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

Morales, Cesar A.; Oakley, Brian B.; Garrish, Johnna K.; Siragusa, Gregory R.; Ard, Mary B.; Seal, Bruce S.

doi:10.1007/s00705-011-1218-2

Cited by 19 publications

(15 citation statements)

References 21 publications

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“…Similar to the siphoviral C. perfringens bacteriophages previously reported from our laboratories, the holin gene is most likely downstream of the lysin gene. This placement is unique to the other clostridial bacteriophages [19], including the podoviruses we recently reported [21], [22]. BLAST analysis of the predicted amidases from ΦZP2, ΦCPV4 and ΦCP7R revealed similarity to a prophage LambdaCh01-like amidase encoded by C. botulinum isolates [YP_002863851; YP_001782467; YP_001392135], C. sporogenes [ZP_02994334] and C. tetani [NP_783826].…”

Section: Resultsmentioning

confidence: 83%

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

et al. 2012

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Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium responsible for human food-borne disease as well as non-food-borne human, animal and poultry diseases. Because bacteriophages or their gene products could be applied to control bacterial diseases in a species-specific manner, they are potential important alternatives to antibiotics. Consequently, poultry intestinal material, soil, sewage and poultry processing drainage water were screened for virulent bacteriophages that lysed C. perfringens. Two bacteriophages, designated ΦCPV4 and ΦZP2, were isolated in the Moscow Region of the Russian Federation while another closely related virus, named ΦCP7R, was isolated in the southeastern USA. The viruses were identified as members of the order Caudovirales in the family Podoviridae with short, non-contractile tails of the C1 morphotype. The genomes of the three bacteriophages were 17.972, 18.078 and 18.397 kbp respectively; encoding twenty-six to twenty-eight ORF's with inverted terminal repeats and an average GC content of 34.6%. Structural proteins identified by mass spectrometry in the purified ΦCP7R virion included a pre-neck/appendage with putative lyase activity, major head, tail, connector/upper collar, lower collar and a structural protein with putative lysozyme-peptidase activity. All three podoviral bacteriophage genomes encoded a predicted N-acetylmuramoyl-L-alanine amidase and a putative stage V sporulation protein. Each putative amidase contained a predicted bacterial SH3 domain at the C-terminal end of the protein, presumably involved with binding the C. perfringens cell wall. The predicted DNA polymerase type B protein sequences were closely related to other members of the Podoviridae including Bacillus phage Φ29. Whole-genome comparisons supported this relationship, but also indicated that the Russian and USA viruses may be unique members of the sub-family Picovirinae.

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Section: Resultsmentioning

confidence: 83%

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

et al. 2012

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“…perfringens cells have been described (Nariya et al 2011; Schmitz et al 2011; Simmons et al 2010; Zimmer et al 2002). A number have also been identified by sequencing and genome mining, producing a rich resource of information for such enzymes (Morales et al 2012; Oakley et al 2011; Schmitz et al 2011; Volozhantsev et al 2012; Volozhantsev et al 2011). …”

Section: Introductionmentioning

confidence: 99%

Expression and delivery of an endolysin to combat Clostridium perfringens

Gervasi

Horn

Wegmann

et al. 2013

Appl Microbiol Biotechnol

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Clostridium perfringens is a cause for increasing concern due to its responsibility for severe infections both in humans and animals, especially poultry. To find new control strategies to treat C. perfringens infection, we investigated the activity and delivery of a bacteriophage endolysin. We identified a new endolysin, designated CP25L, which shows similarity to an N-acetylmuramoyl-l-alanine amidase domain and is distinct from other C. perfringens endolysins whose activity has been demonstrated in vitro. The cp25l gene was cloned and expressed in Escherichia coli, and the gene product demonstrated lytic activity against all 25 C. perfringens strains tested. The probiotic strain Lactobacillus johnsonii FI9785 was engineered to deliver the endolysin to the gastrointestinal tract. The integration of the nisRK two-component regulatory system from the Lactococcus lactis nisin A biosynthesis operon into the chromosome of L. johnsonii allowed constitutive expression of the endolysin under the control of the nisA promoter (PnisA), while the use of a signal peptide (SLPmod) led to successful secretion of the active endolysin to the surrounding media. The high specificity and activity of the endolysin suggest that it may be developed as an effective tool to enhance the control of C. perfringens by L. johnsonii in the gastrointestinal tract.

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“…These enzymes digest peptidoglycan of the bacterial cell wall and could be considered potential therapeutics to control C. perfringens (Volozhantsev et al, 2011). Another bacteriophage, ΦCP24R, was isolated from raw sewage at a US waste treatment plant and lytic activity was observed against a type A C. perfringens isolate (Morales et al, 2012). Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, noncontractile tail, indicative of a member of the family Podoviridae .…”

Section: Resultsmentioning

confidence: 99%

“…Out of 22 predicted genes in the genome, 10 had significant sequence similarity to proteins of known functions. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in the viral kingdom (Morales et al, 2012). …”

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).…”

Section: Methodsmentioning

confidence: 99%

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

Cited by 19 publications

References 21 publications

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

Expression and delivery of an endolysin to combat 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

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