Characterization of the ends and target sites of the novel conjugative transposon Tn5397 from Clostridium difficile: excision and circularization is mediated by the large resolvase, TndX

Tn5397 is a conjugative transposon that was originally isolated from Clostridium difficile. Previous analysis had shown that the central region of Tn5397 was closely related to the conjugative transposon Tn916. However, in this work we obtained the DNA sequence of the ends of Tn5397 and showed that they are completely different to those of Tn916. Tn5397 did not contain the int and xis genes, which are required for the excision and integration of Tn916. Instead, the right end of Tn5397 contained a gene, tndX, that appears to encode a member of the large resolvase family of site-specific recombinases. TndX is closely related to the TnpX resolvase from the mobilizable but nonconjugative chloramphenicol resistance transposons, Tn4451 from Clostridium perfringens and Tn4453 from C. difficile. Like the latter elements, inserted copies of Tn5397 were flanked by a direct repeat of a GA dinucleotide. The Tn5397 target sites were also shown to contain a central GA dinucleotide. Excision of the element in C. difficile completely regenerated the original target sequence. A circular form of the transposon, in which the left and right ends of the element were separated by a GA dinucleotide, was detected by PCR in both Bacillus subtilis and C. difficile. A Tn5397 mutant in which part of tndX was deleted was constructed in B. subtilis. This mutant was nonconjugative and did not produce the circular form of Tn5397, indicating that the TndX resolvase has an essential role in the excision and transposition of Tn5397 and is thus the first example of a member of the large resolvase family of recombinases being involved in conjugative transposon mobility. Finally, we showed that introduction of Tn916 into a strain containing Tn5397 induced the loss of the latter element in 95.6% of recipients.     

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.     

The conjugative transposon Tn5397 has a strong preference for integration into its Clostridium difficile target site

Tn5397 is a conjugative transposon, originally isolated from Clostridium difficile. The Tn5397 transposase TndX is related to the phage-encoded serine integrases and the Clostridium perfringens Tn4451 transposase TnpX. TndX is required for the insertion and excision of the transposon. Tn5397 inserts at one locus, attB(Cd), in C. difficile but at multiple sites in Bacillus subtilis. Apart from a conserved 5' GA dinucleotide at the recombination site, there appears to be little sequence conservation between the known target sites. To test the target site preference of Tn5397, attB(Cd) was introduced into the B. subtilis genome. When Tn5397 was transferred into this strain, 100% of the 50 independent transconjugants tested had Tn5397 inserted into attB(Cd). This experiment was repeated using a 50-bp attB(Cd) with no loss of target preference. The mutation of the 5' GA to 5' TC in the attB(Cd) target site caused a switch in the polarity of insertion of Tn5397, which is consistent with this dinucleotide being at the crossover site and in keeping with the mechanism of other serine recombinases. Tn5397 could also transpose into 50-bp sequences encoding the end joints attL and attR but, surprisingly, could not recombine into the circular joint of Tn5397, attTn. Purified TndX was shown to bind specifically to 50-bp attB(Cd), attL, attR, attTn, and attB(Bs)(3) with relative binding affinities attTn approximately attR > attL > attB(Cd) > attB(Bs3). We conclude that TndX has a strong preference for attB(Cd) over other potential recombination sites in the B. subtilis genome and therefore behaves as a site-specific recombinase.     

The large resolvase TndX is required and sufficient for integration and excision of derivatives of the novel conjugative transposon Tn5397

Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and from Bacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role of tndX, the gene was cloned and the protein was expressed in Escherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion in E. coli.     

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.     

Transfer of Tn916 and Tn916ΔE into Clostridium difficile: demonstration of a hot-spot for these elements in the C. difficile genome

The conjugative transposon Tn916 and a derivative Tn916 delta E was transferred from Bacillus subtilis into Clostridium difficile CD37 by filter mating. All the C. difficile transconjugants appeared to contain one copy of the transposon integrated into the same position in the genome. Transposition from the original site of integration was not observed. Like Tn916 the transferable tetracycline resistance determinant (Tc-CD) of C. difficile has a preferred site of integration in C. difficile and is homologous with Tn916 along the whole length of Tn916. However comparisons of the distribution of TaqI and Sau3AI sites in the homologous regions of the two elements did not demonstrate any hybridizing fragments in common.     

Requirements for intron-mediated enhancement of gene expression in Arabidopsis

To explore possible mechanisms of intron-mediated enhancement of gene expression, the features of PAT1 intron 1 required to elevate mRNA accumulation were systematically tested in transgenic Arabidopsis. This intron is remarkably resilient, retaining some ability to increase mRNA accumulation when splicing was prevented by mutation of 5' and 3' splice sites, branchpoint sequences, or when intron U-richness was reduced. Enhancement was abolished by simultaneously eliminating branchpoints and the 5' splice site, structures involved in the first two steps of spliceosome assembly. Although this suggests that the splicing machinery is required, intron splicing is clearly not enough to enhance mRNA accumulation. Five other introns were all efficiently spliced but varied widely in their ability to increase mRNA levels. Furthermore, PAT1 intron 1 was spliced but lost the ability to elevate mRNA accumulation when moved to the 3' UTR. These findings demonstrate that splicing per se is neither necessary nor sufficient for an intron to enhance mRNA accumulation, and suggest a mechanism that requires intron recognition by the splicing machinery but also involves nonconserved intron sequences.     

The effect of intron location on the splicing of BmKK2 in 293T cells

Previously reported results showed that the BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found in the second exon. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression level of the toxin-GFP fusion protein (Cao et al., J Biochem Mol Toxicol 2006;20:1-6). In this investigation, the BmKK2's intron with 79 nucleotides length was artificially shifted from the 49th nt (the 17th Gly codon between the first base and the second base) to the 100th nt (the 34th Gly codon between the first base and the second base). Based on the constructed intron-splicing system, the results of RT-PCR and the western blotting analysis showed that the BmKK2's shifted-intron (named BmKK2-s) was not recognized and spliced correctly, but the cryptic splicing site of BmKK2 gene was still spliced in the second exon, which possibly indicated that locations of introns were very important to the recognition and splicing of introns, and splicing of introns was very much associated with the corresponding upstream and downstream exons. This result possibly provides evidence for splice-site recognition across the exons.     

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.     

The development of Clostridium difficile genetic systems

Clostridum difficile is a major cause of healthcare-associated disease in the western world, and is particularly prominent in the elderly. Its incidence is rising concomitant with increasing longevity. More effective countermeasures are required. However, the pathogenesis of C. difficile infection is poorly understood. The lack of effective genetic tools is a principal reason for this ignorance. For many years, the only tools available for the transfer of genes into C. difficile have been conjugative transposons, such as Tn916, delivered via filter mating from Bacillus subtilis donors. They insert into a preferred site within the genome. Therefore, they may not be employed for classical mutagenesis studies, but can be employed to modulate gene function through the delivery of antisense RNA. Attempts to develop transformation procedures have so far met with little success. However, in recent years the situation has been dramatically improved through the demonstration of efficient conjugative transfer of both replication-proficient and replication-deficient plasmids from Escherichia coli donors. This efficient transfer can only be achieved in certain strains through negation of the indigenous restriction barrier, and is generally most effective when the plasmid employed is based on the replicon of the C. difficile plasmid, pCD6.     

The Frequency of Conjugative Transposition of Tn916 Is Not Determined by the Frequency of Excision

Excision and formation of a covalently closed circular transposon molecule are required for conjugative transposition of Tn916 but are not the only factors that limit the frequency of conjugative transposition from one host to another. We found that in gram-positive bacteria, an increase in the frequency of excision and circularization of Tn916 caused by expression of integrase (Int) and excisionase (Xis) from a xylose-inducible promoter does not lead to an increase in the frequency of conjugative transposition. We also found that the concentration of Int and Xis in the recipient cell does not limit the frequency of conjugative transposition and that increased excision does not result in increased expression of transfer functions required to mobilize a plasmid containing the Tn916 origin of transfer. We conclude that in gram-positive hosts in which the Tn916 functions Int and Xis are overexpressed, the frequency of conjugative transposition is limited by the availability of transfer functions.     

Behavior and target site selection of conjugative transposon Tn916 in two different strains of toxigenic Clostridium difficile

The insertion sites of the conjugative transposon Tn916 in the anaerobic pathogen Clostridium difficile were determined using Illumina Solexa high-throughput DNA sequencing of Tn916 insertion libraries in two different clinical isolates: 630ΔE, an erythromycin-sensitive derivative of 630 (ribotype 012), and the ribotype 027 isolate R20291, which was responsible for a severe outbreak of C. difficile disease. A consensus 15-bp Tn916 insertion sequence was identified which was similar in both strains, although an extended consensus sequence was observed in R20291. A search of the C. difficile 630 genome showed that the Tn916 insertion motif was present 100,987 times, with approximately 63,000 of these motifs located in genes and 35,000 in intergenic regions. To test the usefulness of Tn916 as a mutagen, a functional screen allowed the isolation of a mutant. This mutant contained Tn916 inserted into a gene involved in flagellar biosynthesis.