High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors.
Vectors have been developed for inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum in which expression of the gene of interest is driven by strong, regulated promoters from bacteriocin operons found in L. sakei strains. The activity of these promoters is controlled via a two-component signal transduction system, which responds to an externally added peptide pheromone. The vectors have a modular design, permitting easy exchange of all essential elements: the inducible promoter, the cognate regulatory system, the gene of interest, the antibiotic resistance marker and the replicon. Various variants of these so-called 'pSIP' vectors were constructed and tested, differing in terms of the bacteriocin regulon from which the regulatory elements were derived (sakacin A or sakacin P), the regulated promoter selected from these regulons, and the replicon (derived from p256 or pSH71). Using beta-glucuronidase (GusA) and aminopeptidase N (PepN) as reporters, it was shown that the best vectors permitted inducible, pheromone-dose-dependent gene expression at very high levels, while displaying moderate basal activities when not induced. The most effective set-up was obtained using a vector containing the pSH71 replicon, the orfX promoter from the sakacin P regulon, and the cognate regulatory genes, in a L. sakei host. GusA levels obtained with this set-up were approximately ten times higher than the levels obtained with prototype pSIP versions, whereas PepN levels amounted to almost 50% of total cellular protein.
Sørvig
Elisabeth
E
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.|MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
Mathiesen
Geir
G
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.
Naterstad
Kristine
K
MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
Eijsink
Vincent G H
VGH
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO Box 5003, N-1432 Ås, Norway.
Axelsson
Lars
L
MATFORSK, Norwegian Food Research Institute, Osloveien 1, N-1430 Ås, Norway.
D001430:Bacteriocins; D018826:CD13 Antigens / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D007778:Lactobacillus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic
D001430:Bacteriocins; C087217:sakacin P protein, Lactobacillus; D005966:Glucuronidase; D018826:CD13 Antigens