Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions

Seal, Bruce S.; Fouts, Derrick E.; Simmons, Mustafa; Garrish, Johnna K.; Kuntz, Robin L.; Woolsey, Rebekah; Schegg, Kathleen M.; Kropinski, Andrew M.; Ackermann, Hans-W.; Siragusa, Gregory R.

doi:10.1007/s00705-010-0812-z

Cited by 32 publications

(37 citation statements)

References 86 publications

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“…restriction enzymes HindIII and AccI (New England Biolabs, Ipswich, MA) and treated with Taq polymerase to be subsequently cloned into vector pCR-XL-TOPO (Invitrogen, Carlsbad, CA) [8]. Complete, 6-fold coverage of the UCP24R genome was obtained by sequencing the cloned fragments, followed by custom primer design and direct sequencing to fill gaps.…”

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Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

et al. 2012

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Bacteriophage ΦCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The ΦCP24R genome described herein is only the second Clostridium perfringens podovirus genome reported to date.

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Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

et al. 2012

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“…The first viruses isolated from broiler chicken offal washes and poultry feces were designated ΦCP39O and ΦCP26F that produced clear plaques on host strains (Seal et al, 2011). Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm noncontractile tails characteristic of members of the family Siphoviridae in the order Caudovirales (Figure 1B).…”

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confidence: 99%

“…Based on phylogenetic analysis of the large terminase protein, these phages are predicted to be pac-type, using a head-full DNA packaging strategy. Importantly, no toxin-encoding genes were identified among the bacteriophages and the phage genomes probably do not integrate into the host genome (Seal et al, 2011). …”

Section: Resultsmentioning

confidence: 99%

“…This has been observed during our investigations, wherein most bacteriophages virulent for C. perfringens have a restricted host range for a specific isolate of the bacterium (Figure 3). Host specificity has routinely been observed relative to the bacteriophages isolated from various C. perfringens isolates that is most likely due to evolution of the receptor and anti-receptor molecules (Seal et al, 2011; Oakley et al, 2011; Volozhantsev et al, 2011, 2012). Therefore, selection of appropriate bacteriophage cocktails may not necessarily be effective against many of the various bacterial isolates that exist in the environment.…”

Section: Resultsmentioning

confidence: 99%

“…Additionally, the viruses were purified by isopycnic centrifugation and analyzed by SDS-PAGE followed by mass spectrometry of the proteins. Subsequent identification of the phage genomic open reading frame ( ORF ) descriptive bacteriophage structure and identification of potential lytic proteins was completed by full-genome nucleotide sequencing, genome annotations, and BLAST analyses as described in detail by the investigators (Seal et al, 2011; Oakley et al, 2011; Volozhantsev et al, 2011, 2012; Morales et al, 2012). Recombinant proteins were produced by cloning bacteriophage genes encoding putative lysins such as N -acetylmuramoyl-l-alanine amidases and expression in E. coli (Simmons et al, 2010, 2012).…”

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Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

Seal

2013

Poultry Science

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There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as nonfoodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.

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Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

Schmitz

Ossiprandi

Rumah

et al. 2010

Appl Microbiol Biotechnol

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With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low µg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or nonclostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.

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Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions

Cited by 32 publications

References 86 publications

Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

Lytic enzyme discovery through multigenomic sequence analysis in Clostridium perfringens

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