Sequence of two plasmids from Clostridium perfringens chicken necrotic enteritis isolates and comparison with C. perfringens conjugative plasmids

Abstract

Twenty-six isolates of Clostridium perfringens of different MLST types from chickens with necrotic enteritis (NE) (15 netB-positive) or from healthy chickens (6 netB-positive, 5 netB-negative) were found to contain 1-4 large plasmids, with most netB-positive isolates containing 3 large and variably sized plasmids which were more numerous and larger than plasmids in netB-negative isolates. NetB and cpb2 were found on different plasmids consistent with previous studies. The pathogenicity locus NELoc1, which includes netB, was largely conserved in these plasmids whereas NeLoc3, present in the cpb2 containing plasmids, was less well conserved. A netB-positive and a cpb2-positive plasmid were likely to be conjugative, and the plasmids were completely sequenced. Both plasmids possessed the intact tcp conjugative region characteristic of C. perfringens conjugative plasmids. Comparative genomic analysis of nine CpCPs, including the two plasmids described here, showed extensive gene rearrangements including pathogenicity locus and accessory gene insertions around rather than within the backbone region. The pattern that emerges from this analysis is that the major toxin-containing regions of the variety of virulence-associated CpCPs are organized as complex pathogenicity loci. How these different but related CpCPs can co-exist in the same host has been an unanswered question. Analysis of the replication-partition region of these plasmids suggests that this region controls plasmid incompatibility, and that CpCPs can be grouped into at least four incompatibility groups.

Figure 1. PFGE analyses of plasmids from NE C. perfringens poultry strains.

Agarose plugs containing DNA from each specified isolate were digested with NotI and subjected to PFGE and staining with ethidium bromide. Line numbers indicate isolate numbers M: Mid-Range II PFG molecular DNA ladder (Kb).

Figure 2. PFGE Southern blot of plasmids from NE C. perfringens poultry strains.

Southern blotting of PFGE (Figure 1) was performed with DIG-labelled probes for netB and hdhA. Results from both netB and hdhA probes are shown overlayed. In all lanes with two bands, the upper band represents netB and the lower band hdhA. M: Mid-Range II PFG molecular DNA ladder (Kb).

Figure 3. Genetic maps of the sequenced NE plasmids pNetB-NE10 and pCpb2-CP1.

The circles represent (from inner to outer most): (i) G + C skew; (ii) G + C content and (iii) open reading frames; arrows indicate the direction of transcription.

Figure 4. Comparative analysis of C. perfringens conjugative plasmids.

Comparative analysis of the sequenced NE plasmids pNetB-NE10 and pCpb2-CP1 and the published Cp plasmids pCPF5609, pCPF4969, pJIR3535, pJIR3844, pCPPB1, p8533etx and pCW3. Conserved regions within the analysed plasmids, pNetB (JQ655731), pCpb2 (JQ655732), pCPF5603 (AB236337), pCPF4969 (NC_007772), pJIR3535 (JN689219), pJIR3844 (JN689217), pCPPB-1 (AB604032), pCP8533etx (NC_011412) and pCW3 (NC_010937) are highlighted by grey boxes. Similarities between plasmids were calculated using the M-GCAT tool and visualised using PerlScript.

Figure 5. Comparative analysis of central control region of C. perfringens conjugative plasmids.

Comparative genomic analysis of the central control region of C. perfringens plasmids starting from regB regulatory gene. Identical colors designate similar function on pNetB-NE10, pCpb2-CP1, pCPF5609, pCPF4969, pJIR3535, pJIR3844, pCPPB1, pCP8533etx and pCW3.

Figure 6. Phylogenetic tree of nine C. perfringens conjugative plasmids.

The phylogenetic tree was inferred using the Neighbor-joining algorithm . The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. All positions containing gaps were eliminated from the dataset (Pairwise deletion option). Phylogenetic analyses were conducted in MEGA5.