Proteases HtrA and HtrB for α-amylase secreted from Bacillus subtilis in secretion stress

ABSTRACT Transcriptome analysis was used to investigate the global stress response of the gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon, which responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example, citM, ylxF, yloA, ykoJ, and several genes of the flgB operon. However, high-affinity CssR binding was observed only for htrA, htrB, and, possibly, citM. In addition, the DNA macroarray approach revealed that several genes of the sporulation pathway are downregulated by AmyQ overexpression and that a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow-cytometric analyses demonstrate that, upon overproduction of AmyQ as well as of a nonsecretable variant of the α-amylase, the process of sporulation is severely inhibited. Similar experiments were performed to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction.

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A Novel Class of Heat and Secretion Stress-Responsive Genes Is Controlled by the Autoregulated CssRS Two-Component System of Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.184.20.5661-5671.2002 ◽

2002 ◽

Vol 184 (20) ◽

pp. 5661-5671 ◽

Cited By ~ 113

Author(s):

Elise Darmon ◽

David Noone ◽

Anne Masson ◽

Sierd Bron ◽

Oscar P. Kuipers ◽

...

Keyword(s):

Bacillus Subtilis ◽

Heat Stress ◽

Transcriptional Activation ◽

Cellular Response ◽

Regulatory Region ◽

Regulatory System ◽

Secretion Stress ◽

Two Component ◽

High Level ◽

Inducible Genes

ABSTRACT Bacteria need dedicated systems that allow appropriate adaptation to the perpetual changes in their environments. In Bacillus subtilis, two HtrA-like proteases, HtrA and HtrB, play critical roles in the cellular response to secretion and heat stresses. Transcription of these genes is induced by the high-level production of a secreted protein or by a temperature upshift. The CssR-CssS two-component regulatory system plays an essential role in this transcriptional activation. Transcription of the cssRS operon is autoregulated and can be induced by secretion stress, by the absence of either HtrA or HtrB, and by heat stress in a HtrA null mutant strain. Two start sites are used for cssRS transcription, only one of which is responsive to heat and secretion stress. The divergently transcribed htrB and cssRS genes share a regulatory region through which their secretion and heat stress-induced expression is linked. This study shows that CssRS-regulated genes represent a novel class of heat-inducible genes, which is referred to as class V and currently includes two genes: htrA and htrB.

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

Applied and Environmental Microbiology ◽

10.1128/aem.01176-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 6876-6885 ◽

Cited By ~ 24

Author(s):

Elise Darmon ◽

Ronald Dorenbos ◽

Jochen Meens ◽

Roland Freudl ◽

Haike Antelmann ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stress Response ◽

Disulfide Bond ◽

Disulfide Bonds ◽

Protein Quality ◽

Secretory Protein ◽

Secretory Proteins ◽

Biotechnological Production ◽

Secretion Stress ◽

High Level

ABSTRACT 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.

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Transcriptome analysis of the secretion stress response of Bacillus subtilis

Applied Microbiology and Biotechnology ◽

10.1007/s00253-005-1898-1 ◽

2005 ◽

Vol 67 (3) ◽

pp. 389-396 ◽

Cited By ~ 52

Author(s):

Hanne-Leena Hyyryläinen ◽

Matti Sarvas ◽

Vesa P. Kontinen

Keyword(s):

Bacillus Subtilis ◽

Stress Response ◽

Transcriptome Analysis ◽

Secretion Stress

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The extracellular proteome of Bacillus subtilis under secretion stress conditions

Molecular Microbiology ◽

10.1046/j.1365-2958.2003.03565.x ◽

2003 ◽

Vol 49 (1) ◽

pp. 143-156 ◽

Cited By ~ 80

Author(s):

Haike Antelmann ◽

Elise Darmon ◽

David Noone ◽

Jan-Willem Veening ◽

Helga Westers ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stress Conditions ◽

Extracellular Proteome ◽

Secretion Stress

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Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase

Microbial Cell Factories ◽

10.1186/s12934-019-1203-0 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 6

Author(s):

Ane Quesada-Ganuza ◽

Minia Antelo-Varela ◽

Jeppe C. Mouritzen ◽

Jürgen Bartel ◽

Dörte Becher ◽

...

Keyword(s):

Bacillus Subtilis ◽

Stress Response ◽

Alpha Amylase ◽

Amylase Secretion ◽

Secretion Stress ◽

Final Yield ◽

Alpha Amylases

Abstract Background PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. Results In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. Conclusions In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA–amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

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Signal Perception by the Secretion Stress-Responsive CssRS Two-Component System in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.05767-11 ◽

2012 ◽

Vol 194 (7) ◽

pp. 1800-1814 ◽

Cited By ~ 13

Author(s):

D. Noone ◽

E. Botella ◽

C. Butler ◽

A. Hansen ◽

I. Jende ◽

...

Keyword(s):

Bacillus Subtilis ◽

Two Component System ◽

Component System ◽

Signal Perception ◽

Secretion Stress ◽

Two Component

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Faculty Opinions recommendation of A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001863.18106 ◽

2001 ◽

Author(s):

Tracy Raivio

Keyword(s):

Bacillus Subtilis ◽

Regulatory System ◽

Secretion Stress ◽

Two Component

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Use of a Promoter Trap System in Bacillus anthracis and Bacillus subtilis for the Development of Recombinant Protective Antigen-Based Vaccines

Infection and Immunity ◽

10.1128/iai.71.2.801-813.2003 ◽

2003 ◽

Vol 71 (2) ◽

pp. 801-813 ◽

Cited By ~ 34

Author(s):

O. Gat ◽

I. Inbar ◽

R. Aloni-Grinstein ◽

E. Zahavy ◽

C. Kronman ◽

...

Keyword(s):

Bacillus Subtilis ◽

Bacillus Anthracis ◽

Protective Antigen ◽

Expression Plasmid ◽

Secretion Stress ◽

Trap System ◽

Promoter Trap ◽

Ability To Drive ◽

Practical Implications ◽

Recombinant Protective Antigen

ABSTRACT We have recently reported Bacillus anthracis attenuated live vaccine strains efficiently expressing recombinant protective antigen (rPA) and have shown a direct correlation between the level of rPA secreted by these cells and efficacy (S. Cohen, I. Mendelson, Z. Altboum, D. Kobiler, E. Elhanany, T. Bino, M. Leitner, I. Inbar, H. Rosenberg, Y. Gozes, R. Barak, M. Fisher, C. Kronman, B. Velan, and A. Shafferman, Infect. Immun. 68:4549-4558, 2000). To isolate more potent Bacillus promoters for a further increase in the production of rPA, we developed a promoter trap system based on various gfp reporter genes adapted for use in both Bacillus subtilis and B. anthracis backgrounds. Accordingly, a B. anthracis library of 6,000 clones harboring plasmids with chromosomal B. anthracis DNA fragments inserted upstream from gfpuv was constructed. Based on fluorescence intensity, 57 clones carrying potentially strong promoters were identified, some of which were DNA sequenced. The most potent B. anthracis promoter identified (Pntr; 271 bp) was 500 times more potent than the native pagA promoter and 70 times more potent than the α-amylase promoter (Pamy). This very potent promoter was tested along with the other promoters (which are three, six, and eight times more potent than Pamy) for the ability to drive expression of rPA in either B. subtilis or B. anthracis. The number of cell-associated pre-PA molecules in B. anthracis was found to correlate well with the strength of the promoter. However, there appeared to be an upper limit to the amount of mature PA secreted into the medium, which did not exceed that driven by Pamy. Furthermore, the rPA constructs fused to the very potent promoters proved to be deleterious to the bacterial hosts and consequently led to genetic instability of the PA expression plasmid. Immunization with attenuated B. anthracis expressing rPA under the control of promoters more potent than Pamy was less efficient in eliciting anti-PA antibodies than that attained with Pamy. The results are consistent with the notion that overexpression of PA leads to severe secretion stress and have practical implications for the design of second-generation rPA-based vaccines.

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