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Random mutagenesis identifies novel host factors involved in the secretion of antimicrobial cell wall lytic enzymes by Lactococcus lactis

Tan, Yu Pei, Giffard, Philip M., Barry, Daniel, Huston, Wilhelmina M., & Turner, Mark S. (2008) Random mutagenesis identifies novel host factors involved in the secretion of antimicrobial cell wall lytic enzymes by Lactococcus lactis. Applied and Environmental Microbiology, 74(24), pp. 7490-7496.

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Abstract

Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.

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3 citations in Scopus
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3 citations in Web of Science®

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ID Code: 16983
Item Type: Journal Article
Additional Information: For more information, please refer to the journal's website (see hypertext link) or contact the author.
Keywords: Lysostaphin, Mutagenesis, Insertional, Lactococcus lactis, Bacterial Proteins, Gene Order, DNA Transposable Elements, Chromosomes, Bacterial
DOI: 10.1128/AEM.00767-08
ISSN: ISSN: 0099-2240
Subjects: Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > GENETICS (060400) > Genetics not elsewhere classified (060499)
Australian and New Zealand Standard Research Classification > BIOLOGICAL SCIENCES (060000) > BIOCHEMISTRY AND CELL BIOLOGY (060100) > Cell Metabolism (060104)
Divisions: Past > QUT Faculties & Divisions > Faculty of Science and Technology
Current > Institutes > Institute of Health and Biomedical Innovation
Past > Schools > School of Life Sciences
Copyright Owner: Copyright 2008 American Society for Microbiology
Deposited On: 05 Jan 2009 14:49
Last Modified: 29 Feb 2012 23:53

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