Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids

Parreira VR, Costa M, Eikmeyer FG, Blom J, Prescott JF. Sequence of Two Plasmids from Clostridium perfringens Chicken Necrotic Enteritis Isolates and Comparison with C. perfringens Conjugative Plasmids. PLoS ONE. 2012;7(11): e49753.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

Phylogenetic tree of nine <i>C. perfringens</i> conjugative plasmids.

<p>The phylogenetic tree was inferred using the Neighbor-joining algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049753#pone.0049753-Saitou1" target="_blank">[35]</a>. 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.</p

Effect of the strain <em>Bacillus amyloliquefaciens</em> FZB42 on the microbial community in the rhizosphere of lettuce under field conditions analyzed by whole metagenome sequencing.

Application of the plant associated bacterium Bacillus amyloliquefaciens FZB42 on lettuce (Lactuca sativa) confirmed its capability to promote plant growth and health by reducing disease severity (DS) caused by the phytopathogenic fungus Rhizoctonia solani. Therefore this strain is commercially applied as an eco-friendly plant protective agent. It is able to produce cyclic lipopeptides (CLP) and polyketides featuring antifungal and antibacterial properties. Production of these secondary metabolites led to the question of a possible impact of strain FZB42 on the composition of microbial rhizosphere communities after its application. Rating of DS and lettuce growth during a field trial confirmed the positive impact of strain FZB42 on the health of the host plant. To verify B. amyloliquefaciens as an environmentally compatible plant protective agent, its effect on the indigenous rhizosphere community was analyzed by metagenome sequencing. Rhizosphere microbial communities of lettuce treated with B. amyloliquefaciens FZB42 and non-treated plants were profiled by high-throughput metagenome sequencing of whole community DNA. Fragment recruitments of metagenome sequence reads on the genome sequence of B. amyloliquefaciens FZB42 proved the presence of the strain in the rhizosphere over 5 weeks of the field trial. Comparison of taxonomic community profiles only revealed marginal changes after application of strain FZB42. The orders Burkholderiales, Actinomycetales and Rhizobiales were most abundant in all samples. Depending on plant age a general shift within the composition of the microbial communities that was independent of the application of strain FZB42 was observed. In addition to the taxonomic profiling, functional analysis of annotated sequences revealed no major differences between samples regarding application of the inoculant strain

PFGE Southern blot of plasmids from NE <i>C. perfringens</i> poultry strains.

<p>Southern blotting of PFGE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049753#pone-0049753-g001" target="_blank">Figure 1</a>) was performed with DIG-labelled probes for <i>netB</i> and <i>hdhA</i>. Results from both <i>netB</i> and <i>hdhA</i> probes are shown overlayed. In all lanes with two bands, the upper band represents <i>netB</i> and the lower band <i>hdhA</i>. M: Mid-Range II PFG molecular DNA ladder (Kb).</p

PFGE analyses of plasmids from NE <i>C. perfringens</i> poultry strains.

<p>Agarose plugs containing DNA from each specified isolate were digested with <i>Not</i>I and subjected to PFGE and staining with ethidium bromide. Line numbers indicate isolate numbers M: Mid-Range II PFG molecular DNA ladder (Kb).</p

Properties of <i>Clostridium perfringens</i> strains.

1<p>Number of plasmids showed by PFGE analysis. Numbers indicate the approximate size of the plasmid (in kb).</p>2<p>Genes detected by PCR amplification. (−) negative;</p

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

<p>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.</p

Comparative analysis of central control region of <i>C. perfringens</i> conjugative plasmids.

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