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Initially a SuperCos 1 (Stratagene) cosmid library was constructed from C. difficile 630 genomic DNA. Selection for Tn5397 positive clones was carried out by selecting for tetracycline resistant colonies and by Southern colony hybridisations. The resulting clones from these experiments were either equal in size to a previous cosmid clone, pC D Cl (Mullany et al., 1996) or internal to it, as determined by restriction analysis. These clones were not investigated further.

3.2.1.2 Determination of the Sequences at the End of Tn5JP7

Primers were designed to amplify the joint o f a circular form o f Tn53P7. Previous hybridisation analysis had shown that the central region o f TnP7d is homologous to

Tn5397 (Hachler et a l, 1987a; Mullany et a l, 1990). Primers were therefore designed

from the Tn916 homologous region that would read into the Tn5397 specific regions. Primer AAB (appendix 1) was designed from the left end o f TnP7d. Primer 5397 (Figure 3.1, Appendix 1) was designed from the right end o f Tn55P7. (Mullany et a l, 1990;

Mullany et a l, 1996) A 1265 bp product was produced and subsequent sequence analysis o f this demonstrated that it consisted o f the ligated ends o f a circular form o f Tn5iP7, thus enabling the joint o f the left and right ends in the circular form to be identified. Based on the sequence o f the left end o f the Tn55P7, a primer (LEO) reading out o f the transposon was designed. Primers 5397 and LEO were used to prime genomic sequencing reactions on DNA from C. difficile 630. The resulting genomic sequence was used to design primers LEI and REI. These were used to prime genomic sequencing reactions in strains that contained TnJ5P7 and isogenic strains in which the transposon had been lost. Primer pairs 5397 and REI and LEO and LEI were used to amplify the junctions o f

Tn5397 and the genomic DNA. Primer pair LEI and REI was used to amplify the empty

target site in strains that had lost TnJ5P7. (Figure 3.1). PCR reactions to detect the circular form o f Tn55P7 and to amplify the junction regions were carried out in C.

difficile strain 630, amplification o f the empty target site was carried out in strain FM I 28

(CD37::TnP7dAE Tn55P7 excisant) and FM136 (CD37::TnP7dAE Tn55P7 excisant), (Wang et a l, 2000a).

L E I ^ _{5397 ►}

Excision and circularisation Regeneration o f

target site after excision 4 REI A A B PCR 5397 i p C R A A B » ^ 4 REI 400 bp 1265 bp <4 REI

Figure 3.1. Strategy used to obtain the DNA sequence o f the ends o f Tn55P7. The unfilled box represents T n 5iP 7, the thin line represents the flanking genom ic D N A. The filled arrows show the position and direction o f binding o f the oligonucleotides (appendix 1) that were used in the PCR and genom ic sequencing reactions. The circular form o f Tn55P7 is also shown together with the region that was amplified by PCR. The vertical line shown in the target represents the joint o f the sequences that were previously on each side o f the transposon. The sizes o f the PCR products are shown underneath the cognate product.

3.2.1.3 Sequencing the Remainder of Tn55P7

The area o f T n5iP7 labelled pCDCl on Figure 3.2 was previously sequenced (Mullany et

a/., 1996). To sequence the remainder o f the element, primers were designed from the

Tn916 sequence data and used in PCR reactions on DNA from donor, recipient and

sequencing C. difficile strain 630, their sequence was also used as a template to design primers for use in PCR reactions. PCR products obtained were sequenced on both strands as detailed in Chapter 2. All primer names and sequences can be found in appendix 1 and their relative positions on Tn5J97 can be seen in Figure 3.2.

p C D C l ► ► 7 2/ 20 /9/g/7 15 14 G r o u p 11 i nt r on 1 4 13 2 5 2 6 te t{ M ) 6 9 10 7 8 tn d X 2 J L 8 10 12 14 16 18 I I I I I I I I I I I 20 F i g u r e 3. 2 P o s it i o n o f th e p r i m e r s th at w e r e d e s i g n e d fro m the T w 9 1 6 s e q u e n c e , S a n g e r c e n t r e s e q u e n c e a n d b y p r i m e r w a l k in g . T h e a r e a la b e lle d p C D C l is th e c o s m i d c lo n e p r e v i o u s l y s e q u e n c e d ( M u l l a n y et al., 1 9 9 5 ). T h e p r i m e r s a re in d ic a te d b y a r r o w s p o i n t i n g in th e d i r e c ti o n o f p r i m i n g .

3.2.2 C om puter Analysis of the Sequence Data.

Sequence analysis was carried out using Gene Jockey, (Apple M acintosh), sequence comparisons and advanced gapped BLAST (Altschul et a l , 1990) searches were carried out using the National Center for Biological Information (NCBI) tools

(w ww.ncbi.nlm.nih.gov/). In silico restriction endonuclease digests were carried out using W ebcutter 2.0 (www.firstmarket.com/cutter/cut2.html) Open reading frame analysis, and protein predictions were carried out using DNASIS (Hitachi, UK). Secondary structures

and RNA folding data was obtained at Michael Zucker’s RNA folding site (bioinfo.math.rpi.edu/~zukerm/ ) (Mathews et a l, 1999).

3.3 Results

The determination o f the entire DNA nucleotide sequence o f Tn53P7 confirmed and extended the previous data (Hachler et al., 1987; Mullany et a l, 1990; Mullany et a l,

1996). The sequence showed that the central region o f this element is very closely related to TnP7d (but with some important differences, discussed below) and the ends o f the two elements are completely different. The int and xis genes o f T nP /d have been replaced by the gene tndX (a member o f the large resolvase family o f site-specific recombinases). The significance o f this observation will be considered in detail in the next chapter. The other major difference is the presence o f a group II intron within Tn5397, this is considered in detail in Chapter 6.

The PCR, which aimed to amplify the ligated ends o f a circular form o f Tn5iP7, resulted in a product o f the expected size (The amplified product from C. difficile 630 can be seen in Figure 4.3, Chapter 4) and DNA sequencing confirmed that it contained the ligated ends o f Tn53P7. The PCR and sequencing strategy outlined above resulted in the DNA sequence o f Tn55P7 being unequivocally determined and the two ends o f the element identified.