|Matson, E - IOWA STATE UNIVERSITY|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 11, 2004
Publication Date: August 1, 2004
Citation: Matson, E.G., Stanton, T.B. 2004. Cloning and characterization of an endolysin of VSH-1, a defective bacteriophage of the swine enteropathogen, Brachyspira hyodysenteriae [abstract]. American Society for Microbiology on the New Phage Biology. Abstract No. 44. Technical Abstract: VSH-1 is a mitomycin C-inducible prophage and host-gene transfer agent of Brachyspira hyodysenteriae (B. hyo.). The small, lambda-like VSH-1 virions package random 7.5-kb fragments of B. hyo. chromosomal DNA and mediate horizontal gene transfer. The release of VSH-1 virions correlates with cell lysis approximately 5 hours after B. hyo. cultures are treated with mitomycin C. This lysis suggests that VSH-1 employs a mechanism for breaching the cell wall to facilitate escape. Analysis of the recently sequenced VSH-1 genome revealed an open reading frame (orf14) near the end of the genome that is transcriptionally active in B. hyo. cells treated with mitomycin C. Orf14 encodes a theoretical protein of 26 kDa with homology to bacteriophage hypothetical endolysins, however, the amino acid sequence was not significantly homologous to enzymes for which murolytic activity was biochemically demonstrated. Consequently, we cloned orf14 with the addition of a hexa-histidine tag into a plasmid vector and expressed the gene in E. coli. Over expression of orf14 was toxic and resulted in E. coli cell lysis. Furthermore, the purified protein demonstrated murolytic activity against peptidoglycan isolated from both E. coli and B. hyo. cells. Treatment of B. hyo. peptidoglycan with the protein resulted in production of carbohydrate-associated reducing groups characteristic of an enzyme that cleaves the peptidoglycan polysaccharide chains. Thus, orf14 encodes a VSH-1-associated lysin (LysV) that is likely a glycosidase that facilitates virion escape from B. hyo. cells. These results provide a basis for determining the specific bond that is cleaved by LysV as well as the enzyme's specificity for peptidoglycan from other bacteria.