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Applied and Environmental Microbiology, December 2008, p. 7490-7496, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00767-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Random Mutagenesis Identifies Novel Genes Involved in the Secretion of Antimicrobial, Cell Wall-Lytic Enzymes by Lactococcus lactis{triangledown}

Yu Pei Tan,1 Philip M. Giffard,2 Daniel G. Barry,1 Wilhelmina M. Huston,1 and Mark S. Turner3*

Infectious Diseases Program, Cells and Tissue Domain, Institute of Health and Biomedical Innovation, Queensland University of Technology,1 School of Land, Crop and Food Sciences, University of Queensland, Brisbane, Queensland,3 Menzies School of Health Research, Darwin, Northern Territory, Australia2

Received 3 April 2008/ Accepted 7 October 2008

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.

* Corresponding author. Mailing address: School of Land, Crop and Food Sciences, University of Queensland, Brisbane, Queensland 4072, Australia. Phone: (61-7) 3365-1171. Fax: (61-7) 3365-1177. E-mail: m.turner2{at}uq.edu.au

{triangledown} Published ahead of print on 17 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7490-7496, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.00767-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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