Circularisation of C difficile CTns

PCR was carried out using primers to amplify the junctions of the circular

intermediates of the CTns from strains 630, R20291, M120, QCD-23M63, QCD-66C26,

QCD-32G58 and ATCC-43255. These PCRs were performed with the Left End Out and

Right End Out (LEO and REO) primers (Table 4.1). In addition, the regenerated target

site in the chromosome was amplified with the Target Site Forward and Target Site

Reverse (TSF and TSR) primers (Table 4.1). The element-chromosome junctions were

amplified with primer pairs LEO + TSF and REO + TSR. Products of the expected size

were obtained for CTn1, CTn2, CTn4, CTn5 and CTn7 from strain 630, Tn6103 from

R20291, Tn6073 and Tn6107 from QCD-23M63, Tn6115 from QCD-63Q42, Tn6110

from QCD-66C26, Tn6111 from QCD-32G58 and Tn6164 from M120. An example for

this is shown for CTn5 in Figure 4.7. These PCR products were sequenced to determine

the direct repeats on either side of the element (Figure 4.8).

Figure 4.7 PCR products to show excision of CTn5 from the chromosome.

PCRs were carried out for the joints of the circular molecule, the regenerated target site and the ends of the transposon in the chromosome. Lane 1 is product of the joint of the circular intermediate of CTn5 using primers CTn5 LEO+REO. Lane 2 is product of the regenerated target site within the chromosome using primers CTn5 TSF+TSR. Lane 3 and 4, respectively, are products of the left and right junctions of the element and the chromosome using primer pairs CTn5 TSF+LEO and TSR+REO. Template in all reactions was DNA of 630 isolated from the exponential growth phase. Lane M is a molecular weight marker. Size of the fragments is shown left of the figure.

CTn1, CTn4 and Tn6073 all encode predicted tyrosine recombinases and an excisionase

which are predicted to be responsible for the excision and integration of these

elements. CTn2, CTn5, CTn7, Tn5397, Tn6103, Tn6104, Tn6107, Tn6110, Tn6111 and

Tn6164 all encode predicted serine recombinases which are probably responsible for

Figure 4.8 Sequences of transposon-chromosome junctions and direct repeat sequences

Sequences of the PCR products of the joint of the circular molecule, the joint of the chromosome-CTn junction and the regenerated target site of each of the CTns which were shown to excise from the chromosome. Bases in red are part of the CTn, bases in blue are part of the chromosome, and bases in black and underlined are the direct repeat sequences at the ends of the elements.

For the putative mobilisable transposon Tn6104, located on Tn6103 in R20291, only

the product for the circular intermediate of the element was amplified (Figure 4.9).

Amplification of the empty target site could not be shown. For the two other putative

mobilisable transposons within Tn6103, Tn6105 and Tn6106, only products for the

joints of the elements in the chromosome could be amplified. This suggests that these

elements did not excise from the chromosome in the bacterial population tested.

Figure 4.9 Excision of Tn6104.

PCR amplification of the joint of the circular intermediate of the putative mobilisable transposon Tn6104 demonstrates the element excises from Tn6103. Tn6103 is shown in blue, Tn6104 is shown in orange, Tn6105 is shown in purple, Tn6106 is shown in green. The elements are not drawn to scale.

The joint of the empty target site or the circular intermediate could not be amplified

for the CTn1-like elements in strains R20291, QCD-66C26 and QCD-32G58, indicating

these elements did not excise from the chromosome in the tested bacterial

populations. Strain ATCC-43255 contains a CTn1-like element which has integrated in

skinCd, see Chapter 3. Although circularization of the CTn1-like element could not be

shown here, circularization of the skinCd, which includes the CTn1-like element was

4.4.3. ClosTron mutagenesis

Using the original ClosTron protocol [241], the intron was retargeted to two locations

in CTn1 in separate isolates of 630∆erm. The first location was in CD0364, an ABC

transporter ATP binding protein, the second in CD0386, a putative cell surface protein.

CTn7 has a homologue of this cell surface protein, CD3392, which could also be

targeted by the same ClosTron construct (Figure 4.10). Sequencing of the intron-target

site junctions of six mutants demonstrated that in half of the mutants, the intron was

inserted in CD0386 and in half of the mutants the intron was inserted in CD3392.

The ClosTron 2.0 protocol [246] was used with 630∆erm to retarget the intron to CTn2

CD0428, a predicted AraC transcriptional regulator. For CTn4, the intron was

retargeted to CD1099, a predicted two component response regulator. For CTn5, the

intron was retargeted to CD1873, a predicted ABC transporter permease protein

(Figure 4.11).

Strain R20291 contains Tn6103, which is 85% identical to CTn5. The intron was

retargeted in this strain using the same construct used for CD1873 on CTn5.

Sequencing of the intron-genome junction confirmed the ClosTron had inserted in ORF

1803, the homologue of CD1873 on CTn5 (Figure 4.12).

PCR was used to confirm that the introduction of the intron did not prevent excision of

Figure 4.10 ClosTron insertions in CTn1 and CTn7.

ORFs that were shown to contain the ClosTron insertion are shown in pink, the integration sites of the ClosTron constructs are indicated by red arrows. ORFs in the recombination module are shown in grey. ORFs on the conjugation module are shown in blue. ORFs on the accessory module are shown in green.

Figure 4.11 ClosTron insertions in CTn2, CTn4 and CTn5.

ORFs that were shown to contain the ClosTron insertion are shown in pink, the integration sites of the ClosTron constructs are indicated by red arrows. ORFs in the recombination module are shown in grey. ORFs in the conjugation module are shown in blue. ORFs in the accessory module are shown in green.

Figure 4.12 ClosTron insertion in Tn6103.

The schematic of Tn6103 is shown as two parts which should be joined at the grey diagonal line. ORF 1803 that was shown to contain the ClosTron insertion is shown in pink, the integration site of the ClosTron construct is indicated by a red arrow. ORFs on the recombination module of Tn6103 and the recombinases of Tn6104, Tn6105 and Tn6106 are shown in grey. ORFs on the conjugation module of Tn6103 are shown in blue. ORFs on the accessory module are shown in green. ORFs that were interrupted by an insertion of a DNA fragment, compared to CTn5, are shown in orange. ORFs of the putative mobilisable elements Tn6104, Tn6105 and Tn6106 are shown in yellow.