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.

Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl–l-alanine amidase as a potential antimicrobial to control the bacterium

“…Also, recombinant DNA produced enzymes as feed additives for food production animals such as phytases and carbohydrases are commercially produced and sold for feed additive purposes during monogastric food–animal production (Adeola and Cowieson 2011). Consequently, research at our laboratory has been the development of bacteriophage or prophage-encoded lysins to specifically lyse pathogenic bacteria that do not affect other potentially beneficial organisms (Simmons et al 2010, 2012). Utilizing a genomics approach another lysin, a putative amidase designated PlyCpAmi, capable of species-specific hydrolysis of C. perfringens was cloned and expressed such that the protein could eventually be utilized as an alternative antimicrobial to control the bacterium.…”

“…DnA was extracted from C. perfringens strain CP509 for amplification of the putative gene as described previously (Simmons et al 2012). PCR products and pET21a plasmid vector (Novagen, Inc) were digested with restriction enzymes, Xho 1 and Nde 1.…”

“…Bacteriophage-encoded lytic enzymes have been reported that lyse C. perfringens (Zimmer et al 2002; Simmons et al 2010; Oakley et al 2011), and at our laboratory, we have also utilized a genomics approach to identify lytic proteins in bacterial genomes (Simmons et al 2012). Consequently, we have taken a genomics approach using previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences to identify putative prophage lysins or autolysins by BLAST analyses encoded by the genomes of C. perfringens isolates (Shimizu et al 2002; Myers et al 2006).…”

Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

“…There is a need to discover innovative pathogen intervention technologies for this bacterium due to resistances to antibiotics and bacteriophages (Shen et al, 2006). Consequently, using our phage lysins as query subjects, bioinformatic analyses were used to identify genes encoding lytic protein sequences in the genomes of L. monocytogenes isolates (Simmons et al, 2012). The PCR primers were designed that amplified nucleotide sequences of a putative N -acetylmuramoyl-L-alanine amidase gene from the genomic DNA of a L. monocytogenes strain 4b isolate.…”

“…Subsequent identification of the phage genomic open reading frame ( ORF ) descriptive bacteriophage structure and identification of potential lytic proteins was completed by full-genome nucleotide sequencing, genome annotations, and BLAST analyses as described in detail by the investigators (Seal et al, 2011; Oakley et al, 2011; Volozhantsev et al, 2011, 2012; Morales et al, 2012). Recombinant proteins were produced by cloning bacteriophage genes encoding putative lysins such as N -acetylmuramoyl-l-alanine amidases and expression in E. coli (Simmons et al, 2010, 2012). …”