Nisin-induced expression of pediocin in dairy lactic acid bacteria

Abstract

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml−1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

“…This phenotype was complemented by introducing the positive allele of pyrB cloned in a plasmid under control of the nisin-inducible promoter. Note that in the medium without nisin partial complementation was observed in keeping with reports about the modest leakiness of this promoter (47). Interestingly, the growth delay was also restored by the addition of uracil to the medium, indicating that even in rich medium the ⌬pyrB mutant is starved for nucleotides (Fig.…”

Nisin-selectable food-grade secretion vector for Lactococcus lactis

“…Several studies have demonstrated that L. lactis is a good host for pediocin expression (Arques et al 2008;Reviriego et al 2007a, b). Nisin-inducible expression of pediocin has also been successfully applied (Renye and Somkuti 2010;Reviriego et al 2007a). However, use of the usp45 signal sequence has not been previously reported for pediocin secretion, and the secretion of pediocin from a food-grade vector with nisin selection is also novel.…”

Zirex: a Novel Zinc-Regulated Expression System for Lactococcus lactis

“…To date, several promoters originally from Lactococcus, regulated by inducers or environmental factors, have been documented, including the dnaJ promoter, induced by heat shock (3); the PA170 promoter, which can be upregulated at a low pH during the transition to stationary phase (4); the prtP promoter, which is regulated by the peptide concentration in the medium (5); and the P Zn zitR promoter, which responds to divalent cation starvation (6). The promoter of nisin, P nisA , is the most widely used promoter for inducible protein expression in L. lactis (1,7) and other Gram-positive bacteria (8). The expression from the P nisA promoter is regulated by the two-component regulatory system NisRK, which is triggered by nisin.…”