The complete genome sequence of Clostridium difficile phage ϕC2 and comparisons to ϕCD119 and inducible prophages of CD630

Abstract

The complete genome sequence of Clostridium difficile phage wC2 and comparisons to wCD119 and inducible prophages of CD630 The complete genomic sequence of a previously characterized temperate phage of Clostridium difficile, wC2, is reported. The genome is 56 538 bp and organized into 84 putative ORFs in six functional modules. The head and tail structural proteins showed similarities to that of C. difficile phage wCD119 and Streptococcus pneumoniae phage EJ-1, respectively. Homologues of structural and replication proteins were found in prophages 1 and 2 of the sequenced C. difficile CD630 genome. A putative holin appears unique to the C. difficile phages and was functional when expressed in Escherichia coli. Nucleotide sequence comparisons of wC2 to wCD119 and the CD630 prophage sequences showed relatedness between wC2 and the prophages, but less so to wCD119. wC2 integrated into a gene encoding a putative transcriptional regulator of the gntR family. wC2, wCD119 and CD630 prophage 1 genomes had a Cdu1-attP-integrase arrangement, suggesting that the pathogenicity locus (PaLoc) of C. difficile, flanked by cdu1, has phage origins. The attP sequences of wC2, wCD119 and CD630 prophages were dissimilar. wC2-related sequences were found in 84 % of 37 clinical C. difficile isolates and typed reference strains. INTRODUCTIONClostridium difficile has emerged as an important intestinal pathogen since the 1970s, continuing to plague hospital settings worldwide and causing recent epidemics in the USA and Canada (Loo et al., 2005;Warny et al., 2005). The major virulence factors of C. difficile are toxins A and B encoded by tcdA and tcdB respectively, which are located on a 19 kb genomic region termed the pathogenicity locus (PaLoc) (Braun et al., 1996;Hammond & Johnson, 1995). The toxins are positively regulated by TcdR (Mani & Dupuy, 2001;Rupnik et al., 2005) and are negatively regulated by TcdC (Hundsberger et al., 1997;Matamouros et al., 2006); they are encoded by tcdR and tcdC respectively, also on the PaLoc. Another toxin-associated gene, tcdE, appears phage related but its function is unknown (Tan et al., 2001). C. difficile acquires antibiotic resistance and virulence genes through plasmids and transposons (Bruggemann, 2005) shown to significantly contribute to genome plasticity (Sebaihia et al., 2006). It is possible that phages also contribute to variance in virulence-associated genes (Lemee et al., 2005) and to the emergence of outbreak strains . However, the prevalence of phage genes within C. difficile genomes is not known. In comparison to phages of Escherichia coli, Staphylococcus aureus and Lactobacillus species, the study of clostridial phages is in its infancy. Only one phage specific for C. difficile, temperate phage wCD119, has been sequenced , while two putative prophage sequences were detected in the recently sequenced genome of C. difficile CD630 (Sebaihia et al., 2006).

Prophage-Stimulated Toxin Production in Clostridium difficile NAP1/027 Lysogens

“…Protein similarity was found between the lysis, lysogeny control, and DNA replication, recombination, and modification modules of phages CD38-2 and CD6356, but their structural genes were unrelated. Also, only a few hits corresponded to gene products from CD27 and CD119, thus confirming that CD38-2 is completely different from all Myoviridae phages of C. difficile described so far (15,17,34,39). The gene organization and modular structure of the CD38-2 genome are similar to those of other C. difficile phages and other temperate phages infecting low-GϩC bacteria (25).…”

“…The two contigs were joined, and gaps were filled after sequencing reactions were performed directly on the phage DNA. The CD38-2 genome is composed of a double-stranded DNA (dsDNA) molecule of 41,090 bp with a GϩC content of 30.83%, which is a little above, but in the range reported for, that of other C. difficile phages (28.7 to 29.4%) (15,17,34) and of C. difficile strain 630 (29.06%) (39). Digestion of the purified phage DNA with various restriction enzymes gave profiles perfectly corresponding to a circular genomic map, except for a faint submolar fragment of ϳ0.9 kb that was observed with EcoRV (see Fig.…”

“…Hence, there is a clear lack of genomic data for this group of phages, especially those of the Siphoviridae family. So far, C. difficile phages have not been found to encode proven virulence factors or to convert nontoxigenic C. difficile isolates into toxin-producing lysogens (15,17,20,34). Nevertheless, two recent studies suggest that phages may somehow contribute to the regulation of toxin production in C. difficile (14,18), but the clear lack of data regarding phages of C. difficile makes it difficult to appreciate the real impact of prophages on C. difficile lifestyle and virulence.…”

Bacteriophage-Mediated Toxin Gene Regulation in Clostridium difficile

“…Following publication of the genome of ⌽CD119 (15), the genome of a second C. difficile temperate phage (⌽C2, a member of the Myoviridae) was published (13). More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11).…”

“…More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11). In addition, the multidrug-resistant C. difficile strain CD630 was found to harbor two highly related prophages (13,39) as part of its mosaic genome, where nearly 11% is made of mobile genetic elements. Thus, it appears that C. difficile strains often harbor temperate phage(s) as part of their genetic makeup.…”