistance to antimicrobial agents is facilitated by mobile genetic elements, such as conjugative plasmids and conjugative transposons. The antibiotic resistance genes in these elements are often located within transposons and/or integrons, elements that facilitate the intracellular movement of genes. Two types of transposons have been found to contain resistance genes. Class I transposons, also known as composite transposons, consist of two insertion sequence (IS) elements that flank additional DNA sequences, such as resistance genes. Class II transposons do not contain recognizable IS elements; instead, the genetic information for their transposition and other phenotypes (including antibiotic resistances) is bordered by 35-to 110-bp inverted repeats (reviewed in reference 10). Integrons also play a major role in the spread of antibiotic resistance genes in gram-negative bacteria (32). Integrons are gene-capturing systems that incorporate gene cassettes and convert them to functional genes (31, 32). Integrons characteristically encode an integrase (intI) that mediates recombination between a sequence in the gene cassette (attC) and an integronassociated sequence (attI). This results in integration of the cassette downstream of a resident promoter to permit expression of the encoded protein. While integrons often are found in plasmids and usually contain antibiotic resistance genes, they can also be located on the chromosome and can contain genes that do not specify resistance to antibiotics (4, 26). To date, three classes of resistance integrons have been described based on similarities in the i...

SXT is representative of a family of conjugative-transposon-like mobile genetic elements that encode multiple antibiotic resistance genes. In recent years, SXT-related conjugative, self-transmissible integrating elements have become widespread in Asian Vibrio cholerae. We have determined the ϳ ϳ100-kb DNA sequence of SXT. This element appears to be a chimera composed of transposon-associated antibiotic resistance genes linked to a variety of plasmid-and phage-related genes, as well as to many genes from unknown sources. We constructed a nearly comprehensive set of deletions through the use of the one-step chromosomal gene inactivation technique to identify SXT genes involved in conjugative transfer and chromosomal excision. SXT, unlike other conjugative transposons, utilizes a conjugation system related to that encoded by the F plasmid. More than half of the SXT genome, including the composite transposon-like structure that contains its antibiotic resistance genes, was not required for its mobility. Two SXT loci, designated setC and setD, whose predicted amino acid sequences were similar to those of the flagellar regulators FlhC and FlhD, were found to encode regulators that activate the transcription of genes required for SXT excision and transfer. Another locus, designated setR, whose gene product bears similarity to lambdoid phage CI repressors, also appears to regulate SXT gene expression.

SummaryVibrio cholerae O139, the ®rst non-O1 serogroup of V. cholerae to give rise to epidemic cholera, is characteristically resistant to the antibiotics sulphamethoxazole, trimethoprim, chloramphenicol and streptomycin. Resistances to these antibiotics are encoded by a 62 kb self-transmissible, conjugative, chromosomally integrating element designated the`SXT element'. We found that the SXT element integrates site speci®cally into both V. cholerae and Escherichia coli K-12 into the 58 end of prfC, the gene encoding peptide chain release factor 3. Integration of the SXT element interrupts the chromosomal prfC gene, but the element encodes a new 58 end of prfC that restores the reading frame of this gene. The recombinant prfC allele created upon element integration is functional. The integration and excision mechanism of the SXT element shares many features with site-speci®c recombination found in lambdoid phages. First, like l, the SXT element forms a circular extrachromosomal intermediate through speci®c recombination of the left and right ends of the integrated element. Second, chromosomal integration of the element occurs via site-speci®c recombination in a 17 bp sequence found in the circular form of the SXT element and a similar 17 bp sequence in prfC. Third, both chromosomal integration and excision of the SXT element were found to require an element-encoded int gene with strong similarities to the l integrase family. Based on the properties of the SXT element, we propose to classify this element as a CONSTIN, an acronym for a conjugative, self-transmissible, integrating element.

The SXT element, a conjugative, self-transmissible, integrating element (a constin) originally derived from a Vibrio cholerae O139 isolate from India, and IncJ element R391, originally derived from a South African Providencia rettgeri isolate, were found to be genetically and functionally related. Both of these constins integrate site specifically into the Escherichia coli chromosome at an identical attachment site within the 5 end of prfC. They encode nearly identical integrases, which are required for chromosomal integration, excision, and extrachromosomal circularization of these elements, and they have similar tra genes. Therefore, these closely related constins have virtually identical mechanisms for chromosomal integration and dissemination. The presence of either element in a recipient cell did not significantly reduce its ability to acquire the other element, indicating that R391 and SXT do not encode surface exclusion determinants. In cells harboring both elements, SXT and R391 were integrated in tandem fashion on the chromosome, and homologous recombination appeared to play little or no role in the formation of these arrays. Interference between R391 and SXT was detected by measuring the frequency of loss of an unselected resident element upon introduction of a second selected element. In these assays, R391 was found to have a stronger effect on SXT stability than vice versa. The level of expression and/or activity of the donor and recipient integrases may play a role in the interference between these two related constins.Similar genetic elements tend not to coexist within the same host cell. Instead, related elements of the same class usually "repel" one another in some fashion. For different types of genetic elements, the molecular bases of incompatibility differ. Plasmid incompatibility, for example, is generally mediated by competition for replication and/or partitioning systems (19). Conjugative plasmids also frequently inhibit host cell entry of related plasmids by altering the host cell's surface (1). Similarly, some bacteriophages alter the surface of host cells to exclude other phages. Phages can also prevent the replication of similar phages through immunity mechanisms (25). Thus, both plasmid and phage incompatibility can depend either on preventing entry of new DNA into a potential host or on inhibiting the replication of new DNA after it has breached the host cell barrier.Chromosomally integrating mobile genetic elements that are transferred between cells via conjugation-often referred to as conjugative transposons-have been found with increasing frequency in both gram-negative and gram-positive bacteria. Unlike the case for phages and plasmids, relatively little is known about whether similar conjugative transposons can coexist within the same host cell. The well-studied conjugative transposon Tn916, which does not integrate site specifically, can be present in more than one copy at different sites in the host cell chromosome, and the presence of Tn916 in a recipient cell does not inhibit...

The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II 536 and PAI III 536 were the most unstable islands followed by PAI I 536 and PAI V 536 , whereas PAI IV 536 was stable. In addition, we found that deletion of PAI II 536 and PAI III 536 was induced by several environmental stimuli. Whereas excision of PAI I 536 , PAI II 536 , and PAI V 536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III 536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II 536 and PAI III 536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.

SummaryThe genome of uropathogenic Escherichia coli isolate 536 contains five well-characterized pathogenicity islands (PAIs) encoding key virulence factors of this strain. Except PAI IV536, the four other PAIs of strain 536 are flanked by direct repeats (DRs), carry intact integrase genes and are able to excise sitespecifically from the chromosome. Genome screening of strain 536 identified a sixth putative asnWassociated PAI. Despite the presence of DRs and an intact integrase gene, excision of this island was not detected. To investigate the role of PAI-encoded integrases for the recombination process the int genes of each unstable island of strain 536 were inactivated. For PAI I 536 and PAI II536, their respective P4-like integrase was required for their excision. PAI III536 carries two integrase genes, intA, encoding an SfX-like integrase, and intB, coding for an integrase with weak similarity to P4-like integrases. Only intB was required for site-specific excision of this island. For PAI V536, excision could not be abolished after deleting its P4-like integrase gene but additional deletion of the PAI II536-specific integrase gene was required. Therefore, although all mediated by P4-like integrases, the activity of the PAI excision machinery is most often restricted to its cognate island. This work also demonstrates for the first time the existence of a cross-talk between integrases of different PAIs and shows that this cross-talk is unidirectional.