An internal hydrophobic helical domain of Bacillus subtilis enolase is essential but not sufficient as a non-cleavable signal for its secretion

Many cytoplasmic proteins without a cleavable signal peptide, including enolase, are secreted during the stationary phase in Bacillus subtilis but the molecular mechanism is not yet clear. We previously identified a highly conserved embedded membrane domain in an internal hydrophobic α-helix of enolase that plays an important role in its secretion. In this study, we examined the role of the helix in more detail for the secretion of enolase. Altering this helix by mutations showed that many mutated forms in this domain were not secreted, some of which were not stable as a soluble form in the cytoplasm. On the other hand, mutations on the flanking regions of the helix or the conserved basic residues showed no deleterious effect. Bacillus enolase with the proper hydrophobic helical domain was also exported extracellularly in Escherichia coli, indicating that the requirement of the helix for the secretion of enolase is conserved in these species. GFP fusions with enolase regions showed that the hydrophobic helix domain itself was not sufficient to serve as a functional secretion signal; a minimal length of N-terminus 140 amino acids was required to mediate the secretion of the fused reporter GFP. We conclude that the internal hydrophobic helix of enolase is essential but is not sufficient as a signal for secretion; the intact long N-terminus including the hydrophobic helix domain is required to serve as a non-cleavable signal for the secretion of Bacillus enolase.     

Optimization of the cell wall microenvironment allows increased production of recombinant Bacillus anthracis protective antigen from B. subtilis.

The stability of heterologous proteins secreted by gram-positive bacteria is greatly influenced by the microenvironment on the trans side of the cytoplasmic membrane, and secreted heterologous proteins are susceptible to rapid degradation by host cell proteases. In Bacillus subtilis, degradation occurs either as the proteins emerge from the presecretory translocase and prior to folding into their native conformation or after the native conformation has been reached. The former process generally involves membrane- and/or cell wall-bound proteases, while the latter involves proteases that are released into the culture medium. The identification and manipulation of factors that influence the folding of heterologous proteins has the potential to improve the yield of secreted heterologous proteins. Recombinant anthrax protective antigen (rPA) has been used as a model secreted heterologous protein because it is sensitive to proteolytic degradation both before and after folding into its native conformation. This paper describes the influence of the microenvironment on the trans side of the cytoplasmic membrane on the stability of rPA. Specifically, we have determined the influence of net cell wall charge and its modulation by the extent to which the anionic polymer teichoic acid is D-alanylated on the secretion and stability of rPA. The potential role of the dlt operon, responsible for D-alanylation, was investigated using a Bacillus subtilis strain encoding an inducible dlt operon. We show that, in the absence of D-alanylation, the yield of secreted rPA is increased 2.5-fold. The function of D-alanylation and the use of rPA as a model protein are evaluated with respect to the optimization of B. subtilis for the secretion of heterologous proteins.     

Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

E xcretion of c ytoplasmic p roteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG‐binding protein, which is secreted via the Sec‐pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan‐binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non‐excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

Efficient secretion of Bacillus subtilis lipase A in Saccharomyces cerevisiae by translational fusion to the Pir4 cell wall protein

Both the secretion and the cell surface display of Bacillus subtilis lipase A (Lip A) in Saccharomyces cerevisiae was investigated using different domains of the cell wall protein Pir4 as translational fusion partners. LipA gene minus its leader peptide was fused inframe in two places of PIR4 to achieve cell wall targeting, or substituting most of the PIR4 sequence, after the signal peptide and the Kex2 processed subunit I of Pir4 to achieve secretion to the growth medium. Expression of the recombinant fusion proteins was investigated in a standard and a glycosylation-deficient strain of S. cerevisiae, grown in selective or rich medium. Fusion proteins intended to be retained at the cell wall were secreted to the growth medium, most likely as result of the degradation of the Pir4 moiety containing the cell wall retention domain, giving low levels of lipase activity. However, the fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 400 IU of lipase activity per milliliter of cell supernatant. This is, to our knowledge, the first report of the efficient production, as a secreted protein, of lipase A of B. subtilis in baker's yeast.     

Bacillus subtilis Vegetative Catalase Is an Extracellular Enzyme

Strong catalase activity was secreted by Bacillus subtilis cells during stationary growth phase in rich medium but not in sporulation-inducing medium. N-terminal sequencing indicated that the secreted activity was due to the vegetative catalase KatA, previously considered an endocellular enzyme. Extracellular catalase protected B. subtilis cells from oxidative assault.     

Cellular lysis in Bacillus subtilis; the affect of multiple extracellular protease deficiencies

Cellular lysis properties of strains of Bacillus subtilis deficient in the synthesis of extracellular proteases was investigated. In all cases, extracellular protease deficiency was found to increase the extent of cellular lysis of batch cultured strains following the transition to stationary phase, the time at which extracellular degradative enzymes are secreted in large quantities. The data indicates that the major extracellular proteases, NprE and AprE, are primarily responsible for the control of this autolytic activity in B. subtilis and has implications for the use of extracellular protease-deficient strains as hosts for the production of heterologous proteins.     

Modulation of thiol-disulfide oxidoreductases for increased production of disulfide-bond-containing proteins in Bacillus subtilis

Disulfide bonds are important for the correct folding, structural integrity, and activity of many biotechnologically relevant proteins. For synthesis and subsequent secretion of these proteins in bacteria, such as the well-known "cell factory" Bacillus subtilis, it is often the correct formation of disulfide bonds that is the greatest bottleneck. Degradation of inefficiently or incorrectly oxidized proteins and the requirement for costly and time-consuming reduction and oxidation steps in the downstream processing of the proteins still are major limitations for full exploitation of B. subtilis for biopharmaceutical production. Therefore, the present study was aimed at developing a novel in vivo strategy for improved production of secreted disulfide-bond-containing proteins. Three approaches were tested: depletion of the major cytoplasmic reductase TrxA; introduction of the heterologous oxidase DsbA from Staphylococcus carnosus; and addition of redox-active compounds to the growth medium. As shown using the disulfide-bond-containing molecule Escherichia coli PhoA as a model protein, combined use of these three approaches resulted in secretion of amounts of active PhoA that were approximately 3.5-fold larger than the amounts secreted by the parental strain B. subtilis 168. Our findings indicate that Bacillus strains with improved oxidizing properties can be engineered for biotechnological production of heterologous high-value proteins containing disulfide bonds.     

Stabilization of cell wall proteins in Bacillus subtilis: a proteomic approach

Even though cell wall proteins of Bacillus subtilis are characterized by specific cell wall retention signals, some of these are also components of the extracellular proteome. In contrast to the majority of extracellular proteins, wall binding proteins disappeared from the extracellular proteome during the stationary phase and are subjected to proteolysis. Thus, the extracellular proteome of the multiple protease-deficient strain WB700 was analyzed which showed an increased stability of secreted WapA processing products during the stationary phase. In addition, stabilization of the WapA processing products was observed also in a sigD mutant strain which is impaired in motility and cell wall turnover. Next, we analyzed if proteins that can be extracted from B. subtilis cell walls are stabilized in the WB700 strain as well as in the sigD mutant. Thus, the cell wall proteome of B. subtilis wild type was defined showing most abundantly cell wall binding proteins (CWBPs) resulting from the WapA and WprA precursor processing. The inactivation of extracellular proteases as well as SigmaD caused an increase of CWBP105 and a decrease of CWBP62 in the cell wall proteome. We conclude that WapA processing products are substrates for the extracellular proteases which are stabilized in the absence of sigD due to an impaired cell wall turnover.     

Complementation of cold shock proteins by translation initiation factor IF1 in vivo.

The cold shock response in both Escherichia coli and Bacillus subtilis is induced by an abrupt downshift in growth temperature and leads to a dramatic increase in the production of a homologous class of small, often highly acidic cold shock proteins. This protein family is the prototype of the cold shock domain (CSD) that is conserved from bacteria to humans. For B. subtilis it has been shown that at least one of the three resident cold shock proteins (CspB to D) is essential under optimal growth conditions as well as during cold shock. Analysis of the B. subtilis cspB cspC double deletion mutant revealed that removal of these csp genes results in pleiotropic alteration of protein synthesis, cell lysis during the entry of stationary growth phase, and the inability to differentiate into endospores. We show here that heterologous expression of the translation initiation factor IF1 from E. coli in a B. subtilis cspB cspC double deletion strain is able to cure both the growth and the sporulation defects observed for this mutant, suggesting that IF1 and cold shock proteins have at least in part overlapping cellular function(s). Two of the possible explanation models are discussed.     

Environmental salinity determines the specificity and need for Tat-dependent secretion of the YwbN protein in Bacillus subtilis

Twin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described "minimal Tat translocase" consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate.     

Production and secretion of collagen-binding proteins from Aeromonas veronii

Collagen-binding protein (CNBP) synthesized by Aeromonas veronii is located conserved within the subcellular fraction. The results of this study show that 98% of the total CNBP produced by Aer. veronii is present in the extracellular medium, and that the remaining CNBP is distributed either on the cell surface, within the periplasm or anchored on the outer membrane. CNBP is specifically secreted from Aer. veronii into the culture medium, because all the beta-lactamase activity was located in the cells and could be released by polymixin B extraction of periplasmic proteins. CNBP was produced at growth temperatures from 12 degrees C to 42 degrees C, but not at 4 degrees C. The findings indicate that the level of CNBP in the medium increases during the exponential growth phase and reaches a maximum during the early stationary phase. There was less CNBP production in poor nutrient MMB medium than in the rich LB nutrient medium. CNBP secretion, in contrast to aerolysin secretion, was unaffected by the exeA mutation of Aer. hydrophila. It is concluded that CNBP secretion from Aer. veronii must be achieved by a mechanism different from that reported for aerolysin secretion.     

Self-assembly product formation of the Bacillus stearothermophilus PV72/p6 S-layer protein SbsA in the course of autolysis of Bacillus subtilis

In order to achieve high level expression and to study the release of a protein capable of self-assembly, the gene encoding the crystalline cell surface (S-layer) protein SbsA of Bacillus stearothermophilus PV72/p6, including its signal sequence, was cloned and expressed in Bacillus subtilis. To obtain high level expression, a tightly regulated, xylose-inducible, stably replicating multicopy-plasmid vector was constructed. After induction of expression, the S-layer protein made up about 15% of the total cellular protein content, which was comparable to the SbsA content of B. stearothermophilus PV72/p6 cells. During all growth stages, SbsA was poorly secreted to the ambient cellular environment by B. subtilis. Extraction of whole cells with guanidine hydrochloride showed that in late stationary growth phase cells 65% of the synthesised SbsA was retained in the peptidoglycan-containing layer, indicating that the rigid cell wall layer was a barrier for efficient SbsA secretion. Electron microscopic investigation revealed that SbsA release from the peptidoglycan-containing layer started in the late stationary growth phase at distinct sites at the cell surface leading to the formation of extracellular self-assembly products which did not adhere to the cell wall surface. In addition, intracellular sheet-like SbsA self-assembly products which followed the curvature of the cell became visible in partly lysed cells. Intracellularly formed self-assembly products remained intact even after complete lysis of the rigid cell envelope layer.     

Secretion of Tat‐dependent halolysin SptA capable of autocatalytic activation and its relation to haloarchaeal growth

Halolysins are Tat‐dependent extracellular subtilases of haloarchaea. Whether halolysins can be activated before transport across the cytoplasmic membrane in a folded state and how haloarchaea minimize the risk of intracellular activation of halolysins and proteolysis of cellular proteins are unknown. Here, we report that both the precursor and proform of halolysin SptA from Natrinema sp. J7‐2 mature autocatalytically, and the SptA maturation proceeds less efficiently in the presence of KCl than NaCl. When produced in Haloferax volcanii, most SptA molecules are secreted into the culture medium, but a small number of molecules can be activated intracellularly, affecting the cell's growth. Furthermore, retardation of SptA secretion in Hfx. volcanii via mutation of the Tat signal peptide leads to intracellular accumulation of the active enzyme and subsequent cell death. Although the Sec signal peptide can mediate SptA secretion in Hfx. volcanii, the secreted protein undergoes proteolysis. In Natrinema sp. J7‐2, SptA is secreted primarily during stationary phase, and the intracellular accumulation of mature enzyme occurs during the stationary and death phases. The growth phase‐dependent synthesis of SptA, highly efficient secretion system, and high intracellular KCl concentration, contribute to the suppression of premature activation of this enzyme in Natrinema sp. J7‐2.     

Post-translational secretion of fusion proteins in the halophilic archaea Haloferax volcanii

Although protein secretion occurs post-translationally in bacteria and is mainly a cotranslational event in Eukarya, the relationship between the translation and translocation of secreted proteins in Archaea is not known. To address this question, the signal peptide-encoding region of the surface layer glycoprotein gene from the Haloarchaea Haloferax volcanii was fused either to the cellulose-binding domain of the Clostridium thermocellum cellulosome or to the cytoplasmic enzyme dihydrofolate reductase from H. volcanii. Signal peptide-cleaved mature versions of both the cellulose-binding domain and dihydrofolate reductase could be detected in the growth medium of transformed H. volcanii cells. Immunoblot analysis revealed, however, the presence of full-length signal peptide-bearing forms of both proteins inside the cytoplasm of the transformed cells. Proteinase accessibility assays confirmed that the presence of cell-associated signal peptide-bearing proteins was not due to medium contamination. Moreover, the pulse-radiolabeled signal peptide cellulose-binding domain chimera could be chased from the cytoplasm into the growth medium even following treatment with anisomycin, an antibiotic inhibitor of haloarchaeal protein translation. Thus, these results provide evidence that, in Archaea, at least some secreted proteins are first synthesized inside the cell and only then translocated across the plasma membrane into the medium.     

Secretion of a peripheral membrane protein, MFG-E8, as a complex with membrane vesicles.

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidin-like domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergent-sensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).     

LytA, major autolysin of Streptococcus pneumoniae, requires access to nascent peptidoglycan

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA. We show that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs.     

Protein traffic for secretion and related machinery of Bacillus subtilis

Gram-positive sporulating Bacillus subtilis secretes high levels of protein. Its complete genome sequence, published in 1997, encodes 4,106 proteins. Bioinformatic searches have predicted that about half of all B. subtilis proteins are related to the cell membrane through export to the extracellular medium, insertion, and attachment. Key features of the B. subtilis protein secretion machinery are the absence of an Escherichia coli SecB homolog and the presence of an SRP (signal recognition particle) that is structurally rather similar to human SRP. In addition, B. subtilis contains five type I signal peptidases (SipS, T, U, V, and W). Our in vitro assay system indicated that co-operation between the SRP-protein targeting system to the cell membrane and the Sec protein translocation machinery across the cytoplasmic membrane constitutes the major protein secretion pathway in B. subtilis. Furthermore, the function of the SRP-Sec pathway in protein localization to the cell membrane and spore was analyzed.