Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118

Abstract

Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

FIG. 1.

(A) RT-PCRs showing nisin induced transcriptional control of the betL gene in UCC118-BetL⁺. Each lane represents the level of betL transcript resulting from 30 PCR cycles with primers XbaIKO (TAAGCGCCACTCTAGACC) and EcoRIKO (GCACGAATTCACCAAGTA) on cDNA generated from total RNA isolated from cells after 30 min of exposure to nonstress conditions in MRS broth (first lane), 0.01% (vol/vol) cell-free supernatant from the nisin-producing strain Lactococcus lactis NZ9700 (second lane) and 25 ng of nisin powder/ml (third lane). Bent arrow, promoter; lollipop, terminator. (B) Growth of L. salivarius UCC118-BetL⁺ (○ and Δ) and UCC118-BetL⁻ (• and ▴) in MRS broth with 7% added NaCl in the presence (circles) and absence (triangles) of added nisin. Each point represents the mean of at least three independent experiments. (C) [¹⁴C]glycine betaine uptake rates in L. salivarius UCC118-BetL⁺ (white bar) and UCC118-BetL⁻ (black bar).

FIG. 2.

Viability of L. salivarius UCC118-BetL⁺ (○ and Δ) and UCC118-BetL⁻ (• and ▴) during storage at 4°C (circles) and 10°C (triangles) in MRS broth. Results are means of four independent experiments.

FIG. 3.

Survival of L. salivarius UCC118-BetL⁺ (○) and UCC118-BetL⁻ (•) following repeated freeze-thaw cycles at −20°C (A) and at −70°C (B). Results are means of four independent experiments.

FIG. 4.

Survival of L. salivarius UCC118-BetL⁺ and UCC118-BetL⁻ following (A) freeze-drying and (B) spray-drying. Results are means of experiments performed in triplicate.

FIG. 5.

High-pressure-induced inactivation of L. salivarius UCC118-BetL⁺ (white bars) and UCC118-BetL⁻ (black bars). All treatments were carried out for 5 min at 20°C. Results are means of experiments performed in triplicate.