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 Transcriptome Analysis Provides Insights into the Mechanism of Astaxanthin Enrichment in a Mutant of the Ridgetail White Prawn Exopalaemon carinicauda

Genes ◽  
2021 ◽  
Vol 12 (5) ◽  
pp. 618
Author(s):  
Yue Jin ◽  
Shihao Li ◽  
Yang Yu ◽  
Chengsong Zhang ◽  
Xiaojun Zhang ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Underlying Mechanism ◽  
Biological Processes ◽  
Wild Type ◽  
Expression Levels ◽  
In The Wild ◽  
Cuticle Proteins ◽  
Apolipoprotein D ◽  
High Level ◽  
Lower Expression

A mutant of the ridgetail white prawn, which exhibited rare orange-red body color with a higher level of free astaxanthin (ASTX) concentration than that in the wild-type prawn, was obtained in our lab. In order to understand the underlying mechanism for the existence of a high level of free astaxanthin, transcriptome analysis was performed to identify the differentially expressed genes (DEGs) between the mutant and wild-type prawns. A total of 78,224 unigenes were obtained, and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant in comparison with the wild-type prawns. Based on Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis, as well as further investigation of annotated DEGs, we found that the biological processes related to astaxanthin binding, transport, and metabolism presented significant differences between the mutant and the wild-type prawns. Some genes related to these processes, including crustacyanin, apolipoprotein D (ApoD), cathepsin, and cuticle proteins, were identified as DEGs between the two types of prawns. These data may provide important information for us to understand the molecular mechanism of the existence of a high level of free astaxanthin in the prawn.

scholarly journals The NADPH Oxidase Nox4 Controls Macrophage Polarization in an NFκB-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
V. Helfinger ◽  
K. Palfi ◽  
A. Weigert ◽  
K. Schröder
Keyword(s):  
Ex Vivo ◽  
Macrophage Polarization ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Superoxide Anions ◽  
Wild Type ◽  
Macrophage Population ◽  
The Family ◽  
High Level

The family of NADPH oxidases represents an important source of reactive oxygen species (ROS) within the cell. Nox4 is a special member of this family as it constitutively produces H2O2 and its loss promotes inflammation. A major cellular component of inflammation is the macrophage population, which can be divided into several subpopulations depending on their phenotype, with proinflammatory M(LPS+IFNγ) and wound-healing M(IL4+IL13) macrophages being extremes of the functional spectrum. Whether Nox4 is expressed in macrophages is discussed controversially. Here, we show that macrophages besides a high level of Nox2 indeed express Nox4. As Nox4 contributes to differentiation of many cells, we hypothesize that Nox4 plays a role in determining the polarization and the phenotype of macrophages. In bone marrow-derived monocytes, ex vivo treatment with LPS/IFNγ or IL4/IL13 results in polarization of the cells into M(LPS+IFNγ) or M(IL4+IL13) macrophages, respectively. In this ex vivo setting, Nox4 deficiency reduces M(IL4+IL13) polarization and forces M(LPS+IFNγ). Nox4-/- M(LPS+IFNγ)-polarized macrophages express more Nox2 and produce more superoxide anions than wild type M(LPS+IFNγ)-polarized macrophages. Mechanistically, Nox4 deficiency reduces STAT6 activation and promotes NFκB activity, with the latter being responsible for the higher level of Nox2 in Nox4-deficient M(LPS+IFNγ)-polarized macrophages. According to those findings, in vivo, in a murine inflammation-driven fibrosarcoma model, Nox4 deficiency forces the expression of proinflammatory genes and cytokines, accompanied by an increase in the number of proinflammatory Ly6C+ macrophages in the tumors. Collectively, the data obtained in this study suggest an anti-inflammatory role for Nox4 in macrophages. Nox4 deficiency results in less M(IL4+IL13) polarization and suppression of NFκB activity in monocytes.

scholarly journals Genes Involved in SkfA Killing Factor Production Protect a Bacillus subtilis Lipase against Proteolysis

2005 ◽  
Vol 71 (4) ◽  
pp. 1899-1908 ◽  
Author(s):  
Helga Westers ◽  
Peter G. Braun ◽  
Lidia Westers ◽  
Haike Antelmann ◽  
Michael Hecker ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protease Inhibitor ◽  
Bactericidal Activity ◽  
Industrial Applications ◽  
High Potential ◽  
Proteolytic Degradation ◽  
Wild Type ◽  
Factor Production ◽  
Protease Deficient ◽  
High Level

ABSTRACT Small lipases of Bacillus species, such as LipA from Bacillus subtilis, have a high potential for industrial applications. Recent studies showed that deletion of six AT-rich islands from the B. subtilis genome results in reduced amounts of extracellular LipA. Here we demonstrate that the reduced LipA levels are due to the absence of four genes, skfABCD, located in the prophage 1 region. Intact skfABCD genes are required not only for LipA production at wild-type levels by B. subtilis 168 but also under conditions of LipA overproduction. Notably, SkfA has bactericidal activity and, probably, requires the SkfB to SkfD proteins for its production. The present results show that LipA is more prone to proteolytic degradation in the absence of SkfA and that high-level LipA production can be improved significantly by employing multiple protease-deficient B. subtilis strains. In conclusion, our findings imply that SkfA protects LipA, directly or indirectly, against proteolytic degradation. Conceivably, SkfA could act as a modulator in LipA folding or as a protease inhibitor.

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.

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 Bacillus subtilis Phosphorylated PhoP: Direct Activation of the EσA- and Repression of the EσE-Responsive phoB-PS+V Promoters during Pho Response

2005 ◽  
Vol 187 (15) ◽  
pp. 5166-5178 ◽  
Author(s):  
Wael R. Abdel-Fattah ◽  
Yinghua Chen ◽  
Amr Eldakak ◽  
F. Marion Hulett
Keyword(s):  
Alkaline Phosphatase ◽  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Pho Regulon ◽  
Dependent Manner ◽  
Wild Type ◽  
Phosphate Deprivation

ABSTRACT The phoB gene of Bacillus subtilis encodes an alkaline phosphatase (PhoB, formerly alkaline phosphatase III) that is expressed from separate promoters during phosphate deprivation in a PhoP-PhoR-dependent manner and at stage two of sporulation under phosphate-sufficient conditions independent of PhoP-PhoR. Isogenic strains containing either the complete phoB promoter or individual phoB promoter fusions were used to assess expression from each promoter under both induction conditions. The phoB promoter responsible for expression during sporulation, phoB-PS, was expressed in a wild-type strain during phosphate deprivation, but induction occurred >3 h later than induction of Pho regulon genes and the levels were approximately 50-fold lower than that observed for the PhoPR-dependent promoter, phoB-PV. EσE was necessary and sufficient for PS expression in vitro. PS expression in a phoPR mutant strain was delayed 2 to 3 h compared to the expression in a wild-type strain, suggesting that expression or activation of σE is delayed in a phoPR mutant under phosphate-deficient conditions, an observation consistent with a role for PhoPR in spore development under these conditions. Phosphorylated PhoP (PhoP∼P) repressed PS in vitro via direct binding to the promoter, the first example of an EσE-responsive promoter that is repressed by PhoP∼P. Whereas either PhoP or PhoP∼P in the presence of EσA was sufficient to stimulate transcription from the phoB-PV promoter in vitro, roughly 10- and 17-fold-higher concentrations of PhoP than of PhoP∼P were required for PV promoter activation and maximal promoter activity, respectively. The promoter for a second gene in the Pho regulon, ykoL, was also activated by elevated concentrations of unphosphorylated PhoP in vitro. However, because no Pho regulon gene expression was observed in vivo during Pi -replete growth and PhoP concentrations increased only threefold in vivo during phoPR autoinduction, a role for unphosphorylated PhoP in Pho regulon activation in vivo is not likely.

scholarly journals Bacillus subtilis functional genomics: global characterization of the stringent response by proteome and transcriptome analysis

2002 ◽  
Vol 184 (9) ◽  
pp. 2500-2520 ◽  
Author(s):  
Christine Eymann ◽  
Georg Homuth ◽  
Christian Scharf ◽  
Michael Hecker
Keyword(s):  
Protein Synthesis ◽  
Bacillus Subtilis ◽  
Profile Analysis ◽  
Bacterial Species ◽  
Stringent Response ◽  
Dependent Manner ◽  
Wild Type ◽  
Stringent Control ◽  
In The Wild ◽  
Growth And Reproduction

ABSTRACT The stringent response in Bacillus subtilis was characterized by using proteome and transcriptome approaches. Comparison of protein synthesis patterns of wild-type and relA mutant cells cultivated under conditions which provoke the stringent response revealed significant differences. According to their altered synthesis patterns in response to dl-norvaline, proteins were assigned to four distinct classes: (i) negative stringent control, i.e., strongly decreased protein synthesis in the wild type but not in the relA mutant (e.g., r-proteins); (ii) positive stringent control, i.e., induction of protein synthesis in the wild type only (e.g., YvyD and LeuD); (iii) proteins that were induced independently of RelA (e.g., YjcI); and (iv) proteins downregulated independently of RelA (e.g., glycolytic enzymes). Transcriptome studies based on DNA macroarray techniques were used to complement the proteome data, resulting in comparable induction and repression patterns of almost all corresponding genes. However, a comparison of both approaches revealed that only a subset of RelA-dependent genes or proteins was detectable by proteomics, demonstrating that the transcriptome approach allows a more comprehensive global gene expression profile analysis. The present study presents the first comprehensive description of the stringent response of a bacterial species and an almost complete map of protein-encoding genes affected by (p)ppGpp. The negative stringent control concerns reactions typical of growth and reproduction (ribosome synthesis, DNA synthesis, cell wall synthesis, etc.). Negatively controlled unknown y-genes may also code for proteins with a specific function during growth and reproduction (e.g., YlaG). On the other hand, many genes are induced in a RelA-dependent manner, including genes coding for already-known and as-yet-unknown proteins. A passive model is preferred to explain this positive control relying on the redistribution of the RNA polymerase under the influence of (p)ppGpp.

scholarly journals Mitochondrial active Ras2 protein promotes apoptosis and regulated cell death in a cAMP/PKA pathway-dependent manner in budding yeast.

2021 ◽  
Author(s):  
Barbara Bonomelli ◽  
Enzo Martegani ◽  
Sonia Colombo
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Death ◽  
Acetic Acid ◽  
Dependent Manner ◽  
Ras Proteins ◽  
Wild Type ◽  
High Affinity ◽  
Regulated Cell Death ◽  
Molecular Machinery ◽  
Pka Pathway

In previous papers, using the eGFP-RBD3 probe, which binds Ras-GTP with high affinity, we showed that activated Ras proteins are localized to the plasma membrane and in the nucleus in wild-type Saccharomyces cerevisiae cells growing exponentially on glucose, while an aberrant accumulation of activated Ras in mitochondria correlates to mitochondrial dysfunction, accumulation of ROS and an increase of apoptosis. In this paper, we show that lack of TPS1, which is known to trigger apoptosis in S. cerevisiae, induces localization of active Ras proteins in mitochondria, confirming the above-mentioned correlation. Next, by characterizing the ras1Δ and ras2Δ mutants concerning localization of active Ras proteins and propensity to undergo cell death, we show that active Ras2 proteins, which accumulate in the mitochondria following addition of acetic acid, a well-known pro-apoptotic stimulus, might be the GTPases involved in regulated cell death, while active Ras1 proteins, constitutively localized in mitochondria, might be involved in a pro-survival molecular machinery. Finally, by characterizing the gpa2Δ and cyr1Δ mutants concerning the propensity to undergo cell death, we show that active mitochondrial Ras proteins promote apoptosis through the cAMP/PKA pathway.

scholarly journals Transcriptional Regulation of MDR1, Encoding a Drug Efflux Determinant, in Fluconazole-Resistant Candida albicans Strains through an Mcm1p Binding Site

2006 ◽  
Vol 5 (12) ◽  
pp. 1957-1968 ◽  
Author(s):  
Perry J. Riggle ◽  
Carol A. Kumamoto
Keyword(s):  
Candida Albicans ◽  
Binding Site ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Drug Efflux ◽  
Dependent Manner ◽  
Wild Type ◽  
High Level Expression ◽  
Mobility Shift ◽  
High Level

ABSTRACT Constitutive, high-level transcription of the gene encoding the drug efflux facilitator Mdr1p is commonly observed in laboratory and clinical strains of Candida albicans that are resistant to the antifungal drug fluconazole (FLC). In five independently isolated FLCR laboratory strains, introduction of a wild-type MDR1 promoter fragment fused to the yeast enhanced green fluorescent protein (yEGFP) reporter gene resulted in high-level expression of GFP, demonstrating that overexpression of MDR1 is dependent on a trans-acting factor. This study identified a 35-bp MDR1 promoter element, termed the MDRE, that mediates high-level MDR1 transcription. When inserted into a heterologous promoter, the MDRE was sufficient to mediate high-level expression of the yEGFP reporter gene specifically in MDR1 trans-activation strains. The MDRE promoted transcription in an orientation-independent and dosage-dependent manner. Deletion of the MDRE in the full-length promoter did not abolish MDR1 trans-activation, indicating that elements upstream of the MDRE also contribute to transcription of MDR1 in these overexpression strains. Analysis of the MDRE sequence indicated that it contains an Mcm1p binding site very similar in organization to the site seen upstream of the Saccharomyces cerevisiae MFA1 and STE2 genes. Electrophoretic mobility shift analysis demonstrated that both wild-type, FLC-sensitive and MDR1 trans-activated, FLC-resistant strains contain a factor that binds the MDRE. Depletion of Mcm1p, by use of a strain in which MCM1 expression is under the control of a regulated promoter (44), resulted in a loss of MDRE binding activity. Thus, the general transcription factor Mcm1p participates in the regulation of MDR1 expression.

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