Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338

Abstract

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52 degrees C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54 degrees C) and lactobacilli (at 60 degrees C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.

FIG. 1.

2D-PAGE used to investigate the mechanisms involved in the development of thermotolerance for probiotic L. paracasei NFBC 338. The electrophoretograms shown are representative of two independent trials. IEF, isoelectric focusing.

FIG. 2.

(A) Schematic representation of the groESL operon under control of the NICE system. RBS, ribosome binding site. (B and C) SDS-PAGE illustrating the controlled expression of GroEL and GroES using the NICE system in L. lactis (B) using vector pNZ8048 and in L. paracasei NFBC 338 (C) using vector pMSP3535. Lanes for both gels: 1 and 10, protein standard marker; 2, plasmid control at time zero; 3, plasmid control plus nisin after 1 h; 4, plasmid control plus nisin after 3 h; 5, plasmid control plus nisin after 5 h; 6, GroESL-overproducing strain at time zero; 7, GroESL-overproducing strain plus nisin after 1 h; 8, GroESL overproducing strain plus nisin after 3 h; 9, GroESL-overproducing strain plus nisin after 5 h.

FIG. 3.

Stress tolerance in GroESL-overproducing strains of L. lactis during (A) heat stress (54°C for 30 min), (B) salt stress (5 M NaCl for 1 h), and (C) butanol stress (0.5% [vol/vol] butanol for 6 h). Symbols: ▴, nisin-induced L. lactis(pNZ8048); ▵, L. lactis(pNZ8048); •, nisin-induced, heat-adapted L. lactis(pNZ8048); □, L. lactis(pGRO1); ▪, nisin-induced L. lactis(pGRO1); ○, nisin-induced, heat-adapted L. lactis(pGRO1). The values shown are the means ± standard errors (error bars) for three challenge trials.

FIG. 4.

Stress tolerance in GroESL-overproducing strains of L. paracasei NFBC 338 during (A) heat stress (60°C for 30 min), (B) salt stress (5 M NaCl for 1 h), and (C) butanol stress (0.5% [vol/vol] butanol for 6 h). Symbols: ▴, nisin-induced L. paracasei NFBC 338(pMSP3535); ▵, L. paracasei NFBC 338(pMSP3535); •, nisin-induced, heat-adapted L. paracasei NFBC 338(pMSP3535); □, L. paracasei NFBC 338(pGRO2); ▪, nisin-induced L. paracasei NFBC 338(pGRO2); ○, nisin-induced, heat-adapted L. paracasei NFBC 338(pGRO2). The values shown are the means ± standard errors (error bars) for three challenge trials.