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

2006 ◽  
Vol 72 (11) ◽  
pp. 6876-6885 ◽  
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.

scholarly journals Modulation of Thiol-Disulfide Oxidoreductases for Increased Production of Disulfide-Bond-Containing Proteins in Bacillus subtilis

2008 ◽  
Vol 74 (24) ◽  
pp. 7536-7545 ◽  
Author(s):  
Thijs R. H. M. Kouwen ◽  
Jean-Yves F. Dubois ◽  
Roland Freudl ◽  
Wim J. Quax ◽  
Jan Maarten van Dijl
Keyword(s):  
Bacillus Subtilis ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Structural Integrity ◽  
Downstream Processing ◽  
Cell Factory ◽  
Biotechnological Production ◽  
Redox Active ◽  
Model Protein

ABSTRACT 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 ∼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.

scholarly journals Production and Secretion Stress Caused by Overexpression of Heterologous α-Amylase Leads to Inhibition of Sporulation and a Prolonged Motile Phase in Bacillus subtilis

2007 ◽  
Vol 73 (16) ◽  
pp. 5354-5362 ◽  
Author(s):  
Andrzej T. Lulko ◽  
Jan-Willem Veening ◽  
Girbe Buist ◽  
Wiep Klaas Smits ◽  
Evert Jan Blom ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Secretion ◽  
Dependent Manner ◽  
Wild Type ◽  
Positive Bacterium ◽  
Expression Levels ◽  
High Affinity ◽  
Flow Cytometric ◽  
Secretion Stress ◽  
High Level

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.

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

2002 ◽  
Vol 184 (20) ◽  
pp. 5661-5671 ◽  
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.

Transcriptome analysis of the secretion stress response of Bacillus subtilis

2005 ◽  
Vol 67 (3) ◽  
pp. 389-396 ◽  
Author(s):  
Hanne-Leena Hyyryläinen ◽  
Matti Sarvas ◽  
Vesa P. Kontinen
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Transcriptome Analysis ◽  
Secretion Stress

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

2019 ◽  
Vol 18 (1) ◽  
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.

scholarly journals Disulfide Transfer between Two Conserved Cysteine Pairs Imparts Selectivity to Protein Oxidation by Ero1

2006 ◽  
Vol 17 (5) ◽  
pp. 2256-2266 ◽  
Author(s):  
Carolyn S. Sevier ◽  
Chris A. Kaiser
Keyword(s):  
Disulfide Bond ◽  
Oxidase Activity ◽  
Disulfide Bonds ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Secretory Proteins ◽  
Mixed Disulfide ◽  
Redox Active

The membrane-associated flavoprotein Ero1p promotes disulfide bond formation in the endoplasmic reticulum (ER) by selectively oxidizing the soluble oxidoreductase protein disulfide isomerase (Pdi1p), which in turn can directly oxidize secretory proteins. Two redox-active disulfide bonds are essential for Ero1p oxidase activity: Cys100-Cys105 and Cys352-Cys355. Genetic and structural data indicate a disulfide bond is transferred from Cys100-Cys105 directly to Pdi1p, whereas a Cys352-Cys355 disulfide bond is used to reoxidize the reduced Cys100-Cys105 pair through an internal thiol-transfer reaction. Electron transfer from Cys352-Cys355 to molecular oxygen, by way of a flavin cofactor, maintains Cys352-Cys355 in an oxidized form. Herein, we identify a mixed disulfide species that confirms the Ero1p intercysteine thiol-transfer relay in vivo and identify Cys105 and Cys352 as the cysteines that mediate thiol-disulfide exchange. Moreover, we describe Ero1p mutants that have the surprising ability to oxidize substrates in the absence of Cys100-Cys105. We show the oxidase activity of these mutants results from structural changes in Ero1p that allow substrates increased access to Cys352-Cys355, which are normally buried beneath the protein surface. The altered activity of these Ero1p mutants toward selected substrates leads us to propose the catalytic mechanism involving transfer between cysteine pairs evolved to impart substrate specificity to Ero1p.

Assembly Characteristics and Structural Motifs in an Aquatic Insect's Biopolymer

1990 ◽  
Vol 218 ◽  
Author(s):  
Steven T. Case ◽  
Susan E. Wellman ◽  
Stavros Hamodrakas
Keyword(s):  
Circular Dichroism ◽  
Physical Properties ◽  
Disulfide Bonds ◽  
Protein Complexes ◽  
Structure Constant ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Α Helix ◽  
Circular Dichroïsm

AbstractSecretory proteins of Chironomus tentans larvae form insoluble fibers that are spun into threads used to construct underwater feeding and pupation tubes. The physical properties of spun fibers should reflect their protein constituents.The disassembly and reassembly of secretory protein complexes in vitro were studied by solution turbidity, electron microscopy and circular dichroism spectroscopy. Secretory proteins were able to reform complexes with a similar morphology and dichroic spectrum to that of native complexes. These complexes are stabilized by electrostatic and disulfide bonds.cDNA and genomic cloning indicate that spIs, the 1000-kDa secretory proteins, primarily consist of alternating “constant” and “subrepeat” regions. We synthesized and purified synthetic peptides for each region and used circular dichroism and infrared spectroscopy to measure their secondary structure. Constant peptide consists of α-helix and β-turn. Subrepeat peptide consists of poly(Gly) II-type helix and β-turn.We conclude that spIs form the fibrous backbone of a novel biopolymer with alternating α-helices and poly(Gly)II helices punctuated by β-turns. Such fibers may have unique physical properties.

scholarly journals The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis

2000 ◽  
Vol 350 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Keith STEPHENSON ◽  
Christina L. JENSEN ◽  
Steen T. JØRGENSEN ◽  
Jeremy H. LAKEY ◽  
Colin R. HARWOOD
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Culture Medium ◽  
Sequence Data ◽  
Chemical Properties ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Bacterium Bacillus Subtilis ◽  
Bacterium Bacillus ◽  
Late Stages

Following their secretion across the cytoplasmic membrane, processed secretory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture medium. The rate and efficiency of folding are critical in determining the yields of intact secretory proteins. The B. subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan and anionic polymers. The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage in the export process. To determine the influence of charge on late stages in the secretion of proteins from this bacterium, we have used sequence data from two related α-amylases, to engineer the net charge of AmyL, an α-amylase from Bacillus licheniformis that is normally secreted efficiently from B. subtilis. While AmyL has a pI of 7.0, chimaeric enzymes with pI values of 5.0 and 10.0 were produced and characterized. Despite the engineered changes to their physico-chemical properties, the chimaeric enzymes retained many of the enzymic characteristics of AmyL. We show that the positively charged protein interacts with the cell wall in a manner that influences its secretion.

scholarly journals On the Functional Interchangeability, Oxidant versus Reductant, of Members of the Thioredoxin Superfamily

2000 ◽  
Vol 182 (3) ◽  
pp. 723-727 ◽  
Author(s):  
Laurent Debarbieux ◽  
Jon Beckwith
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Signal Sequence ◽  
Cell Envelope ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Thioredoxin 1 ◽  
High Level

ABSTRACT Escherichia coli thioredoxin 1 has been characterized in vivo and in vitro as one of the most efficient reductants of disulfide bonds. Nevertheless, under some conditions, thioredoxin 1 can also act in vivo as an oxidant, promoting formation of disulfide bonds in the cytoplasm (E. J. Stewart, F. Åslund, and J. Beckwith, EMBO J. 17:5543–5550, 1998). We recently showed that when a signal sequence is attached to thioredoxin 1 it is exported to the periplasm, where it can also act as an oxidant, replacing the normal periplasmic catalyst of disulfide bond formation, DsbA, in oxidizing cell envelope proteins (L. Debarbieux and J. Beckwith, Proc. Natl. Acad. Sci. USA 95:10751–10756, 1998). Here we report pulse-chase studies of the efficiency of disulfide bond formation in strains exporting thioredoxin 1 and more-oxidizing variants of it. While the exported thioredoxin 1 itself substantially speeds up the kinetics of disulfide bond formation, a version of this protein containing the DsbA active site exhibits kinetics that are indistinguishable from those of the DsbA protein itself. Further, we confirm the findings of Jonda et al. (S. Jonda, M. Huber-Wunderlich, R. Glockshuber, and E. Mössner, EMBO J. 18:3271–3281, 1999), who found that DsbB is responsible for the oxidation of exported thioredoxin 1, and we report the detection of a disulfide-bonded DsbB-thioredoxin 1 complex. Finally, we have found that under conditions of high-level expression of exported thioredoxin 1, the protein can act as both an oxidant and a reductant.

scholarly journals The CssRS two-component regulatory system controls a general secretion stress response in Bacillus subtilis

FEBS Journal ◽  
2006 ◽  
Vol 273 (16) ◽  
pp. 3816-3827 ◽  
Author(s):  
Helga Westers ◽  
Lidia Westers ◽  
Elise Darmon ◽  
Jan Maarten van Dijl ◽  
Wim J. Quax ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Stress Response ◽  
Regulatory System ◽  
Secretion Stress ◽  
Two Component
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