Relative contributions of non-essential Sec pathway components and cell envelope-associated proteases to high-level enzyme secretion by Bacillus subtilis

Abstract

Background: Bacillus subtilis is an important industrial workhorse applied in the production of many different commercially relevant proteins, especially enzymes. Virtually all of these proteins are secreted via the general secretion (Sec) pathway. Studies from different laboratories have demonstrated essential or non-essential contributions of various Sec machinery components to protein secretion in B. subtilis. However, a systematic comparison of the impact of each individual Sec machinery component under conditions of high-level protein secretion was so far missing.

Results: In the present study, we have compared the contributions of non-essential Sec pathway components and cell envelope-associated proteases on the secretion efficiency of three proteins expressed at high level. This concerned the α-amylases AmyE from B. subtilis and AmyL from Bacillus licheniformis, and the serine protease BPN' from Bacillus amyloliquefaciens. We compared the secretion capacity of mutant strains in shake flask cultures, and the respective secretion kinetics by pulse-chase labeling experiments. The results show that secDF, secG or rasP mutations severely affect AmyE, AmyL and BPN' secretion, but the actual effect size depends on the investigated protein. Additionally, the chaperone DnaK is important for BPN' secretion, while AmyE or AmyL secretion are not affected by a dnaK deletion. Further, we assessed the induction of secretion stress responses in mutant strains by examining AmyE- and AmyL-dependent induction of the quality control proteases HtrA and HtrB. Interestingly, the deletion of certain sip genes revealed a strong differential impact of particular signal peptidases on the magnitude of the secretion stress response.

Conclusions: The results of the present study highlight the importance of SecDF, SecG and RasP for protein secretion and reveal unexpected differences in the induction of the secretion stress response in different mutant strains.

Keywords: Bacillus subtilis; DnaK; HtrA; HtrB; Protein production; Protein translocation; RasP; SecDF; SecG; Secretion stress.

Fig. 1

Secretion of AmyL, AmyE and BPN’ upon 16, 20 or 24 h of growth. Cells were separated from the growth medium by centrifugation after 16, 20 or 24 h of growth in MBU medium at 37 °C. Subsequently, proteins in the growth medium fractions were precipitated with TCA, separated by LDS-PAGE, and visualized with SimplyBlue SafeStain (upper panel). Prior to TCA precipitation and gel loading, the samples were corrected for the OD₆₀₀ of the respective cultures as listed in the bottom panel. To assess the extent of cell lysis during culturing, the extracellular levels of the cytoplasmic marker protein TrxA were assessed by Western blotting with specific antibodies (middle panel). Molecular weights of marker proteins are indicated (in kDa) on the left side of the gel segment

Fig. 2

Secretion of AmyE, AmyL or BPN’ by strains lacking individual non-essential secretion machinery components. AmyE-, AmyL- or BPN’-producing strains lacking the dnaK, secDF, secG, sipS, sipT, sipU, sipV or sipW genes, as well as the respective wild-type (wt) control, were grown for 16 h in MBU medium at 37 °C. Next, cells and growth media were separated by centrifugation and proteins in the growth medium fractions were analyzed by LDS-PAGE and SimplyBlue SafeStaining as described for Fig. 1

Fig. 3

Secretion of AmyE, AmyL or BPN’ by strains lacking individual cell envelope-associated proteases. AmyE-, AmyL- or BPN’-producing strains lacking the sppA, tepA, rasP, prsW, wprA, yqeZ, htrA or htrB genes, as well as the respective wild-type (wt) control, were grown for 16 h in MBU medium at 37 °C. Next, cells and growth media were separated by centrifugation, and proteins in the growth medium fractions were analyzed by LDS-PAGE and SimplyBlue SafeStaining as described for Fig. 1. *, the effects of a rasP deletion were previously described [26]

Fig. 4

Kinetics of AmyE and AmyL precursor processing, and BPN’ secretion in secDF, secG or dnaK mutant strains. Processing of AmyE or AmyL precursors (p) to the respective mature forms (m) was analyzed by pulse-chase labeling. Cells grown in MBU medium at 37 °C were labeled with [³⁵S]-methionine for 30 s prior to chase with excess non-radioactive methionine. Samples were withdrawn at the indicated time points after the chase and mixed with ice-cold TCA. Subsequently, (pre-)AmyE or (pre-)AmyL were immunoprecipitated with specific antibodies against AmyE or AmyL, separated by LDS-PAGE, and visualized by autoradiography. The secretion of BPN’ was also analyzed by pulse-chase labeling of cells grown in MBU at 37 °C with [³⁵S]-methionine for 30 s prior to chase with excess non-radioactive methionine. However, in this case, samples withdrawn at the indicated time points after the chase were chilled on ice and, subsequently, cells were separated from the growth medium by centrifugation. The appearance of BPN’ in the growth medium fractions was then analyzed by immunoprecipitation with antibodies against BPN’, LDS-PAGE and autoradiography. The position of mature BPN’ (m) is indicated

Fig. 5

Expression of HtrA and HtrB upon AmyE or AmyL production. Wild-type cells producing AmyE or AmyL were separated from the growth medium by centrifugation after 16 h of growth in MBU medium at 37 ℃. Subsequently, proteins in the cells and growth medium fractions were separated by LDS-PAGE, and visualized with SimplyBlue SafeStain as described for Figure 1 (upper panel). The presence of HtrA and HtrB in the cell and growth medium fractions was analyzed by Western blotting using polyclonal antibodies against HtrA (middle panel) or HtrB (lower panel). The extracellular proteolytically processed forms of HtrA and HtrB are marked with a star. Major cell-associated degradation products are marked with a ‘D’. Molecular weights of marker proteins are indicated (in kDa) on the left side of each gel and Western blot

Fig. 6

Analysis of HtrA and HtrB levels in strains lacking individual non-essential secretion machinery components or cell envelope-associated proteases upon production of AmyE or AmyL. The levels of full-size HtrA (a) or HtrB (b) in wild-type or mutant cells producing AmyE or AmyL was assessed by Western blotting with specific antibodies as described for Fig. 5. The relative levels of HtrA or HtrB compared to the respective levels in the wild-type strain were assessed by ImageJ analysis. Black bars represent the HtrA or HtrB levels in non-producing strains, grey bars relate to the HtrA or HtrB levels in AmyE-producing strains, and white bars to HtrA or HtrB levels in AmyL-producing strains. The error bars represent the standard error of the mean for three independent experiments