Gene Cloning and Expression and Secretion of Listeria monocytogenes Bacteriophage-Lytic Enzymes in Lactococcus lactis

Abstract

Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SP slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SP slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3 -end coding sequence of SP slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511⌬(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SP SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511⌬C into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

Molecular Aspects and Comparative Genomics of Bacteriophage Endolysins

“…lactis bacteriophage LL-H endolysin can be removed without destroying the lytic activity (85). C-terminal deletions of the Staphylococcus aureus bacteriophage endolysins PlyTW and Ply187, L. monocytogenes bacteriophage endolysin Ply511, Bacillus amyloliquefaciens phage endolysin Morita2001, Bacillus anthracis prophage endolysin PlyL, and Bacillus cereus phage endolysin Ply21 even increase the muralytic activity (50,83,(86)(87)(88). This pattern is also present in endolysins with dual lytic domains, for which, in theory, both should be equally active.…”

Genomic Sequence of Bacteriophage ATCC 8074-B1 and Activity of Its Endolysin and Engineered Variants against Clostridium sporogenes

“…Truncation of the endolysin to just the proposed catalytic domain produced an active lysin but did not affect the specificity. Other studies reported the maintenance of endolysin activity upon removal of C-terminal domains; in many cases, this truncation led to a notable increase in activity (9,14,28,32), while in others, activity remained similar to or was lower than that of the full-length endolysin (10,21). In several cases, endolysin catalytic domains retained the same or a similar host range upon removal of the C-terminal domain (9,18,28,32), despite the fact that their peptidoglycan target is usually shared with a number of other species.…”

Bacteriophage biocontrol of foodborne pathogens

“…Another technique of using lyisn is via lysin-secreting recombinant bacteria (Borysowski et al 2006). This was demonstrated in the case of recombinant Lactococcus lactis cells containing listerial lysin encoding genes to lyse L. monocytogenes in the surrounding medium (Gaeng et al 2000). This study also showed that the expression of functional lysin by L. lactis was detected in the presence of lactose that is used in milk fermentation.…”