Final Discussions
8.0 Final conclusions and scope for further work
The determination o f the entire nucleotide sequence o f the conjugative transposon
Tn5397 from C difficile has been completed. Analysis o f the sequence has revealed a
mosaic structure composed o f different functional modules that show homology to genes from different genetic elements that are themselves found in different bacterial hosts. The data obtained have given insights into the molecular evolution o f these elements. It is clear that Tn5397 is comprised o f conjugation, resistance and regulation modules that are related to Tn916. The integration/excision module is related to that o f the non-
conjugative but mobilisable transposons Tn4451 and Tn4453 originally found in C.
perfringens and C. difficile respectively. It is possible therefore that recombination
reactions between these two types o f elements may have resulted in a hybrid element such
as Tn5397. As the G+C content ofTn.5397 is different to that o f the C. difficile genome it
seems unlikely that Tn5397 is a native element in C. difficile. Therefore the
recombination reactions that resulted in the formation o f this element may have occurred in a different, unrelated host and subsequently transferred to C. difficile. This may explain how the group II intron, which is most closely related to the group II intron from B.
megaterium transposon Tr\MERIl (Huang et al, 1999), was acquired. Alternatively, all of
the component modules present on Tn55P7 may have arrived in the C. difficle cell independently and subsequently recombined to form Tn5397. In support o f the latter o f these two explanations for the formation o f Tn55P7, both Tn916 (Wang et al, 2001) and
hypotheses are not mutually exclusive, Tn5397 may have resulted from a combination of both. It is also important that putative, alternative start codons have been identified for some o f the Tn916 orfs. These alternative start codons were found as a direct result o f comparing the entire nucleotide sequences o f Tn55P7 and Tn916. More analysis is
needed to identify exactly the length o f these genes as this could provide valuable insights into the functionality o f the proteins concerned. More work is needed to understand the interactions o f this family o f mobile elements, both with other members o f the Tn916
family and completely unrelated mobile genetic elements such as plasmids and phages. Understanding the interactions between different mobile elements may explain why
Tn916 modules or individual genes are so ubiquitous in the bacterial world.
This work has also contributed to a fuller understanding o f the mechanisms o f excision and insertion o f Tn55P7. A hypothesis was proposed based on studies o f the tndX mutant and the sequence o f the transposon - genome junction regions and the target sites in both the C. difficile and B. subtilis genomes. The proposed model for the TndX mediated excision and insertion o f Tn55P7 is mechanistically identical to that thought to occur for the clostridial mobilisable transposons Tn4451 and Tn.4453. An interesting observation is the target site for insertion o f both Tn5iP7 and TnP7d is highly conserved in C. difficile.
This raises questions about the suitability o f conjugative transposons for use as genetic tools. While they would not be useful as insertional mutagens they would be very useful for inserting DNA into a specific region o f the genome. However, considerations such as the maximum amount o f DNA that can be ‘inserted’ into a conjugative transposon and
the overall host range o f these elements need to be addressed before their full potential as genetic tools can be realised.
Transcription from the 3 ' end o f tetQsA) has been shown to be inducible by addition of tetracycline to the growth medium. Analysis o f the nucleotide sequence o f Tn55P7 has revealed a major deletion when compared to homologous region in TnP7d which is thought to be involved in regulating the transcription o f tet(M) and downstream genes (Su
et al, 1995). Despite this deletion, two hypotheses have been put forward to explain the
inducible nature o f the transcription seen in the RT-PCR experiments. Both hypotheses differ to that put forward for TnP7(5 but are still based on the secondary structure o f the primary RNA transcript and the formation o f terminator stem-loop structures. Both hypotheses offer a plausible explanation o f what might be happening at the molecular level when tetracycline is present. That some degree o f control is present even after such a deletion event is expected because the continued transcription o f the tet(M) gene in the absence o f tetracycline would be metabolically wasteful and therefore the deletant would probably be selected out o f a population. The main focus o f future work needs to be the understanding o f the regulation o f gene expression in both TnP7d and Tn55P7. Only with a detailed knowledge o f the mechanisms that control the expression o f the
integration/excision genes and the conjugation-related genes can we begin to control the spread o f these elements in our environment. Efforts also need to be directed towards discovering the mechanisms o f conjugative transfer, as remarkably little is known about how the transfer o f the DNA actually occurs.
The effect o f the group II intron on the mobility o f Tn55P7 has been investigated. The intron has been shown to splice from the pre-mRNA transcripts o f the orfl 4 gene in both
C. difficile and B. suhtilis. However, it has been shown that splicing is not essential for
conjugative transposition o f TnJJP7. Host factors in B. subtilis may complement the function o f O rfl 4, alternatively the portion o f Orfl 4 translated may be sufficient for function in the cell, as the intron has entered the gene very close to the 3' end. It is interesting that the intron has retained the ability to splice when it appears that splicing is not necessary. Due to the retained ability o f the intron to splice, it may be able to reverse- transcribe into ectopic sites affording itself more chance o f dissemination.
Our knowledge o f the host range o f Tn5JP7 has been extended. A streptococcal recipient has been identified within a microcosm dental plaque. This now provides us with data that shows transfer o f conjugative transposons within oral and gut communities (Doucet- Populaire et al, 1991), the two sites where large numbers of different bacteria are able to come together in close contact. Future work in this area should concentrate on
determining the likelihood and frequency o f transfer o f conjugative transposons from oral communities to the enteric organisms and subsequently into the environment.
It is now becoming accepted that conjugative transposons, such as Tn55P7 and TnP7d, are responsible for the majority o f the observed spread o f antibiotic resistance among bacteria. Therefore, if the problem o f the spread o f antibiotic resistance genes is ever going to be properly addressed and ultimately controlled, continued work on Tn5397 and other conjugative transposons is essential.
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