Possible origins of CTnBST, a conjugative transposon found recently in a human colonic Bacteroides strain

A previous survey of Bacteroides isolates suggested that the ermB gene entered Bacteroides spp. recently. Previously, ermB had been found almost exclusively in gram-positive bacteria. In one Bacteroides strain, ermB was located on 100-kb conjugative transposon (CTn) CTnBST. To assess the possible origin of this CTn, we obtained the full DNA sequence of CTnBST and used this information to investigate its possible origins. Over one-half of CTnBST had high sequence identity to a putative CTn found in the genome of Bacteroides fragilis YCH46. This included the ends of the CTn and genes involved in integration, transfer, and excision. However, the region around the ermB gene contained genes that appeared to originate from gram-positive organisms. In particular, a 7-kb segment containing the ermB gene was 100% identical to an ermB region found in the genome of the gram-positive bacterium Arcanobacterium pyogenes. A screen of Bacteroides isolates whose DNA cross-hybridized with a CTnBST probe revealed that several isolates did not carry the 7-kb region, implying that the acquisition of this region may be more recent than the acquisition of the entire CTnBST element by Bacteroides spp. We have also identified other Bacteroides isolates that carry a slightly modified 7-kb region but have no other traces of CTnBST. Thus, it is possible that this 7-kb region could itself be part of a mobile element that has inserted in a Bacteroides CTn. Our results show that CTnBST is a hybrid element which has acquired a portion of its coding region from gram-positive bacteria but which may originally have come from Bacteroides spp. or some related species.     

Characterization of a conjugative transposon integrase, IntDOT

Sequence analysis revealed that the integrase of the Bacteroides conjugative transposon CTnDOT (IntDOT) might be a member of the tyrosine recombinase family because IntDOT has five of six highly conserved residues found in the catalytic domains of tyrosine recombinases. Yet, IntDOT catalyses a reaction that appears to differ in some respects from well-studied tyrosine recombinases such as that of phage lambda. To assess the importance of the conserved residues, we changed residues in IntDOT that align with conserved residues in tyrosine recombinases. Some substitutions resulted in a complete loss or significant decrease of integration activity in vivo. The ability of the mutant proteins to cleave and ligate CTnDOT attachment site (attDOT) DNA in vitro in general paralleled the in vivo results, but the H345A mutant, which had a wild-type level of integration in vivo, exhibited a slightly lower level of cleavage and ligation in vitro. Our results confirm the hypothesis that IntDOT belongs to the tyrosine recombinase family, but the catalytic core of the protein seems to have somewhat different organization. Previous DNA sequence analyses showed that CTnDOT att sites contain 5 bp non-homologous coupling sequences which were assumed to define the putative staggered sites of cleavage. However, cleavage assays showed that one of the cleavage sites is 2 bp away from the junction of CTnDOT and coupling sequence DNA. The site is in a region of homology that is conserved in CTnDOT att sites.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

A newly discovered Bacteroides conjugative transposon,CTnGERM1, contains genes also found in gram-positive bacteria

Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.     

Transfer region of a bacteroides conjugative transposon, CTnDOT

Bacteroides species harbor large self-transmissible integrated elements called conjugative transposons (CTns). In this paper, we report the first complete sequence analysis of the transfer region of a Bacteroides CTn. The transfer region contained 17 genes (designated orfA-orfQ). Only 2 of the genes shared sequence similarity with genes in the databases and only 1 of these genes was associated with self-transmissible elements.     

Tn1545: a conjugative shuttle transposon

Summary Tn1545, from Streptococcus pneumoniae BM4200, confers resistance to kanamycin (aphA-3), erythromycin (ermAM) and tetracycline (tetM). The 25.3 kb element is self-transferable to various Gram-positive bacterial genera where it transposes. Tn1545 was cloned in its entirety in the recombination deficient Escherichia coli HB101 where it was unstable. The three resistance genes aphA-3, ermAM and tetM were expressed but were not transferable to other E. coli cells. Tn1545 transposed from the hybrid plasmid to multiple sites of the chromosome of its new host. The element re-transposed, at a frequency of 5×10^-9, from the chromosome to various sites of a conjugative plasmid where it could be lost by apparently clean excision. The element transformed and transposed to the chromosome of Bacillus subtilis. The properties of the conjugative shuttle transposon Tn1545 may account for the recent emergence of genes from Gram-positive bacteria in Gramnegative organisms.     

Tn5381, a conjugative transposon identifiable as a circular form in Enterococcus faecalis.

We have identified two 19-kb conjugative transposons (Tn5381 and Tn5383) in separate strains of multiply resistant Enterococcus faecalis. These transposons confer resistance to tetracycline and minocycline via a tetM gene, are capable of both chromosomal and plasmid integration in a Rec- environment, and transfer between strains in the absence of detectable plasmid DNA at frequencies ranging from < 1 x 10(-9) to 2 x 10(-5) per donor CFU, depending on the donor strain and the growth conditions. Hybridization studies indicate that these transposons are closely related to Tn916. We have identified bands of ca. 19 kb on agarose gel separations of alkaline lysis preparations from E. faecalis strains containing chromosomal copies of Tn5381, which we have confirmed to be a circularized form of this transposon. This phenomenon has previously been observed only when Tn916 has been cloned in Escherichia coli. Overnight growth of donor strains in the presence of subinhibitory concentrations of tetracycline results in an approximately 10-fold increase in transfer frequency of Tn5381 into enterococcal recipients and an increase in the amount of the circular form of Tn5381 as detectable by hybridization. These results suggest that Tn5381 is a Tn916-related conjugative transposon for which the appearance of a circular form and the conjugative-transfer frequency are regulated by a mechanism(s) affected by the presence of tetracycline in the growth medium.     

Integration and excision of a Bacteroides conjugative transposon, CTnDOT

Bacteroides conjugative transposons (CTns) are thought to transfer by first excising themselves from the chromosome to form a nonreplicating circle, which is then transferred by conjugation to a recipient. Earlier studies showed that transfer of most Bacteroides CTns is stimulated by tetracycline, but it was not known which step in transfer is regulated. We have cloned and sequenced both ends of the Bacteroides CTn, CTnDOT, and have used this information to examine excision and integration events. A segment of DNA that contains the joined ends of CTnDOT and an adjacent open reading frame (ORF), intDOT, was necessary and sufficient for integration into the Bacteroides chromosome. Integration of this miniature form of the CTn was not regulated by tetracycline. Excision of CTnDOT and formation of the circular intermediate were detected by PCR, using primers designed from the end sequences. Sequence analysis of the PCR products revealed that excision and integration involve a 5-bp coupling sequence-type mechanism possibly similar to that used by CTn Tn916, a CTn found originally in enterococci. PCR analysis also demonstrated that excision is a tetracycline-regulated step in transfer. The integrated minielement containing intDOT and the ends of CTnDOT did not excise, nor did a larger minielement that also contained an ORF located immediately downstream of intDOT designated orf2. Thus, excision involves other genes besides intDOT and orf2. Both intDOT and orf2 were disrupted by single-crossover insertions. Analysis of the disruption mutants showed that intDOT was essential for excision but orf2 was not. Despite its proximity to the integrase gene, orf2 appears not to be essential for excision.     

Transfer of a conjugative transposon, Tn5397 in a model oral biofilm

A tetracycline resistance profile was established from a microcosm dental plaque in a constant depth film fermenter. The fermenter was inoculated with a Bacillus subtilis strain which contained the conjugative transposon, Tn5397, which confers tetracycline resistance upon its host. After 6 hour and 24 hour the tetracycline resistance profile of the biofilm was redetermined and a tetracycline resistant Streptococcus species was isolated. A molecular analysis of this strain confirmed that Tn5397 was present in the genomic DNA of the isolate. These data represent the first report, to our knowledge, of intergeneric transfer of a conjugative transposon in a mixed species biofilm and demonstrates the ability of conjugative transposons to disseminate antibiotic resistance genes in a mixed species environment.     

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853.

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.     

Integration and excision of a newly discovered bacteroides conjugative transposon, CTnBST

Conjugative transposons (CTns) are major contributors to the spread of antibiotic resistance genes among Bacteroides species. CTnBST, a newly discovered Bacteroides conjugative transposon, carries an erythromycin resistance gene, ermB, and previously has been estimated to be about 100 kbp in size. We report here the locations and sequencing of both of its ends. We have also located and sequenced the gene that catalyzes the integration of CTnBST, intBST. The integrase gene encodes a 377-amino-acid protein that has the C-terminal R-K-H-R-H-Y motif that is characteristic of members of the tyrosine recombinase family of integrases. DNA sequence comparisons of the ends of CTnBST, the joined ends of the circular intermediate, and the preferred site into which the circular form of CTnBST had integrated revealed that the preferred integration site (attB1) contained an 18-bp sequence of identity to the crossover region, attBST, on CTnBST. Although this site was used in about one-half of the integration events, sequence analysis of these integration events revealed that both CTnBST and a miniature form of CTnBST (miniBST) integrated into a variety of other sites in the chromosome. All of the sites had two conserved regions, AATCTG and AAAT. These two regions flanked a 2-bp sequence, bp 10 and bp 11 of the 18-bp sequence, that varied in some of the different sites and sometimes in the attBST sequences. Our results suggest that CTnBST integrates site selectively and that the crossover appears to occur within a 12-bp region that contains the two regions of conserved sequences.     

Sequence analysis of termini of conjugative transposon Tn916.

Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.     

Conditions for conjugative transposon transfer in Lactococcus lactis

Three different techniques for bacterial mating were applied to wild type and culture collection strains of Lactococcus lactis harbouring transposons: direct plate conjugation, filter mating and mating on milk agar. Efficiencies and frequencies of transfer were compared. Transconjugants were characterized by marker properties and molecular assays. Transposon-coded Suc+ Nis+ phenotype as well as Suc+ Bac+ Nis- phenotype were transferred with frequencies ranging between 10-9 and 10-6. Milk agar plate mating was the best technique for obtaining gene transfer events involving wild type lactococci.     

Genetic organization of the bacterial conjugative transposon Tn916.

Tn916, which encodes resistance to tetracycline, is a 16.4-kilobase conjugative transposon originally identified on the chromosome of Streptococcus faecalis DS16. The transposon has been cloned in Escherichia coli on plasmid vectors, where it expresses tetracycline resistance; it can be reintroduced into S. faecalis via protoplast transformation. We have used a lambda::Tn5 bacteriophage delivery system to introduce Tn5 into numerous sites within Tn916. The Tn5 insertions had various effects on the behavior of Tn916. Some insertions eliminated conjugative transposition but not intracellular transposition, and others eliminated an excision step believed to be essential for both types of transposition. A few inserts had no effect on transposon behavior. Functions were mapped to specific regions on the transposon.     

Identification of genes required for excision of CTnDOT, a Bacteroides conjugative transposon

Integrated self-transmissible elements called conjugative transposons have been found in many different bacteria, but little is known about how they excise from the chromosome to form the circular intermediate, which is then transferred by conjugation. We have now identified a gene, exc, which is required for the excision of the Bacteroides conjugative transposon, CTnDOT. The int gene of CTnDOT is a member of the lambda integrase family of recombinases, a family that also contains the integrase of the Gram-positive conjugative transposon Tn916. The exc gene was located 15 kbp from the int gene, which is located at one end of the 65 kbp element. The exc gene, together with the regulatory genes, rteA, rteB and rteC, were necessary to excise a miniature form of CTnDOT that contained only the ends of the element and the int gene. Another open reading frame (ORF) in the same operon and upstream of exc, orf3, was not essential for excision and had no significant amino acid sequence similarity to any proteins in the databases. The deduced amino acid sequence of the CTnDOT Exc protein has significant similarity to topoisomerases. A small ORF (orf2) that could encode a small, basic protein comparable with lambda and Tn916 excision proteins (Xis) was located immediately downstream of the CTnDOT int gene. Although Xis proteins are required for excision of lambda and Tn916, orf2 had no effect on excision of the element. Excision of the CTnDOT mini-element was not affected by the site in which it was integrated, another difference from Tn916. Our results demonstrate that the Bacteroides CTnDOT excision system is tightly regulated and appears to be different from that of any other known integrated transmissible element, including those of some Bacteroides mobilizable transposons that are mobilized by CTnDOT.     

Autonomous plasmid‐like replication of a conjugative transposon

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra‐chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid‐like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1‐encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid‐like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.     

Nisin biosynthesis genes are encoded by a novel conjugative transposon

Summary Genes for biosynthesis of the lactococcal peptide antibiotic nisin were shown to be encoded by a novel chromosomally located transposon Tn5301. The element is 70 kb in size and lacks inverted repeats at its termini. Although a copy of the insertion sequence IS904 is located near to one end, this did not appear to be involved in the transposition process. The integrated element is flanked by the directly repeated sequence 5-TTTTTG-3. Analysis of ten independent transconjugants revealed that Tn5301 integration is site-specific; two chromosomal targets were identified and shown to have some sequence homology. The element shares features with the Tn916 family of conjugative transposons and with Tn554 but is also exhibits some unique properties. Tn5301 is thus considered to be the prototype of a novel class of conjugative transposon.     

Generation of Tn5 insertions in streptococcal conjugative transposon Tn916.

A method has been developed for the introduction of Tn5 into Escherichia coli plasmid chimeras containing Streptococcus faecalis DNA. Tn5 could be introduced via a lambda::Tn5 delivery vehicle. The system proved to be particularly efficient and facilitated insertions at numerous sites on DNA containing the 16-kilobase conjugative transposon Tn916. It was possible to introduce some of the resulting Tn916::Tn5 derivatives back into S. faecalis by using a recently developed protoplast transformation procedure. A presumed zygotic induction resulted in insertion of the Tn916 derivatives at multiple sites in the S. faecalis chromosome.     

A group II intron in a conjugative transposon from the gram-positive bacterium,Clostridium difficile

We have been studying the conjugative transposon Tn5397, originally isolated from the Gram-positive pathogen Clostridium difficile. Physical analysis of this transposon demonstrated that it contained a group II intron. This is the first report of an intron in a conjugative transposon and the first report of a group II intron in Gram-positive bacteria. The intron interrupted a gene in Tn5397 that is almost identical to orf14 from Tn916. DNA hybridisation analysis showed that elements related to Tn5397, containing the group II intron, were present in five other C. difficile strains from different geographical locations suggesting that the element is likely to be widely distributed.