|Simmons, Mustafa -|
Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Abstract Only
Publication Acceptance Date: July 1, 2010
Publication Date: September 22, 2010
Citation: Simmons, M., Seal, B.S. 2010. Recombinant Expression of a Genome-encoded N-acetylmuramoyl-L-alanine Amidase that Synergistically Lyses Listeria monocytogenes Biofilms with a Protease. Federation of European Microbiological Societies Microbiology Letters. Technical Abstract: Listeria monocytogenes plays a significant role in human food-borne disease caused by eating food contaminated with the bacterium and although incidence is low it is a leading cause of life-threatening, bacterial food-borne disease in humans. L. monocytogenes serotypes 1/2a and 4b can form mixed-culture biofilms and this is an important aspect affecting control of the bacterium for food safety. Consequently, genomics BLAST analyses were used to identify potential lytic enzyme sequences in the genomes of L. monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of an N-acetylmuramoyl-L-alanine amidase gene from L. monocytogenes strain 4b. The resultant amplification products were cloned into expression vectors, propagated in E. coli Rosetta strains and the recombinant protein was purified to homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequence of the recombinant protein was homologous to N-acetylmuramoyl-L-alanine amidases. The recombinant lytic enzyme was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot and turbidity reduction 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 amidase to digest a L. monocytogenes biofilm. Protease or lysozyme digestion alone did not significantly reduce the biofilm. Although the amidase alone reduced the biofilm by only twenty percent, complete digestion of the biofilm was accomplished in combination with a protease.