Reproducible transfer of the PaLoc has been demonstrated here for the first time.
Although the PaLoc has been hypothesised to be a mobile genetic element or a phage
remnant [32], the transfer events described here are unlikely to be PaLoc specific as
variable amounts of 630 DNA have transferred. Analysing the transconjugants
containing the PaLoc could not indicate a specific MGE with which the PaLoc co-
transfers, although co-transfer of the PaLoc with CTn1, CTn5 and Tn5397 was
demonstrated. Although Tn5397 had transferred on the same transferred fragment as
the PaLoc, CTn1 and CTn5 never transferred on the PaLoc fragment and transfer of
these elements could be a separate event. Because of the presence of CTn4 and CTn7-
like elements in CD37, co-transfer of these two elements cannot be excluded.
The transfer mechanism of the PaLoc was not determined, although a number of
known mechanisms are considered to be highly unlikely. Transformation of free DNA
has never been observed in C. difficile despite multiple attempts in our laboratory and
others [105,107]. However, to rule out this possibility, filter-matings will be repeated in
our lab on agar plates containing DNase.
The PaLoc could have been transferred via transduction however, the size of the DNA
fragments that were shown to transfer makes this unlikely as the transferred DNA is
over three times the size of the phage genomes of C630-1 and C630-2 of strain 630. Furthermore, CD37 was shown not to be susceptible to the two prophages of 630 and
The transfer of the PaLoc could be facilitated by a high frequency recombination (Hfr)-
type mechanism. During Hfr, a conjugative element mobilises the chromosome in cis
by integrating into the chromosome or in trans by mobilising another element within
the chromosome after which (part of) the chromosome is transferred into a recipient
cell [114,115,260]. The transferred fragment of DNA will recombine with the
chromosome of the recipient cell through a double-crossover event replacing part of
the recipient genome, a process which depends on the recombination protein RecA
[104,114,115]. The mechanism has been described in a number of Gram negative
bacteria but in Gram positive bacteria it has only been described in L. lactis [261]. The
L. lactis sex factor is chromosomally located and can mobilise the chromosome but it can also transpose through site-specific recombination into non-conjugative plasmids
in order to mobilise these [120,121] (Chapter 1).
To determine if the transfer event is Hfr, a number of experiments need to be
performed. As Hfr is a RecA dependent event in the recipient strain, a CD37 recA
mutant could be constructed and used as recipient in filter-matings. Hfr in C. difficile
630 would most likely be mobilisation in cis by one of the CTns. In cis mobilisation
would involve using the oriT of the CTn that facilitated the transfer which means the
element rarely comes across completely as part of the element would be the last
sequence to be transferred to the transconjugant. Part of the element that does come
across may be lost during the double crossover event that integrates the transferred
DNA fragment into the chromosome. Furthermore, if the element responsible for
transfer is unique to 630 then recombination would not occur with this sequence and
the transferred fragment of the responsible element would be lost. As the whole
two of the transconjugants, it is unlikely that the oriT of this element is used as the
whole of the chromosome would have to have been transferred into the recipient cell.
However, CTn1 and CTn2 are located close to this element and were not present in any
of the transconjugants on the integrated DNA fragment (Figure 5.5). Therefore, one of
these two elements may facilitate the Hfr transfer. In order to test this hypothesis,
mutants could be made for genes that are essential for conjugative transfer of these
elements. If genes which are essential for one of the elements are also essential for
Hfr, then the element responsible for Hfr is identified. However, more than one of the
CTns may be capable of mobilising the genome in this manner.
The PaLoc contains the two major virulence factors that have been identified in C.
difficile, making transfer of this locus clinically important. C. difficile has been shown here, and in previous studies, to exchange DNA with a number of species including B.
subtilis [164], E. faecalis [163] and S. aureus [170]. Transfer of the PaLoc into any of these species is therefore theoretically possible and when the transfer mechanism of
the PaLoc transfer is determined, experiments should be carried out in order to
determine the host range. If the transfer mechanism is shown to be Hfr, transfer into
unrelated species would be less likely.
The cytotoxicity assay showed that the CD37 transconjugant has the same level of
cytotoxicity as the donor strain. Although horizontally acquired DNA can be silenced
after transfer, the PaLoc encodes two hypothetical regulators, tcdC and tcdD, which
may also regulate toxin expression in transconjugants [52,53]. Furthermore, a recent
paper by Dingle et al. [89] described MLST data which suggests that a toxigenic
the PaLoc. This data suggests that the PaLoc transfer that was described here may
have also occurred in vivo.
Protective colonization with non-toxigenic C. difficile strains is a new strategy to
prevent colonization by toxigenic C. difficile. Protective colonization in hamsters was
shown to lead to colonization resistance of toxigenic strains and to prevent disease
symptoms [262]. Currently, human phase II trials are being conducted [263] however,
if in vivo transfer of the PaLoc can occur, this treatment strategy may be less effective
than expected and the effect on C. difficile epidemiology cannot be predicted.
5.5.2. Conclusions
The data in this chapter describes the transfer of the PaLoc from a toxigenic donor
strain to a non-toxigenic recipient strain. The recipient strain becomes cytotoxic and
produces similar levels of toxin as the wildtype donor strain.
The transfer mechanism of the PaLoc transfer is still unknown although a number of
transfer methods have been ruled out. Transfer via transformation is unlikely due to
the natural inability of C. difficile to take up DNA under laboratory conditions.
Furthermore, the size of the transferred DNA fragment in some of the transconjugants
is more than three times larger than the phage genomes of C630-1 and C630-2 of strain 630, making transduction of the DNA fragment highly unlikely. As the size of the
DNA fragment that was incorporated in the recipient chromosome varies between the
transconjugants, the donor DNA could be incorporated by non-specific recombination.
This leads to the hypothesis that a Hfr-like transfer mechanism could be responsible