Recombinant expression of two bacteriophage proteins that lyse clostridium perfringens and share identical sequences in the C-terminal cell wall binding domain of the molecules but are dissimilar in their N-terminal active domains.
Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium capable of producing four major toxins that are responsible for disease symptoms and pathogenesis in a variety of animals, humans, and poultry. The organism is the third leading cause of human foodborne bacterial disease, and C. perfringens is the presumptive etiologic agent of necrotic enteritis among chickens, which in the acute form can cause increased mortality among broiler flocks. Countries that have complied with the ban on antimicrobial growth promoters (AGP) in feeds have had increased incidences of C. perfringens-associated necrotic enteritis in poultry. To address this issue, new antimicrobial agents, putative lysins from the genomes of bacteriophages, are identified. Two putative phage lysin genes (ply) from the clostridial phages phiCP39O and phiCP26F were cloned and expressed in Escherichia coli , and the resultant proteins were purified to near homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequences of the two recombinant proteins were homologous to N-acetylmuramoyl-l-alanine amidases. The proteins were identical in the C-terminal putative cell-wall binding domain, but only 55% identical to each other in the presumptive N-terminal catalytic domain. Both recombinant lysins were 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. The observed reduction in turbidity was correlated with up to a 3 log cfu/mL reduction in viable C. perfringens on brain-heart infusion agar plates. However, other member species of the clostridia were resistant to the lytic activity by both assays.
Simmons
Mustafa
M
Poultry Microbiology Safety Research Unit, Agricultural Research Service, US Department of Agriculture, Richard B Russell Agricultural Research Center, 950 College Station Road, Athens, Georgia 30605, USA.
Donovan
David M
DM
Siragusa
Gregory R
GR
Seal
Bruce S
BS
D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002473:Cell Wall / Q000378:metabolism; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000378:metabolism / Q000821:virology*; D015870:Gene Expression; D008969:Molecular Sequence Data; D009088:Mucoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D009088:Mucoproteins; D011994:Recombinant Proteins; C010393:lysin, gastropoda
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