Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Tools for gene manipulation, expression and detection in lactic acid bacteria

One important problem for genetic manipulation of natural strains of lactic acid bacteria (LAB) is that some of them are refringent to electrophoration. An alternative DNA transfer system is conjugation. Utilisation of this type of horizontal transfer from gram-positive and gram-negative bacteria to LAB has been investigated during the development of this project. Methods for plasmid transfer by mobilization to Lactobacillus, Lactococcus lactis and Oenococcus oeni have been standardised. Moreover, transfer by one of the methods of pMV158GFP plasmid has allowed standardisation of fluorescent detection of L. lactis in cheese matrix.

In addition, a method for transfer by electrophoration and detection of the pCIT264 plasmid encoding the citrate transport system has been developed. The method is based on the acid resistance conferred by the citrate transport and metabolism. The new recombinant strain CRL30[pCIT264] has been characterised and the results revealed that indeed is a new L. lactis food grade-strain expressing the citrate transport system.

Furthermore, in this study there have been developed and/or validated expression vectors. A food-grade expression system has been developed for Lactobacillus casei. A food-grade strain with nisRK stably integrated into the genome was constructed, in order to implement the nisin-controlled expression system (NICE) in this bacteria. Expression of b-glucuronidase (gus) reporter gene was employed to optimize the system, which has been successfully used to produce the main antigenic protein from Norwalk virus.

Moreover, the studies performed in this study have shown that the pLS1RGFP vector is suitable for gene inducible overexpression in L. lactis. The usage of pLS1RGFP in L. lactis was standardised, and its utililisation for controlled expression of housekeeping enzymes validated by cloning of the lactococcal rnc gene in it, and characterization of the RNase III encoded by the constructed recombinant plasmid.

Finally, the vector has been validated for expression of genes of industrial interest.
The gtf of the Pediococcus parvulus 2.6, which synthesises an exopolysaccharide with prebiotic properties, was cloned in the vector under the control of the PM promoter. Immunological and enzymatic studies revealed that overexpression of the gene conferred to Gram-positive bacteria, including L. lactis, the ability to synthesise and secrete the exopolysaccharide. In addition, they demonstrated that indeed the gtf gene product is a glycosyltransferase bound to the cytoplasmic membrane.

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Consejo Superior de Investigaciones Científicas
Centro de Investigaciones Biológicas, Ramiro de Maeztu 9
28040 Madrid
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