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United States Department of Agriculture

Agricultural Research Service

Research Project: INTERVENTIONS AND METHODOLOGIES TO REDUCE HUMAN FOOD-BORNE BACTERIAL PATHOGENS IN CHICKENS

Location: Poultry Microbiological Safety Research

Title: Lytic Clostridium perfringens Bacteriophage 39-O Genomic

Authors
item Seal, Bruce
item Siragusa, Gregory
item Fouts, Derrick - J. CRAIG VENTER INST
item Woolsey, Rebekah - U OF NEVADA
item Schegg, Kathleen - U OF NEVADA

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 6, 2008
Publication Date: July 26, 2008
Citation: Seal, B.S., Siragusa, G.R., Fouts, D.E., Woolsey, R., Schegg, K.M. 2008. Lytic Clostridium perfringens Bacteriophage 39-O Genomic. Abstract at Edinburgh International Phage Conference, Session 2-1, Z-30.

Technical Abstract: Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations were initially characterized by transmission electron microscopy along with isolation of genomic DNA. From the collection a highly lytic virus was isolated from broiler chicken offal wash designated phage Cp39-O that produced clear plaques following limit dilution plating of the virus on the host. Bacteriophage Cp39-O had an icosohedral head with an approximate 100 nm tail characteristic of the Siphoviridae family in the order Caudovirales. The double-strand DNA genome of bacteriophage 39-O was 38,753 base pairs with a 30.4% GC content encoding sixty-two (62) predicted open reading frames (ORF), of which all but one were predicted to be encoded on one strand. Predicted ORF’s included common bacteriophage proteins such as the pre-neck protein with an exonuclease domain, tail protein, a tape-measure protein and portal protein along with other potential structural components. Several putative proteins including a DNA primase, single-strand DNA binding proteins, integrase, terminase, thymidylate synthase, and antirepressor or transcription factor genes were also present in the genome of phage Cp39-O. Potential lytic enzymes such as a fibronectin-binding autolysin, amidase/hydrolase along with a potential holin and peptidase with colicin-like sequences were identified in the viral genome. Several ORF’s encoded predicted similar proteins from Clostridium spp. or other Gram-positive bacterial and bacteriophage genomes along with unknown Collinsella aerofaciens ORF’s. Proteomics analyses of the purified virus were completed using two-dimensional gel electrophoresis followed by obtaining peptide molecular masses by MALDI-TOF-TOF MS to identify virion proteins. This resulted in identification of a putative pre-neck/appendage and an endopeptidase protein encoded by large open reading frames. Variants of the portal protein were identified and several mycobacteriophage gp6-like protein variants were detected in the largest amounts relative to other virion proteins. The endopeptidase is presumably the tail protein while the gp-6-like protein is most likely the major capsid protein of the virus. Several smaller proteins with no known homologous function were also purified along with the intact bacteriophage.

Last Modified: 8/1/2014
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