title: Recombinant Expression of a Putative Amidase Cloned from the Genome of Listeria monocytogenes that Lyses the Bacterium and its Monolayer in Conjunction with a Protease.
abstract: Listeria monocytogenes is a Gram-positive, non-spore forming, catalase-positive rod that is a major bacterial food-borne disease agent associated with uncooked meats, including poultry, uncooked vegetables, soft cheeses, and unpasteurized milk. The bacterium may be carried by animals without signs of disease, can replicate at refrigeration temperatures, and is frequently associated with biofilms. There is a need to discover innovative pathogen intervention technologies for this bacterium. Consequently, bioinformatic analyses were used to identify genes encoding lytic protein sequences in the genomes of L. monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of a putative N-acetylmuramoyl-L-alanine amidase gene from L. monocytogenes strain 4b. The resultant amplification product was cloned into an expression vector, propagated in Escherichia coli Rosetta strains, and the recombinant protein was purified to homogeneity. Gene and protein sequencing confirmed that the predicted and chemically determined amino acid sequence of the recombinant protein designated PlyLM was a putative N-acetylmuramoyl-L-alanine amidase. The recombinant lytic protein was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot and MIC/MIB assays, but was not active against other bacteria beyond the genus. A microtiter plate assay was utilized to assay for the ability of the recombinant lysin protein to potentially aid with digestion of a L. monocytogenes biofilm. Protease or lysozyme digestion alone did not significantly reduce the L. monocytogenes biofilm. Although the recombinant protein alone reduced the biofilm by only 20%, complete digestion of the bacterial monolayer was accomplished in conjunction with a protease.
authors.affiliation: Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, 950 College Station Road, 30605, Athens, GA, USA.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Simmons
authors.affiliation: Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, 950 College Station Road, 30605, Athens, GA, USA.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Morales
authors.affiliation: Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, 950 College Station Road, 30605, Athens, GA, USA.
authors.forename: Brian B
authors.initials: BB
authors.lastname: Oakley
authors.affiliation: Poultry Microbiology Safety Research Unit, Richard B. Russell Agricultural Research Center, Agricultural Research Service, USDA, 950 College Station Road, 30605, Athens, GA, USA. bruce.seal@ars.usda.gov.
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Seal
keywords: Antimicrobial; Biofilm; Endolysin; Enzybiotic; Food safety; Peptidoglycan hydrolase