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Applied and Environmental Microbiology, December 2002, p. 6152-6161, Vol. 68, No. 12
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.12.6152-6161.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Novel Food-Grade Plasmid Vector Based on Melibiose Fermentation for the Genetic Engineering of Lactococcus lactis

Isabelle Boucher,¹ Marc Parrot,^1, Hélène Gaudreau,² Claude P. Champagne,² Christian Vadeboncoeur,¹ and Sylvain Moineau^1*

Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Groupe de Recherche en Écologie Buccale (GREB), Faculté de Médecine Dentaire, Université Laval, Québec, Canada G1K 7P4,¹ Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Québec, Canada, J2S 8E3²

Received 12 September 2002/ Accepted 13 September 2002

The -galactosidase gene (aga) and a gene coding for a putative transcriptional regulator from the LacI/GalR family (galR) of Lactococcus raffinolactis ATCC 43920 were cloned and sequenced. When transferred into Lactococcus lactis and Pediococcus acidilactici strains, aga modified the sugar fermentation profile of the strains from melibiose negative (Mel^-) to melibiose positive (Mel⁺). Analysis of galA mutants of L. lactis subsp. cremoris MG1363 indicated that the putative galactose permease GalA is also needed to obtain the Mel⁺ phenotype. Consequently, GalA may also transport melibiose into this strain. We demonstrated that when aga was associated with the theta-type replicon of a natural L. lactis plasmid, it constituted the selectable marker of a cloning vector named pRAF800. Transcriptional analysis by reverse transcriptase PCR suggests that this vector is also suitable for gene expression. The -galactosidase activity conferred by pRAF800 was monitored in an industrial strain grown in the presence of various carbon sources. The results indicated that the enzymatic activity was induced by galactose and melibiose, but not by glucose or lactose. The gene encoding the phage defense mechanism, AbiQ, was cloned into pRAF800, and the resulting clone (pRAF803) was transferred into an industrial L. lactis strain that became highly phage resistant. The measurements of various growth parameters indicated that cells were not affected by the presence of pRAF803. Moreover, the plasmid was highly stable in this strain even under starter production conditions. The L. raffinolactis aga gene represents the basis of a novel and convenient food-grade molecular tool for the genetic engineering of lactic acid bacteria.

Present address: Centre de Recherche STELA, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Québec, Canada G1K 7P4.

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