Geeske Zanen

Proteomics of Protein Secretion by Bacillus subtilis: Separating the “Secrets” of the Secretome

SUMMARY Secretory proteins perform a variety of important“ remote-control” functions for bacterial survival in the environment. The availability of complete genome sequences has allowed us to make predictions about the composition of bacterial machinery for protein secretion as well as the extracellular complement of bacterial proteomes. Recently, the power of proteomics was successfully employed to evaluate genome-based models of these so-called secretomes. Progress in this field is well illustrated by the proteomic analysis of protein secretion by the gram-positive bacterium Bacillus subtilis, for which ∼90 extracellular proteins were identified. Analysis of these proteins disclosed various“ secrets of the secretome,” such as the residence of cytoplasmic and predicted cell envelope proteins in the extracellular proteome. This showed that genome-based predictions reflect only∼ 50% of the actual composition of the extracellular proteome of B. subtilis. Importantly, proteomics allowed the first verification of the impact of individual secretion machinery components on the total flow of proteins from the cytoplasm to the extracellular environment. In conclusion, proteomics has yielded a variety of novel leads for the analysis of protein traffic in B. subtilis and other gram-positive bacteria. Ultimately, such leads will serve to increase our understanding of virulence factor biogenesis in gram-positive pathogens, which is likely to be of high medical relevance.

Signal peptide hydrophobicity is critical for early stages in protein export by Bacillus subtilis

Signal peptides that direct protein export in Bacillus subtilis are overall more hydrophobic than signal peptides in Escherichia coli. To study the importance of signal peptide hydrophobicity for protein export in both organisms, the α‐amylase AmyQ was provided with leucine‐rich (high hydrophobicity) or alanine‐rich (low hydrophobicity) signal peptides. AmyQ export was most efficiently directed by the authentic signal peptide, both in E. coli and B. subtilis. The leucine‐rich signal peptide directed AmyQ export less efficiently in both organisms, as judged from pulse‐chase labelling experiments. Remarkably, the alanine‐rich signal peptide was functional in protein translocation only in E. coli. Cross‐linking of in vitro synthesized ribosome nascent chain complexes (RNCs) to cytoplasmic proteins showed that signal peptide hydrophobicity is a critical determinant for signal peptide binding to the Ffh component of the signal recognition particle (SRP) or to trigger factor, not only in E. coli, but also in B. subtilis. The results show that B. subtilis SRP can discriminate between signal peptides with relatively high hydrophobicities. Interestingly, the B. subtilis protein export machinery seems to be poorly adapted to handle alanine‐rich signal peptides with a low hydrophobicity. Thus, signal peptide hydrophobicity appears to be more critical for the efficiency of early stages in protein export in B. subtilis than in E. coli.

The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis

Bacillus species are valuable producers of industrial enzymes and biopharmaceuticals, because they can secrete large quantities of high‐quality proteins directly into the growth medium. This requires the concerted action of quality control factors, such as folding catalysts and ‘cleaning proteases’. The expression of two important cleaning proteases, HtrA and HtrB, of Bacillus subtilis is controlled by the CssRS two‐component regulatory system. The induced CssRS‐dependent expression of htrA and htrB has been defined as a protein secretion stress response, because it can be triggered by high‐level production of secreted α‐amylases. It was not known whether translocation of these α‐amylases across the membrane is required to trigger a secretion stress response or whether other secretory proteins can also activate this response. These studies show for the first time that the CssRS‐dependent response is a general secretion stress response which can be triggered by both homologous and heterologous secretory proteins. As demonstrated by high‐level production of a nontranslocated variant of the α‐amylase, AmyQ, membrane translocation of secretory proteins is required to elicit this general protein secretion stress response. Studies with two other secretory reporter proteins, lipase A of B. subtilis and human interleukin‐3, show that the intensity of the protein secretion stress response only partly reflects the production levels of the respective proteins. Importantly, degradation of human interleukin‐3 by extracellular proteases has a major impact on the production level, but only a minor effect on the intensity of the secretion stress response.

Genetic or chemical protease inhibition causes significant changes in the Bacillus subtilis exoproteome

Bacillus subtilis is a prolific producer of enzymes and biopharmaceuticals. However, the susceptibility of heterologous proteins to degradation by (extracellular) proteases is a major limitation for use of B. subtilis as a protein cell factory. An increase in protein production levels has previously been achieved by using either protease‐deficient strains or addition of protease inhibitors to B. subtilis cultures. Notably, the effects of genetic and chemical inhibition of proteases have thus far not been compared in a systematic way. In the present studies, we therefore compared the exoproteomes of cells in which extracellular proteases were genetically or chemically inactivated. The results show substantial differences in the relative abundance of various extracellular proteins. Furthermore, a comparison of the effects of genetic and/or chemical protease inhibition on the stress response triggered by (over) production of secreted proteins showed that chemical protease inhibition provoked a genuine secretion stress response. From a physiological point of view, this suggests that the deletion of protease genes is a better way to prevent product degradation than the use of protease inhibitors. Importantly however, studies with human interleukin‐3 show that chemical protease inhibition can result in improved production of protease‐sensitive secreted proteins even in mutant strains lacking eight extracellular proteases.

FlhF, the Third Signal Recognition Particle-GTPase of Bacillus subtilis, Is Dispensable for Protein Secretion

Bacillus subtilis contains three proteins of the signal recognition particle-GTPase family known as Ffh, FtsY, and FlhF. Here we show that FlhF is dispensable for protein secretion, unlike Ffh and FtsY. Although flhF is located in the fla/che operon, B. subtilis 168 flhF mutant cells assemble flagella and are motile.

The Bacillus secretion stress response is an indicator for α-amylase production levels

Aims:  Overproduced α‐amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two‐component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high‐level α‐amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to α‐amylase production.