Expression of the Staphylococcus hyicus lipase in Lactococcus lactis

Abstract

The extracellular Staphylococcus hyicus lipase was expressed under the control of different promoters in Lactococcus lactis and Bacillus subtilis. Its expression at high and moderate levels is toxic for the former and the latter hosts, respectively. In L. lactis, the lipase was expressed at a high level, up to 30% of the total cellular proteins, under the control of the inducible promoter PnisA. About 80% of the lipase remained associated with the cells. Close to half of this amount remained associated with the inner side of the cytoplasmic membrane as unprocessed pre-pro-lipase. The other half was trapped by the cell wall and partially degraded at the N-terminal end. This result suggests that extracellular proteases degrade the lipase. Surprisingly, the kinetics and the pattern of lipase degradation were different in the two L. lactis subspecies, L. lactis subsp. cremoris and L. lactis subsp. lactis. The extracellular proteolytic systems that degrade lipase are thus different in these closely related subspecies. The incorrect export of the lipase is not due to an inappropriate leader peptide but may be due to an inefficiency of several steps of lipase secretion. We propose that (i) the S. hyicus lipase may require a special accessory system to be correctly exported or (ii) the kinetics of lipase synthesis may be a critical factor for proper folding.

FIG. 1

Strategy used to place the lip gene under the control of a strong constitutive promoter. Chloramphenicol resistance is used to select the first cloning step in L. lactis. PX, P5, P23, P32, P44, or P59.

FIG. 2

Growth (A) and lipolytic activities (B) determined for cell extracts of strains JIM5496 (diamonds), JIM7022 (squares), and JIM7048 (triangles). Strains JIM7022 and JIM7048 were induced by nisin at an OD₆₀₀ of 0.3 (arrows).

FIG. 3

Coomassie blue staining of JIM7022 total cell extracts. The samples (0.1 U, corresponding to the quantity of cells in 100 μl of L. lactis culture at an OD₆₀₀ of 1) were loaded at different times after nisin induction (in minutes) as shown in Fig. 2.

FIG. 4

Western blots of total cell extract (0.1 U) and supernatant (0.5 U) proteins from lactococcal strain JIM5496 with anti-CtermLIP (A), anti-NtermLIP (B), and anti-PepC sera (C). The samples were taken at different stages of growth (OD₆₀₀s of 0.2, 0.6, 1, and 1.4). See the legend to Fig. 3 for an explanation of U.

FIG. 5

Comparison of the degradation products of the S. hyicus lipase produced in JIM7048 (L. lactis subsp. lactis) and JIM7022 (L. lactis subsp. cremoris), as revealed by immunostaining with anti-CtermLIP sera.

FIG. 6

Differences in the kinetics of lipase production and degradation of cell extracts (0.1 U) from JIM7022 (A) and JIM7048 (B), as revealed by immunostaining with anti-CtermLIP sera. Total cell extracts were loaded at different times after nisin induction (in minutes) as shown in Fig. 2. See the legend to Fig. 3 for an explanation of U.

FIG. 7

Localization of the S. hyicus lipase in strain JIM5496 (P23) by Western blotting with anti-CtermLIP sera. Panel 1, total cell extract; panel 2, sample without treatment; panel 3, sample incubated for 30 min at 37°C; panel 4, cell wall and protoplast fractions incubated with trypsin for 30 min at 37°C; panel 5, protoplasts incubated with Triton X-100 and trypsin for 30 min at 37°C; panel 6, total cell extract incubated with Triton X-100 for 30 min at 37°C. Abbreviations: T, total cell extract; W, cell wall fraction; C, cytoplasm fraction; M, membrane fraction; CM, protoplasts; Tryp, trypsin; TX100, Triton X-100.

FIG. 8

Effect of the Usp45 leader peptide on the secretion of lipase. Shown is a Western blot of total cell extract (0.1 U) (A) and supernatant (0.5 U) (B) proteins from lactococcal strains JIM5498 (native leader) and JIM5928 (Usp45 leader). The samples were taken at an OD₆₀₀ of 0.8. See the legend to Fig. 3 for an explanation of U.