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.

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.

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.

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.

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.

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).

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.