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Applied and Environmental Microbiology, July 2000, p. 2951-2958, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

Susanne Gaeng,1 Siegfried Scherer,1 Horst Neve,2 and Martin J. Loessner1,*

Institut für Mikrobiologie, FML Weihenstephan, Technische Universität München, D-85350 Freising,1 and Institut für Mikrobiologie, Bundesanstalt für Milchforschung, D-24103 Kiel,2 Germany

Received 27 January 2000/Accepted 17 April 2000

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 SPslpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SPslpA-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 SPslpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Delta (S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SPSlpA-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-PL511Delta 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.

* Corresponding author. Mailing address: Institut für Mikrobiologie, FML Weihenstephan, Technische Universität München, Weihenstephaner Berg 3, D-85350 Freising, Germany. Phone: 49-8161-71-3859. Fax: 49-8161-71-4492. E-mail: M.J.Loessner{at}Lrz.tum.de.

Applied and Environmental Microbiology, July 2000, p. 2951-2958, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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