Isolation and characterization of temperate bacteriophages of Clostridium difficile

Abstract

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages phiC2, phiC5, phiC6, and phiC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of phiC2 showed nucleotide homology to the sequenced C. difficile strain CD630.

FIG. 1.

Phages φC2, φC8, and φC5 possess contractile sheaths and rigid tails, while φC6 particles have long, flexible tails. Bar, 100 nm.

FIG. 2.

Restriction digests and Southern hybridization of phage DNA. (a) HindIII and XbaI double digest of phage DNA. Lanes: 1, λ, HindIII DIG-labeled marker (Roche); 2, φC2; 3, φC5; 4, φC6; 5, φC8. (b) Southern hybridization of gel shown in panel a with DIG-labeled φC6 genomic probe (25 ng ml⁻¹).

FIG. 3.

Determining the site of chromosomal prophage integration through Southern hybridization of DIG-labeled φC2 to DNA of lysogen(s). (a) Agarose gel of HindIII-digested DNA. Lanes: 1, λ HindIII DIG-labeled marker (Roche); 2, CD839; 3, CD839C2; 4, φC2. (b) A Southern blot of the gel shown in panel a with a φC2 genome probe (25 ng ml⁻¹) revealed differences between the banding patterns of CD839C2 and φC2 in the 1.9- to 2-kb region. (c) Southern hybridization of the 1.9-kb HindIII fragment (2.5 ng ml⁻¹) to DNA of lysogens CD839C2 and CD843C4 resulted in new bands. Lanes: 1, CD839C2; 2, CD843C2; 3, φC2.

FIG. 4.

φC2 prophage nucleotide positions in C. difficile CD630 genome. (a) φC2 sequences (blank boxes) were located between nucleotide positions 765221 and 765669, 1114882 and 1122754, and 1254421 and 1254907. Of the nine sequences, seven were clustered, and six of the seven showed protein homology to phage structural proteins. RW2 and RW25 were located 349,183 and 131,667 bases, respectively, from the cluster. The PaLoc (larger striped box) is shown located between nucleotide positions 786149 and 795379, approximately 20 and 319 kb from RW2 and RW11, respectively. sigK (smaller striped box) was not in close proximity to phage sequences. (b) The φC2 cluster on CD630 with the proposed attP in sequence 1.9 is shown in grey.