Degradation of Extracytoplasmic Catalysts for Protein Folding in Bacillus subtilis

Abstract

The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold into their active and protease-resistant conformations once membrane passage is completed. The lipoprotein PrsA and the membrane proteins HtrA and HtrB facilitate the extracytoplasmic folding and quality control of exported proteins. Among the native exported proteins of B. subtilis are at least 10 proteases that have previously been implicated in the degradation of heterologous secreted proteins. Recently, we have shown that these proteases also degrade many native membrane proteins, lipoproteins, and secreted proteins. The present studies were therefore aimed at assessing to what extent these proteases also degrade extracytoplasmic catalysts for protein folding. To this end, we employed a collection of markerless protease mutant strains that lack up to 10 different extracytoplasmic proteases. The results show that PrsA, HtrA, and HtrB are indeed substrates of multiple extracytoplasmic proteases. Thus, improved protein secretion by multiple-protease-mutant strains may be related to both reduced proteolysis and improved posttranslocational protein folding and quality control.

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

“…Of note, HtrA and HtrB induction can also be detected in the growth medium ( Fig. 5) but, as previously shown, the extracellular levels of their proteolytically processed forms depend critically on the levels of RasP and the eight secreted proteases of B. subtilis, especially WprA [30,34,37,39,48]. Hence, the cellular HtrA and HtrB levels are reflecting secretion stress induction more reliably than the extracellular levels and, importantly, they directly reflect the levels of the main effectors regulated by the secretion stress response.…”

“…N-terminally cleaved forms of HtrA and HtrB can also be encountered in the growth medium, but they are subject to degradation by secreted proteases of B. subtilis [34][35][36]. Of note, htrA and htrB are CssRS-dependently cross-regulated, which means that one is upregulated when the other is deleted [37,38]. This indicates that basal levels of HtrA and HtrB production are needed to avoid secretion stress.…”

“…This indicates that basal levels of HtrA and HtrB production are needed to avoid secretion stress. Intriguingly, the protease WprA serves an important function at the membrane-cell wall interface controlling not only the levels of secretory proteins but also of the protein folding catalyst PrsA [37,39,40].…”

Enhanced heterologous protein productivity by genome reduction in Lactococcus lactis NZ9000

“…The ybdD mutant strain shows increased levels of exported proteins [18]. The multiple protease mutant strains were also reported in Laxmi’s work [19]. In his study, not only the degradation of heterologous protein was reduced, but also the levels of cell-associated protein-folding catalysts were elevated in the multiple protease mutants.…”

“…The plasmid pLEB690- lecC [19] was digested with Hin dIII and Bam HI. The fragment (P nisZ +Usp45- lecC + lecI ) was obtained with DNA gel extraction kit and was cloned into Hin dIII- Bam HI digested pLEB124, yielding pLEB124- lecC (containing the fragment P45+P nisZ +Usp45- lecC + lecI ).…”