The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis.

BACKGROUND: Heterologous gene expression is well established for various prokaryotic model systems. However, low yield, incorrect folding and instability still impede the production of soluble, bioactive proteins. To improve protein production with the Gram-positive host Bacillus subtilis, a secretory expression system was designed that enhances translocation, folding and stability of heterologous proteins, and simplifies purification. Based on the theta-replication plasmid pHT01, a B. subtilis secretory expression vector was constructed that encodes a fusion protein consisting of a signal peptide and a StrepII-tag linked to a SUMO-tag serving as a folding catalyst. The gene of a protein of interest can be translationally fused to the SUMO cassette and an additional 6xHis-tag encoding region. In order to maximize secretory expression of the construct by fitting the signal peptide to the StrepII-SUMO part of the fusion protein, a B. subtilis signal-peptide library was screened with the Escherichia coli alkaline phosphatase PhoA as a reporter. RESULTS: The YoaW signal peptide-encoding region (SPyoaW) was identified with highest secretory expression capacity in context with the StrepII-SUMO-tag fusion in a B. subtilis eightfold extracellular protease deletion strain. PhoA activity and fusion protein production was elevated by a factor of approximately five when compared to an α-amylase (AmyQ) signal peptide construct. Replacement of PhoA with a single-chain variable fragment antibody specific for GFP or the B. amyloliquefaciens RNase barnase, respectively, resulted in a similar enhancement of secretory expression, demonstrating universality of the YoaW signal peptide-StrepII-SUMO encoding cassette for secretory expression in B. subtilis. Optimisation of codon usage and culture conditions further increased GFP-specific scFv fusion-protein production, and a simple affinity purification strategy from culture supernatant with removal of the StrepII-SUMO-tag by SenP-processing yielded 4 mg of pure, soluble and active GFP-specific scFv from 1 l of culture under standard laboratory conditions. CONCLUSIONS: The new expression system employing a YoaW signal peptide-StrepII-SUMO fusion will simplify secretory protein production and purification with B. subtilis. It can obviate the need for time consuming individual signal-peptide fitting to maximize yield for many different heterologous proteins of interest.

Two proteolytic modules are involved in regulated intramembrane proteolysis of Bacillus subtilis RsiW.

Stress-induced degradation of the Bacillus subtilis anti-sigma factor RsiW results in the induction of genes controlled by the extracytoplasmic function sigma factor sigma(W). RsiW is cleaved by the mechanism of regulated intramembrane proteolysis at site-1 and -2 by PrsW and RasP respectively, and is then further degraded by cytoplasmic Clp peptidases. In a reconstituted Escherichia coli system, PrsW removes 40 amino acids from RsiW by cleaving between Ala168 and Ser169 of the extracytoplasmic domain, thereby generating RsiW-S1. Further trimming of RsiW-S1's C-terminus by the periplasmic tail-specific protease Tsp is crucial for subsequent RasP-catalysed clipping. In B. subtilis, mutation of RsiW at Ala168 severely impairs site-1 processing. RsiW-S1 is undetectable in wild-type B. subtilis and knockout strains lacking various extracytoplasmic proteases. While it can be stabilized by C-terminal tagging, even this fusion protein is still attacked. Thus, several peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Overall, the RsiW degradation pathway can be subdivided into two modules each consisting of a site-specific peptidase that prepares RsiW for further degradation by downstream proteases.

Stress-responsive systems set specific limits to the overproduction of membrane proteins in Bacillus subtilis.

Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the sigma(W) regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

Identification of sigma(V)-dependent genes of Bacillus subtilis.

The chromosome of Bacillus subtilis codes for seven extracytoplasmic function sigma factors the activity of which is modulated normally by a cognate anti-sigma factor. While inducing factors and genes for four of them (sigma(M), sigma(W), sigma(X), and sigma(Y)) have been identified, those of the remaining three sigma factors including sigma(V) remain elusive. The objective of the present study was the unequivocal identification of its anti-sigma factor and of genes controlled by sigma(V). In many cases reported so far the gene coding for the anti-sigma factor is located immediately downstream of the gene coding for the sigma factor, and both form a bicistronic operon. We could show by two different experimental approaches that this is also the case for sigV and rsiV. Under conditions of overproduction of sigma(V), 13 genes could be identified being induced several-fold by the DNA macroarray technique. Induction of three of them was confirmed by Northern blots, and the potential promoter of sigV was identified by primer extension. This led to the deduction of a consensus sequence recognized by sigma(V).

Analysis of a DNA-binding motif of the Bacillus subtilis HrcA repressor protein.

The hrcA gene of Bacillus subtilis encodes a transcriptional repressor protein which negatively controls the heat shock operons dnaK and groESL. Alignment of the HrcA protein with repressor proteins from the NCBI database revealed that it exhibits a striking homology near its N-terminal part with proteins of the DeoR family. This region contains a helix-turn-helix motif and has been shown to be involved in DNA binding. To investigate whether this is also true for the HrcA protein, three critical amino acid residues were changed within or adjacent to the recognition helix. While single amino acid replacements barely influenced the binding activity, alteration of two consecutive amino acid residues within the recognition helix completely abolished the binding activity. When this mutant hrcA allele was expressed together with the wild-type allele within the same cell, it conferred a dominant-negative phenotype to the cells underlining that these amino acid residues are crucial for specific DNA binding and that HrcA binds to DNA in an oligomeric form.

Analysis of orthologous hrcA genes in Escherichia coli and Bacillus subtilis.

The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species. At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system. We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis. While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E. coli, but that of T. maritima turned out to repress expression of the reporter gene in B. subtilis. All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

Definition of the σ(W) regulon of Bacillus subtilis in the absence of stress.

Bacteria employ extracytoplasmic function (ECF) sigma factors for their responses to environmental stresses. Despite intensive research, the molecular dissection of ECF sigma factor regulons has remained a major challenge due to overlaps in the ECF sigma factor-regulated genes and the stimuli that activate the different ECF sigma factors. Here we have employed tiling arrays to single out the ECF σ(W) regulon of the Gram-positive bacterium Bacillus subtilis from the overlapping ECF σ(X), σ(Y), and σ(M) regulons. For this purpose, we profiled the transcriptome of a B. subtilis sigW mutant under non-stress conditions to select candidate genes that are strictly σ(W)-regulated. Under these conditions, σ(W) exhibits a basal level of activity. Subsequently, we verified the σ(W)-dependency of candidate genes by comparing their transcript profiles to transcriptome data obtained with the parental B. subtilis strain 168 grown under 104 different conditions, including relevant stress conditions, such as salt shock. In addition, we investigated the transcriptomes of rasP or prsW mutant strains that lack the proteases involved in the degradation of the σ(W) anti-sigma factor RsiW and subsequent activation of the σ(W)-regulon. Taken together, our studies identify 89 genes as being strictly σ(W)-regulated, including several genes for non-coding RNAs. The effects of rasP or prsW mutations on the expression of σ(W)-dependent genes were relatively mild, which implies that σ(W)-dependent transcription under non-stress conditions is not strictly related to RasP and PrsW. Lastly, we show that the pleiotropic phenotype of rasP mutant cells, which have defects in competence development, protein secretion and membrane protein production, is not mirrored in the transcript profile of these cells. This implies that RasP is not only important for transcriptional regulation via σ(W), but that this membrane protease also exerts other important post-transcriptional regulatory functions.

Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis.

Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.

Regulated intramembrane proteolysis in the control of extracytoplasmic function sigma factors.

There is growing evidence that proteolytic degradation of membrane-spanning regulatory proteins such as anti-sigma factors is involved in a variety of important transmembrane signaling processes in bacteria. This mechanism of regulated intramembrane proteolysis (RIP) enables them to respond to extracellular signals and stresses. Here, we summarize current knowledge of RIP controlling extracytoplasmic function sigma factors.

The Bacillus subtilis ABC transporter EcsAB influences intramembrane proteolysis through RasP.

The Bacillus subtilis sigma(W) regulon is induced by different stresses that most probably affect integrity of the cell envelope. The activity of the extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by the transmembrane anti-sigma factor RsiW, which undergoes stress-induced degradation in a process known as regulated intramembrane proteolysis, finally resulting in the release of sigma(W) and the transcription of sigma(W)-controlled genes. Mutations in the ecsA gene, which encodes an ATP binding cassette (ABC) of an ABC transporter of unknown function, block site-2 proteolysis of RsiW by the intramembrane cleaving protease RasP (YluC). In addition, degradation of the cell division protein FtsL, which represents a second RasP substrate, is blocked in an ecsA-negative strain. The defect in sigma(W) induction of an ecsA-knockout strain could be partly suppressed by overproducing RasP. A B. subtilis rasP-knockout strain displayed the same pleiotropic phenotype as an ecsA knockout, namely defects in processing alpha-amylase, in competence development, and in formation of multicellular structures known as biofilms.

YpdC determines site-1 degradation in regulated intramembrane proteolysis of the RsiW anti-sigma factor of Bacillus subtilis.

Genes of Bacillus subtilis controlled by the alternative extracytoplasmic function family sigma factor sigmaW constitute an antibiosis regulon. Its activity is modulated by RsiW, a transmembrane anti-sigma factor that sequesters and inactivates sigmaW. Upon a stress signal, RsiW is degraded by a mechanism of regulated intramembrane proteolysis. To identify genes which influence RsiW degradation, a transposon screen with a reporter fusion of the green fluorescent protein to RsiW was performed. Among several gene loci identified, the ypdC (prsW) gene displayed a strong effect on RsiW stability. In a ypdC null mutant, induction of sigmaW-controlled genes is abolished and site-1 proteolysis of RsiW is completely blocked. Transcriptional analysis revealed that ypdC is a monocistronic gene, and the defect of sigmaW induction of the null mutant was complemented by ectopically integrated ypdC under xylose control. Orthologues of YpdC can be found in a variety of different bacteria. Its membrane topology was analysed by alkaline phosphatase fusions, revealing that YpdC contains five transmembrane segments and two larger extracytoplasmic loops. In the first loop, two invariantly conserved glutamate residues can be found. In an Escherichia coli system, the cloned ypdC is the only determinant of efficient degradation of RsiW; however, YpdC does not display plain similarities to known proteases, suggesting that it either controls the activity of site-1 proteolysis of RsiW or represents a new type of protease.

Involvement of Clp protease activity in modulating the Bacillus subtilissigmaw stress response.

The induction of Bacillus subtilis genes controlled by the extracytoplasmic function alternative sigma factor sigmaW is strongly impaired in a strain deleted for the ClpP peptidase gene and in a double knockout of the ClpX and ClpE ATPase genes. Truncated soluble forms of the sigmaW anti-sigma factor RsiW are stabilized in a clpP minus strain as revealed by the green fluorescent reporter protein fused to the N-terminus of RsiW and by pulse-chase experiments. Conserved alanine residues are present in the transmembrane region of RsiW, and mutations in these positions abolish induction of sigmaW-controlled genes. Following alkaline shock, a truncated cytoplasmic form of RsiW is detectable in a strain expressing a triple alanine mutant allele of rsiW. These data point to a mechanism where the trans-membrane segment of RsiW contains a cryptic proteolytic tag that is uncovered as a result of intramembrane proteolysis of RsiW by RasP (YluC). After RasP-clipped RsiW is detached from the membrane, this proteolytic tag becomes crucial for the complete degradation of RsiW by cytoplasmic proteases and the release of sigmaW. ClpXP plays a major role in this third proteolytic step of stress-induced degradation of RsiW. Overexpression of SsrA-tagged green fluorescent protein as a ClpXP substrate protein reduces alkali induction of a sigmaW-controlled gene by a factor of about three, indicating that a titration mechanism is able to tune the sigmaW-mediated stress response to the cellular state.

Isolation and analysis of mutant alleles of the Bacillus subtilis HrcA repressor with reduced dependency on GroE function.

The hrcA gene of Bacillus subtilis codes for a transcriptional repressor protein that negatively regulates expression of the heptacistronic dnaK and the bicistronic groE operon by binding to an operator-element called CIRCE. Recently, we have published data suggesting that the activity of HrcA is modulated by the GroE chaperonin system. Biochemical analyses of the HrcA protein have been hampered so far by its strong tendency to aggregate. Here, a genetic method was used to isolate mutant forms of HrcA with increased activity under conditions of decreased GroE function. One of these mutant forms (HrcA114) containing five amino acid replacements exhibited enhanced solubility when overexpressed. HrcA114 purified under native conditions produced two retarded CIRCE-containing DNA fragments in band shift experiments. The amount of the larger fragment increased after addition of GroEL, GroES, and ATP but decreased when ATP was replaced by the nonhydrolyzable ATP analog ATPgammaS. DNase I footprinting experiments exhibited full protection of the CIRCE element and neighboring nucleotides in an asymmetric way. An in vitro binding assay using affinity chromatography showed direct and specific interaction between HrcA114 and GroEL. All these experimental data are in full agreement with our previously published model that HrcA needs the GroE chaperonin system for activation.

Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis.

Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3' terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein(+), yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.

The Bacillus subtilis sigmaW anti-sigma factor RsiW is degraded by intramembrane proteolysis through YluC.

The Bacillus subtilis sigma(W) regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor sigma(W) is modulated by a specific anti-sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two-hybrid system suggested a direct interaction of sigma(W) with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that sigma(W) induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse-chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a sigma(W)-controlled promoter after alkaline shock and accumulated a membrane-bound truncated form of RsiW, suggesting that the activity of sigma(W) is controlled by the proteolysis of RsiW by at least two different proteolytic steps.

The absence of FtsH metalloprotease activity causes overexpression of the sigmaW-controlled pbpE gene, resulting in filamentous growth of Bacillus subtilis.

FtsH is a membrane-bound and energy-dependent metalloprotease in bacteria which is involved in the posttranslational control of the activity of a variety of important transcription factors and in the degradation of uncomplexed integral membrane proteins. For Bacillus subtilis, little is known about the target proteins of FtsH protease. Its gene is not essential, but knockout strains display a pleiotropic phenotype including sensitivity toward salt and heat stress, defects in sporulation and competence, and largely filamentous growth. Comparison of the intracellular proteomes of wild-type and ftsH knockout strains revealed that at least nine proteins accumulated in the absence of ftsH, four of which could be identified. Two of these proteins turned out to be members of the sigma(W) regulon. Accumulation of one of these sigma(W)-controlled proteins, the penicillin-binding protein PBP4*, was analyzed in more detail. We could show that PBP4* is not a proteolytic substrate of FtsH and that its overproduction is due to the enhanced transcription of its gene (pbpE) in ftsH null mutants. The filamentous growth phenotype of DeltaftsH strains was abolished in a DeltaftsH DeltapbpE double knockout. In ftsH wild-type strains with the pbpE gene under regulatable control, pbpE overexpression caused filamentation of the cells. DNA macroarray analysis revealed that most genes of the sigma(W) regulon are transcribed at elevated levels in an ftsH mutant. The influence of FtsH on sigma(W)-controlled genes is discussed.

Regulation of the Bacillus subtilis heat shock gene htpG is under positive control.

The heat shock genes of Bacillus subtilis are assigned to four classes on the basis of their regulation mechanisms. While classes I and III are negatively controlled by two different transcriptional repressors, class II is regulated by the alternative sigma factor sigma(B). All heat shock genes with unidentified regulatory mechanisms, among them htpG, constitute class IV. Here, we show that expression of htpG is under positive control. We identified a DNA sequence (GAAAAGG) located downstream of the sigma(A)-dependent promoter of htpG. The heat inducibility of the promoter could be destroyed by inversion, nucleotide replacements, or removal of this DNA sequence. Fusion of this sequence to the vegetative lepA promoter conferred heat inducibility. Furthermore, we were able to show that the heat induction factor is dependent on the absolute temperature rather than the temperature increment and that nonnative proteins within the cytoplasm fail to induce htpG.