Genetic organisation, mobility and predicted functions of genes on integrated, mobile genetic elements in sequenced strains of Clostridium difficile

Abstract

Background: Clostridium difficile is the leading cause of hospital-associated diarrhoea in the US and Europe. Recently the incidence of C. difficile-associated disease has risen dramatically and concomitantly with the emergence of 'hypervirulent' strains associated with more severe disease and increased mortality. C. difficile contains numerous mobile genetic elements, resulting in the potential for a highly plastic genome. In the first sequenced strain, 630, there is one proven conjugative transposon (CTn), Tn5397, and six putative CTns (CTn1, CTn2 and CTn4-7), of which, CTn4 and CTn5 were capable of excision. In the second sequenced strain, R20291, two further CTns were described.

Results: CTn1, CTn2 CTn4, CTn5 and CTn7 were shown to excise from the genome of strain 630 and transfer to strain CD37. A putative CTn from R20291, misleadingly termed a phage island previously, was shown to excise and to contain three putative mobilisable transposons, one of which was capable of excision. In silico probing of C. difficile genome sequences with recombinase gene fragments identified new putative conjugative and mobilisable transposons related to the elements in strains 630 and R20291. CTn5-like elements were described occupying different insertion sites in different strains, CTn1-like elements that have lost the ability to excise in some ribotype 027 strains were described and one strain was shown to contain CTn5-like and CTn7-like elements arranged in tandem. Additionally, using bioinformatics, we updated previous gene annotations and predicted novel functions for the accessory gene products on these new elements.

Conclusions: The genomes of the C. difficile strains examined contain highly related CTns suggesting recent horizontal gene transfer. Several elements were capable of excision and conjugative transfer. The presence of antibiotic resistance genes and genes predicted to promote adaptation to the intestinal environment suggests that CTns play a role in the interaction of C. difficile with its human host.

Figure 1. Detection of excision of the elements from the genome.

a) Schematic of primer binding sites on the element and the genome. The chromosomal region is shown in lilac and the element in blue, the circular form of the element is also shown (centre), as is the regenerated target after excision (bottom). Oligonucleotide primers and their direction of priming are represented by arrows. Primer pair 1+4 will detect the empty target site, primer pair 2+3 will detect the circular form of the element, primer pairs 1+2 and 3+4 for detection of the junctions between the genome and the element. b) PCR products run on 1% agarose gel for CTn5 excision from 630 genomic DNA. Lane 1; primers 2+3, lane 2; primers 1+4, lane 3; primers 3+4, lane 4; primers 1+2.

Figure 2. Sequences of the joints of circular intermediates element-genome junctions and regenerated target sites after excision.

Sequence in red is part of the transposons, blue is part of the chromosome. Terminal repeats are underlined. a) CTn1, b) CTn2, c) CTn7, d) Tn6103 in R20291, e) Tn6104 in R20291 sequenced across the circular joint and left and right ends in the target site only.

Figure 3. Schematic representation of CTn1-like elements in C. difficile strains.

Blue ORFs have homologues present in CTn1 in strain 630, green ORFs are not present in CTn1. Coloured boxes show regions of homology: red boxes show recombination modules, blue boxes show accessory genes, brown boxes show conjugation modules, yellow boxes show homologues that are not present in 630 CTn1. The element in strain QCD-66C26 is representative of the elements in strains QCD-32G58, QCD-37X79, QCD-76W55 and QCD-97B34.

Figure 4. Schematic representation of CTn2 in strain 630.

ORFs in blue are now predicted to be part of CTn2, ORFs shown in red were previously thought to be part of the element but were present in the target site after excision (see text for more details).

Figure 5. Schematic representation of Tn6104, Tn6105 and Tn6106 in Tn6103.

Comparison of CD1849–CD1853 of CTn5 in strain 630 and the homologous region of Tn6103 in R20291 (see Figure 8 for a diagram of the whole of CTn5). Homologous genes are shown by red boxes. The three separate insertions in Tn6103, are shown by dotted lines.

Figure 6. Sequences of the joints of circular intermediates element-genome junctions and regenerated target sites after excision.

Sequence in red is part of the transposons, blue is part of the chromosome. Terminal repeats are underlined. a) Tn6073 QCD-23M63, b) Tn6107 QCD-23M63, c) Tn6110 QCD-66C26, d) Tn6111 QCD-32G58.

Figure 7. Schematic representation of the CTn1-like element in C. difficile QCD-63Q42.

Blue ORFs have homologues present in CTn1 in strain 630, green ORFs are not present in CTn1. Coloured boxes show regions of homology: red boxes show recombination modules, blue boxes show accessory genes, brown boxes show conjugation modules, yellow boxes show homologues that are not present in CTn1 in strain 630. Comparison of CTn1 in 630, CTn1-like element in QCD-63Q42 and phage 1 of 630. Black arrows indicate gaps in the DNA sequence.

Figure 8. Schematic representation of CTn5-like elements in C. difficile strains.

Blue ORFs are present in strain 630 CTn5. Green ORFs are not present in CTn5. Coloured boxes show regions of homology: red boxes show recombination modules, blue boxes show accessory genes, brown boxes show conjugation modules, yellow boxes show homologues that are not present in CTn5 in strain 630. The element shown for strain QCD-66C26 is representative of elements identified in strains QCD-32G58 and QCD-37X79.

Figure 9. Schematic representation of the CTn4-like element in QCD-23M63.

Blue ORFs are present in strain 630 CTn4, green ORFs are not present in CTn4. Brown boxes show regions of homology.

Figure 10. Schematic representation of the putative mobilisable elements.

a) Comparison of Tn5398 in strain 630 and the novel putative element in strain ATCC-43255. Blue ORFs are present in Tn5398 in strain 630, green ORFs are not present in Tn5398. The insertion site of the erm(B) cassette is shown by dotted lines. Brown boxes show regions of homology. b) Representation of the novel putative mobilisable element in strain QCD-63Q42, Tn6115. Blue ORFs are part of the element, the pink ORF is the insertion site, a homologue of CD1573 in strain 630.