food-grade cloning vector for lactic acid bacteria based on the nisin immunity gene nisI

A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. E...

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Bibliographic Details
Published in:Applied microbiology and biotechnology 2002, Vol.59 (4/5), p.467-471
Main Authors: Takala, T.M, Saris, P.E.J
Format: Article
Language:English
Subjects:
Aminopeptidases - genetics
Anti-Bacterial Agents - immunology
Anti-Bacterial Agents - pharmacology
Bacteria
Bacterial Proteins - genetics
Cheese
cheese starters
Cloning
Cloning, Molecular
Deoxyribonucleic acid
DNA
Drug Resistance, Bacterial
Electroporation
Fermentation
Food
Food Industry - methods
Food Microbiology
Genes
Genetic Vectors
immunity
lactic acid bacteria
Lactobacillus - drug effects
Lactobacillus - enzymology
Lactobacillus - genetics
Lactobacillus helveticus
Lactococcus lactis
Lactococcus lactis - drug effects
Lactococcus lactis - enzymology
Lactococcus lactis - genetics
Lipoproteins - genetics
Membrane Proteins
Microbiology
molecular cloning
nisin
Nisin - immunology
Nisin - pharmacology
Plasmids
Plasmids - genetics
proline
prolyl aminopeptidase
promoter regions
replicon
Transformation, Bacterial
Vectors (Biology)
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Description
Summary:A new food-grade cloning vector for lactic acid bacteria was constructed using the nisin immunity gene nisI as a selection marker. The food-grade plasmid, pLEB590, was constructed entirely of lactococcal DNA: the pSH71 replicon, the nisI gene, and the constitutive promoter P45 for nisI expression. Electroporation into Lactococcus lactis MG1614 with 60 international units (IU) nisin/ml selection yielded approximately 10(5) transformants/microgram DNA. MG1614 carrying pLEB590 was shown to be able to grow in medium containing a maximum of 250 IU nisin/ml. Plasmid pLEB590 was successfully transformed into an industrial L. lactis cheese starter carrying multiple cryptic plasmids. Suitability for molecular cloning was confirmed by cloning and expressing the proline iminopeptidase gene pepI from Lactobacillus helveticus in L. lactis and Lb. plantarum. These results show that the food-grade expression system reported in this paper has potential for expression of foreign genes in lactic acid bacteria in order to construct improved starter bacteria for food applications.
ISSN:0175-7598
1432-0614
DOI:10.1007/s00253-002-1034-4