High-level, inducible gene expression in Lactobacillus sakei and Lactobacillus plantarum using versatile expression vectors

Abstract

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 b-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. INTRODUCTIONMany Lactobacillus species are important in the food industry, where they are used in a variety of products. Lactobacillus sakei is used in meat and vegetable fermentations and has a broad range of possible habitats. The versatility of L. sakei can partly be explained by its ability to survive and grow under adverse conditions, such as low temperature and pH, high salt concentration, smoke, ethanol, low water activity and radiation. Lactobacillus plantarum is also found in many habitats, including dairy and meat products, plant and vegetable fermentations, and in the gastrointestinal tract and oral cavity of humans (Axelsson & Ahrné, 2000). Some L. plantarum strains have probiotic effects on human health (Alander et al., 1999;Schultz et al., 2002;Mercenier et al., 2003). Lactobacilli have great potential as food-grade cell factories and as delivery vehicles for interesting proteins, such as antigens, antibodies and growth factors (Pouwels et al., 1996(Pouwels et al., , 2001Pavan et al., 2000; Krüger et al., 2002;Scheppler et al., 2002). Thus, there is considerable interest in the development of genetic tools for efficient and controllable gene expression in lactobacilli (de Vos, 1999;Mercenier et al., 2000).Many lactic acid bacteria (LAB) produce antimicrobial peptides called bacteriocins, and their production is often regulated via quorum-sensing mechanisms based on a secreted peptide pheromone (Eijsink et al., 2002;Quadri, 2002). The pheromone activates a two-component regulatory s...

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

“…Similar to our result, L. plantarum, L. pentosus and L. brevis usually predominated in the different fermented vegetables. Lactobacillus sakei is usually isolated from fermented vegetables and meat (Andrighetto et al 2001;Kim and Chun 2005); the versatility can partly be explained by its ability to survive and grow under adverse condi- tions, such as low temperature and pH, high salt concentration, ethanol and low water activity (Sorvig et al, 2005). Lactobacillus spicheri is typically isolated from sourdoughs (Meroth et al, 2004), and it is rare in fermented vegetables.…”

Enantioselective Regulation of Lactate Racemization by LarR in Lactobacillus plantarum

“…The bacterial strains and plasmids used in the present study are listed in Table 1. Plasmid constructions were performed in Escherichia coli TOP10 for pUC18Ery (14) and pSIP409 derivatives (15) and in L. lactis NZ3900 (16) for pNZ8048 derivatives (17). E. coli was grown in Luria broth (LB) at 37°C with aeration, Lactococcus lactis was grown in M17 medium (Merck, Germany) supplemented with 0.5% glucose at 28°C at 120 rpm, and Lactobacillus plantarum was grown in MRS (De Man-Rogosa-Sharpe) broth (Difco Laboratories, Inc., Detroit, MI) at 28°C without shaking.…”

Genetically Engineered Lactobacilli for Technological and Functional Food Applications

“…Similar to the NICE system, in L. plantarum (Mathiesen et al, 2004) and L. sakei (Axelsson et al, 2003) vectors have been developed using a pheromone-regulated bacteriocin promoter and the regulatory system of sakacin A production, respectively. The pSIP vector series, based on the genes and promoters involved in sakacin A and P, used erythromycin as selection marker (Sorvig et al, 2005). In order to developed a potential food-grade expression system the erythromycin gene in the pSIP vectors has been replaced by the alr gene, which encodes the alanine racemase enzyme that is essential for cell wall biosynthesis (Nguyen et al, 2011).…”