Strain R20291 contains an element that has previously been reported as unique to
ribotype 027 strains, designated CTn027, and reported to contain a 20 kb phage island
called Stoke Mandeville Phage Island (SMPI) [14]. Bioinformatics shows that only a part
of the SMPI sequence is present in other ribotype 027 strains such as QCD-66C26
(Figure 3.5). Here, in-depth analysis of the integration site of the SMPI showed that the
sequence actually consisted of three separate elements integrated into two
homologues of CTn5 CDS (Figure 3.6). The complete element was renamed Tn6103
and the three insertions were designated Tn6104, Tn6105 and Tn6106 as they all
resemble transposons in their own right.
The core element of Tn6103, excluding Tn6104, Tn6105 and Tn6106, has 85%-99%
sequence identity with CTn5. Tn6103 is present in the same target site as CTn5 in 630
and nearly all CDS of CTn5 are present.
At the left end of Tn6104 (Figure 3.6), CDS 1744 and 1745 are homologues of the
serine recombinase tnpX and tnpV of Tn4451 and Tn4453, respectively, mobilisable
transposons of C. perfringens and C difficile [166,216,217]. Although TnpV has some
similarity to Xis from Tn916 (18%), only TnpX is essential in the excision of these
elements from their respective chromosomes and the function of TnpV remains
unknown [166,217]. Furthermore, Tn6104 also contains a mobA/mobL homologue
(CDS 1758). In contrast to the single catP accessory gene present on Tn4451/Tn4453,
Tn6104 encodes thirteen putative accessory proteins including a predicted
transcriptional regulator of the Xre family (CDS 1747), a two-component regulatory
system (CDS 1748-1749), an ABC transporter system (CDS 1750-1752), three σ24 sigma
Figure 3.5 Schematic representation of CTn5 and the CTn5-like elements in strains R20291, QCD-66C26, QCD-63Q42 and QCD-23M63.
CDS are represented by arrows. All blue CDS have homologues in CTn5 in strain 630. Green CDS are not present in CTn5. Red, brown and yellow boxes show regions with sequence similarity. Red boxes are part of the recombination module, blue boxes are part of the regulation and accessory module, brown boxes are part of the conjugation module, yellow boxes show sequence similarity between regions of the elements that are not present in CTn5. Blue boxes are part of the regulation and accessory module but do not show sequence similarity. The element presented for Tn6110 in QCD-66C26 also represents Tn6111 in QCD-32G58 which is 99% identical. A digital version of this image is included on the disc accompanying the thesis.
Both Tn6105 and Tn6106 encode two predicted serine recombinases which could be
involved in excision of these elements from the genome (respectively CDS1771-1772
and 1788) (Figure 3.6). Both elements also encode TnpV homologues (CDS 1765 and
1777) and putative mobilisation proteins (CDS 1768 and 1784). Tn6105 encodes eight
putative accessory genes including a transcriptional regulator (CDS 1766) and a σ70
sigma factor whereas Tn6106 encodes nine accessory proteins including a multidrug
efflux pump protein (CDS 1779) and a transcriptional regulator of the Xre family.
Figure 3.6 Schematic representation of CTn5 and the insertions present in Tn6103.
CTn5 and Tn6103, are compared to show the sites where Tn6104, Tn6105 and Tn6106 have inserted in the sequence. CDS are represented by arrows. All Blue CDS have homologues in CTn5 in strain 630, all green CDS are not present in CTn5, red boxes show sequence similarity. Insertion sites are indicated by red dotted arrows. A digital version of this image is included on the disc accompanying the thesis.
Strains QCD-66C26 and QCD-32G58 each contain a CTn5-like element which have been
shown to excise from their respective chromosomes into circular intermediates
(Chapter 4) and were designated Tn6110 and Tn6111 respectively. Strain QCD-37X79
has a CTn5-like element containing 99% sequence identity with Tn6110. All three
elements have insertions identical to Tn6105 in Tn6103, however, none contains
Tn6104 or Tn6106 (Figure 3.5). All three elements are present in similar target sites,
Strains QCD-63Q42 and BI9 contain CTn5-like elements that are 99% identical to each
other, containing similar accessory modules as CTn5 in 630 but containing a
substitution in the conjugation module (Figure 3.5). Excluding this substitution, there is
approximately 88% sequence identity between the elements in QCD-63Q42 and BI9
and CTn5 in 630. The substituted region in the new elements encodes a predicted
restriction modification endonuclease and two methylase proteins. These could
function as a form of molecular vaccination in which methylation protects the
incoming element from host endonucleases and, following integration, will protect the
host chromosome from endonucleases present on other mobile genetic elements
when these enter the cell [218]. Due to frameshift mutations, there are no functional
homologues of several CDS in the conjugation module and it is therefore conceivable
that the element no longer transfers via conjugation. The CTn5-like elements in strains
QCD-63Q42 and BI9 are both present in tandem with a homologue of CTn7 in the
homologue of the CTn7 target site.
Strains QCD-23M63 (Figure 3.5) and 2007855 (Figure 3.7) both contain CTn5-like
elements which have integrated in the CTn7 target site without homologues of CTn7
being present. The element in QCD-23M63 was shown to excise from the genome and
was designated Tn6107 (Chapter 4). The element in strain 2007855 has an insertion in
the homologue of a conjugation related gene which encodes a predicted serine
recombinase and a MobA mobilisation protein and is therefore hypothesized to be a
Figure 3.7 Schematic representation of CTn5 and the CTn5-like element in strain 2007855.
CDS are represented by arrows. All blue CDS have homologues in CTn5 in strain 630. Green CDS are not present in CTn5. Pink CDS are interrupted by an insertion. Coloured boxes show regions with sequence similarity. The red box is part of the recombination module, the blue box is part of the regulation and accessory module, brown boxes are part of the conjugation module. A digital version of this image is included on the disc at the back of this thesis.
3.4.4. Tn916
Strain M68 contains an element with 98% sequence identity to Tn916 excluding two
insertions (Figure 3.8). The accessory module of Tn916 is present on the element,
including tet(M), which indicates the element may confer tetracycline resistance. The
first of the two insertions is between homologues of Tn916 orf21 and orf22 and is
predicted to encode a protein containing a metallo -lactamase domain, which may play a role in resistance to -lactams [219]. Although C. difficile isolates are generally resistant to a high number of -lactams, not all isolates are resistant and -lactamases may account for some of the resistance that has been recorded [220,221]. The second
insertion replaces orf24 with a homologue of the putative cell surface protein found on
CTn1 (93% DNA sequence identity)(see 3.4.7). The element has integrated in a
predicted exosporium glycoprotein gene (CD0332).
Strain M120 contains a Tn916-like element that has 97% sequence identity to Tn916
which was designated Tn6190 as conjugative transfer of the element has been shown
(Chapter 4). The difference between Tn6190 and Tn916 is the absence of a homologue
of orf12 of on Tn6190 (Figure 3.9). ORF12 is involved in transcriptional regulation of
Figure 3.8 Schematic representation of Tn916 compared the Tn916-like element in strain M68 and CTn1.
CDS are represented by arrows. All blue CDS have homologues in Tn916 in E. faecalis. Green CDS are not present in Tn916. Coloured boxes show regions with sequence similarity. The red boxes are part of the recombination module, blue boxes are part of the regulation and accessory module, brown boxes are part of the conjugation module. A digital version of this image is included on the disc at the back of this thesis.
Figure 3.9 Schematic representation of Tn6190 in M120 compared to Tn916 in E. faecalis.
CDS are represented by arrows. Coloured boxes show regions with similarity. The red box is part of the recombination module. The blue box is part of the regulation and accessory module, the brown box is part of the conjugation module.