A disulfide bond-containing alkaline phosphatase triggers a BdbC-dependent secretion stress response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for alpha-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

A Disulfide Bond-Containing Alkaline Phosphatase Triggers a BdbC-Dependent Secretion Stress Response in Bacillus subtilis

The gram-positive bacterium Bacillus subtilis secretes high levels of proteins into its environment. Most of these secretory proteins are exported from the cytoplasm in an unfolded state and have to fold efficiently after membrane translocation. As previously shown for α-amylases of Bacillus species, inefficient posttranslocational protein folding is potentially detrimental and stressful. In B. subtilis, this so-called secretion stress is sensed and combated by the CssRS two-component system. Two known members of the CssRS regulon are the htrA and htrB genes, encoding potential extracytoplasmic chaperone proteases for protein quality control. In the present study, we investigated whether high-level production of a secretory protein with two disulfide bonds, PhoA of Escherichia coli, induces secretion stress in B. subtilis. Our results show that E. coli PhoA production triggers a relatively moderate CssRS-dependent secretion stress response in B. subtilis. The intensity of this response is significantly increased in the absence of BdbC, which is a major determinant for posttranslocational folding of disulfide bond-containing proteins in B. subtilis. Our findings show that BdbC is required to limit the PhoA-induced secretion stress. This conclusion focuses interest on the BdbC-dependent folding pathway for biotechnological production of proteins with disulfide bonds in B. subtilis and related bacilli.     

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.     

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.     

Translocation of proteins across the cell envelope of Gram-positive bacteria

In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.     

Developments in the use of Bacillus species for industrial production

Bacillus species continue to be dominant bacterial workhorses in microbial fermentations. Bacillus subtilis (natto) is the key microbial participant in the ongoing production of the soya-based traditional natto fermentation, and some Bacillus species are on the Food and Drug Administration's GRAS (generally regarded as safe) list. The capacity of selected Bacillus strains to produce and secrete large quantities (20-25 g/L) of extracellular enzymes has placed them among the most important industrial enzyme producers. The ability of different species to ferment in the acid, neutral, and alkaline pH ranges, combined with the presence of thermophiles in the genus, has lead to the development of a variety of new commercial enzyme products with the desired temperature, pH activity, and stability properties to address a variety of specific applications. Classical mutation and (or) selection techniques, together with advanced cloning and protein engineering strategies, have been exploited to develop these products. Efforts to produce and secrete high yields of foreign recombinant proteins in Bacillus hosts initially appeared to be hampered by the degradation of the products by the host proteases. Recent studies have revealed that the slow folding of heterologous proteins at the membrane-cell wall interface of Gram-positive bacteria renders them vulnerable to attack by wall-associated proteases. In addition, the presence of thiol-disulphide oxidoreductases in B. subtilis may be beneficial in the secretion of disulphide-bond-containing proteins. Such developments from our understanding of the complex protein translocation machinery of Gram-positive bacteria should allow the resolution of current secretion challenges and make Bacillus species preeminent hosts for heterologous protein production. Bacillus strains have also been developed and engineered as industrial producers of nucleotides, the vitamin riboflavin, the flavor agent ribose, and the supplement poly-gamma-glutamic acid. With the recent characterization of the genome of B. subtilis 168 and of some related strains, Bacillus species are poised to become the preferred hosts for the production of many new and improved products as we move through the genomic and proteomic era.     

Protein secretion in Bacillus species.

Bacilli secrete numerous proteins into the environment. Many of the secretory proteins, their export signals, and their processing steps during secretion have been characterized in detail. In contrast, the molecular mechanisms of protein secretion have been relatively poorly characterized. However, several components of the protein secretion machinery have been identified and cloned recently, which is likely to lead to rapid expansion of the knowledge of the protein secretion mechanism in Bacillus species. Comparison of the presently known export components of Bacillus species with those of Escherichia coli suggests that the mechanism of protein translocation across the cytoplasmic membrane is conserved among gram-negative and gram-positive bacteria differences are found in steps preceding and following the translocation process. Many of the secretory proteins of bacilli are produced industrially, but several problems have been encountered in the production of Bacillus heterologous secretory proteins. In the final section we discuss these problems and point out some possibilities to overcome them.     

Bacillus subtilis as cell factory for pharmaceutical proteins: a biotechnological approach to optimize the host organism

Bacillus subtilis is a rod-shaped, Gram-positive soil bacterium that secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. These enzymes are produced commercially and this production represents about 60% of the industrial-enzyme market. Unfortunately, the secretion of heterologous proteins, originating from Gram-negative bacteria or from eukaryotes, is often severely hampered. Several bottlenecks in the B. subtilis secretion pathway, such as poor targeting to the translocase, degradation of the secretory protein, and incorrect folding, have been revealed. Nevertheless, research into the mechanisms and control of the secretion pathways will lead to improved Bacillus protein secretion systems and broaden the applications as industrial production host. This review focuses on studies that aimed at optimizing B. subtilis as cell factory for commercially interesting heterologous proteins.     

Mutations in the alpha-amylase gene of Bacillus amyloliquefaciens, leading to a decrease in the temperature of protein inactivation

Alpha-amylase genes of Bacillus amyloliquefaciens, coding proteins with reduced thermostability, had been obtained as a result of hydroxylamine mutagenesis. Temperature, pH and starch concentration dependences of two mutant alpha-amylases were investigated. The synthesis of the alpha-amylases by several B. subtilis strains with different levels of extracellular proteases was also studied. The mutation containing fragments were localized and the structures of the mutations were determined. It was found that the decrease of thermostability of mutant No 141 was due to Asp to Asn change at the position No 194 of the mature protein, and for mutant No 191--due to Glu to Lys change at the position No 185.     

Protein secretion in gram-positive bacteria.

Gram-positive bacteria often secrete large amounts of proteins into the surrounding medium. This feature makes them attractive as hosts for the industrial production of extracellular enzymes. Compared to Escherichia coli, relatively little is known about the mechanism of protein secretion in these organisms. However, the recent identification of Bacillus subtilis genes whose gene products are highly homologous to some of the Sec (secretion) proteins of E. coli strongly suggests that important principles of protein translocation across the plasma membrane might be highly conserved. In contrast, the steps following the actual translocation event might be different in Gram-positive and Gram-negative bacteria. The scope of this review is to outline the recent progress that has been made in the elucidation of the secretion pathway in Gram-positive bacteria and to discuss potential applications in strain improvement for the industrial production of extracellular proteins.     

Synthesis of OmpA protein of Escherichia coli K12 in Bacillus subtilis

We have inserted a C-terminally truncated gene of the major outer membrane protein OmpA of Escherichia coli downstream from the promoter and signal sequence of the secretory alpha-amylase of Bacillus amyloliquefaciens in a secretion vector of Bacillus subtilis. B. subtilis transformed with the hybrid plasmid synthesized a protein that was immunologically identified as OmpA. All the protein was present in the particulate fraction. The size of the protein compared to the peptide synthesized in vitro from the same template indicated that the alpha-amylase derived signal peptide was not removed; this was verified by N-terminal amino acid sequence determination. The lack of cleavage suggests that there was little or no translocation of OmpA protein across the cytoplasmic membrane. This is an unexpected difference compared with periplasmic proteins, which were both secreted and processed when fused to the same signal peptide. A requirement of a specific component for the export of outer membrane proteins is suggested.     

Improving protein secretion by engineering components of the bacterial translocation machinery.

The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.     

Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria.

Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein. The OmpA protein was found to be translocated across the plasma membrane of both microorganisms. However, the majority of the translocated OmpA was similarly degraded in B. subtilis and S. carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium. The finding that purified OmpA, which was added externally to the culture medium of growing S. carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease. Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety. The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed.     

Functional association of the stress-responsive LiaH protein and the minimal TatAyCy protein translocase in Bacillus subtilis

The bacterial twin-arginine (Tat) pathway serves in the exclusive secretion of folded proteins with bound cofactors. While Tat pathways in Gram-negative bacteria and chloroplast thylakoids consist of conserved TatA, TatB and TatC subunits, the Tat pathways of Bacillus species and many other Gram-positive bacteria stand out for their minimalist nature with the core translocase being composed of essential TatA and TatC subunits only. Here we addressed the question whether the minimal TatAyCy translocase of Bacillus subtilis recruits additional cellular components that modulate its activity. To this end, TatAyCy was purified by affinity- and size exclusion chromatography, and interacting co-purified proteins were identified by mass spectrometry. This uncovered the cell envelope stress responsive LiaH protein as an accessory subunit of the TatAyCy complex. Importantly, our functional studies show that Tat expression is tightly trailed by LiaH induction, and that LiaH itself determines the capacity and quality of TatAyCy-dependent protein translocation. In contrast, LiaH has no role in high-level protein secretion via the general secretion (Sec) pathway. Altogether, our observations show that protein translocation by the minimal Tat translocase TatAyCy is tightly intertwined with an adequate bacterial response to cell envelope stress. This is consistent with a critical need to maintain cellular homeostasis, especially when the membrane is widely opened to permit passage of large fully-folded proteins via Tat.