scholarly journals Antisense Downregulation of ς32 as a Transient Metabolic Controller in Escherichia coli: Effects on Yield of Active Organophosphorus Hydrolase

2000 ◽  
Vol 66 (10) ◽  
pp. 4366-4371 ◽  
Author(s):  
Ranjan Srivastava ◽  
Hyung Joon Cha ◽  
Marvin S. Peterson ◽  
William E. Bentley
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Stress Response ◽  
Heat Shock ◽  
Total Yield ◽  
Fold Increase ◽  
Organophosphorus Hydrolase ◽  
Ethanol Stress ◽  
Antisense Mrna ◽  
Partial Degradation

ABSTRACT Plasmids containing an antisense fragment of the ς32gene were constructed and introduced into Escherichia colicells. Downregulation of the ς32-mediated stress response was evaluated under heat shock and ethanol stress and during the production of organophosphorus hydrolase (OPH). Northern blot analyses revealed that ς32 sense mRNA was virtually undetected in antisense-producing cultures from 5 to 20 min after antisense induction. However, lower-molecular-weight bands were found, presumably due to partial degradation of ς32 mRNA. While a >10-fold increase in ς32 protein level was found under ethanol stress in the control cultures, antisense producing cultures resulted in a <3-fold increase, indicating downregulation of ς32. Correspondingly, antisense synthesis resulted in a decreased level of a ς32 regulated chaperone (GroEL) for the first 2 h after induction relative to control cultures without ς32 antisense mRNA. The total yield of OPH in the presence of ς32 antisense was, on average, 62% of the yield without antisense. However, during ς32 antisense production, a sixfold-higher specific OPH activity was observed compared to non-antisense-producing cultures.

scholarly journals TOR and RAS pathways regulate desiccation tolerance inSaccharomyces cerevisiae

2013 ◽  
Vol 24 (2) ◽  
pp. 115-128 ◽  
Author(s):  
Aaron Z. Welch ◽  
Patrick A. Gibney ◽  
David Botstein ◽  
Douglas E. Koshland
Keyword(s):  
Growth Rate ◽  
Stress Response ◽  
Heat Shock ◽  
Desiccation Tolerance ◽  
Ribosome Biogenesis ◽  
Inverse Correlation ◽  
Fold Increase ◽  
Nutrient Deprivation ◽  
Camp Pathway ◽  
Dividing Cells

Tolerance to desiccation in cultures of Saccharomyces cerevisiae is inducible; only one in a million cells from an exponential culture survive desiccation compared with one in five cells in stationary phase. Here we exploit the desiccation sensitivity of exponentially dividing cells to understand the stresses imposed by desiccation and their stress response pathways. We found that induction of desiccation tolerance is cell autonomous and that there is an inverse correlation between desiccation tolerance and growth rate in glucose-, ammonia-, or phosphate-limited continuous cultures. A transient heat shock induces a 5000–fold increase in desiccation tolerance, whereas hyper-ionic, -reductive, -oxidative, or -osmotic stress induced much less. Furthermore, we provide evidence that the Sch9p-regulated branch of the TOR and Ras-cAMP pathway inhibits desiccation tolerance by inhibiting the stress response transcription factors Gis1p, Msn2p, and Msn4p and by activating Sfp1p, a ribosome biogenesis transcription factor. Among 41 mutants defective in ribosome biogenesis, a subset defective in 60S showed a dramatic increase in desiccation tolerance independent of growth rate. We suggest that reduction of a specific intermediate in 60S biogenesis, resulting from conditions such as heat shock and nutrient deprivation, increases desiccation tolerance.

scholarly journals Association of Constitutive Hyperphosphorylation of Hsf1p with a Defective Ethanol Stress Response in Saccharomyces cerevisiae Sake Yeast Strains

2011 ◽  
Vol 78 (2) ◽  
pp. 385-392 ◽  
Author(s):  
Chiemi Noguchi ◽  
Daisuke Watanabe ◽  
Yan Zhou ◽  
Takeshi Akao ◽  
Hitoshi Shimoi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stress Response ◽  
Heat Shock ◽  
Laboratory Strain ◽  
Underlying Mechanism ◽  
Ethanol Stress ◽  
Heat Shock Element ◽  
Content Type ◽  
Yeast Strains ◽  
Acute Ethanol

ABSTRACTModern sake yeast strains, which produce high concentrations of ethanol, are unexpectedly sensitive to environmental stress during sake brewing. To reveal the underlying mechanism, we investigated a well-characterized yeast stress response mediated by a heat shock element (HSE) and heat shock transcription factor Hsf1p inSaccharomyces cerevisiaesake yeast. The HSE-lacZactivity of sake yeast during sake fermentation and under acute ethanol stress was severely impaired compared to that of laboratory yeast. Moreover, the Hsf1p of modern sake yeast was highly and constitutively hyperphosphorylated, irrespective of the extracellular stress. SinceHSF1allele replacement did not significantly affect the HSE-mediated ethanol stress response or Hsf1p phosphorylation patterns in either sake or laboratory yeast, the regulatory machinery of Hsf1p is presumed to function differently between these types of yeast. To identify phosphatases whose loss affected the control of Hsf1p, we screened a series of phosphatase gene deletion mutants in a laboratory strain background. Among the 29 mutants, a Δppt1mutant exhibited constitutive hyperphosphorylation of Hsf1p, similarly to the modern sake yeast strains, which lack the entirePPT1gene locus. We confirmed that the expression of laboratory yeast-derived functionalPPT1recovered the HSE-mediated stress response of sake yeast. In addition, deletion ofPPT1in laboratory yeast resulted in enhanced fermentation ability. Taken together, these data demonstrate that hyperphosphorylation of Hsf1p caused by loss of thePPT1gene at least partly accounts for the defective stress response and high ethanol productivity of modern sake yeast strains.

scholarly journals Extrathymic production of thymulin induced by oxidative stress, heat shock, apoptosis, or necrosis

2017 ◽  
Vol 30 (1) ◽  
pp. 58-69 ◽  
Author(s):  
Sergey M Lunin ◽  
Maxim O Khrenov ◽  
Olga V Glushkova ◽  
Elena V Vinogradova ◽  
Valery A Yashin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Molecular Weight ◽  
Stress Response ◽  
Heat Shock ◽  
Cell Types ◽  
Cell Nuclei ◽  
Sequence Comparisons ◽  
Blast Search ◽  
Thymic Peptides ◽  
Thymic Peptide

Thymic peptides are immune regulators produced mainly in the thymus. However, thymic peptides such as thymosin-α and thymopoietin have precursors widely expressed outside the thymus, localized in cell nuclei, and involved in vital nuclear functions. In stress-related conditions, they can relocalize. We hypothesized that another thymic peptide, thymulin, could be similarly produced by non-thymic cells during stress and have a precursor therein. Non-thymic cells, including macrophages and fibroblasts, were exposed to oxidative stress, heat, apoptosis, or necrosis. Extracellular thymulin was identified in media of both cell types 2 h after exposure to stress or lethal signals. Therefore, thymulin is released by non-thymic cells. To examine possible thymulin precursors in non-thymic cells, macrophage lysates were analyzed by western blotting. Bands stained with anti-thymulin antibody were detected in two locations, approximately 60 kDa and 10 kDa, which may be a possible precursor and intermediate. All of the exposures except for heat were effective for induction of the 10 kDa protein. BLAST search using thymulin sequence identified SPATS2L, an intranucleolar stress-response protein with molecular weight of 62 kDa, containing thymulin-like sequence. Comparisons of blots stained with anti-thymulin and anti-SPATS2L antibodies indicate that SPATS2L may be a possible candidate for the precursor of thymulin.

scholarly journals Nonnative Proteins Induce Expression of the Bacillus subtilis CIRCE Regulon

1998 ◽  
Vol 180 (11) ◽  
pp. 2895-2900 ◽  
Author(s):  
Axel Mogk ◽  
Andrea Völker ◽  
Susanne Engelmann ◽  
Michael Hecker ◽  
Wolfgang Schumann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Heat Shock ◽  
Bacterial Species ◽  
Negative Control ◽  
Expression Level ◽  
Ethanol Stress ◽  
Transcriptional Fusion ◽  
Repressor Protein ◽  
E Coli

ABSTRACT The chaperone-encoding groESL and dnaKoperons constitute the CIRCE regulon of Bacillus subtilis. Both operons are under negative control of the repressor protein HrcA, which interacts with the CIRCE operator and whose activity is modulated by the GroESL chaperone machine. In this report, we demonstrate that induction of the CIRCE regulon can also be accomplished by ethanol stress and puromycin. Introduction of the hrcA gene and a transcriptional fusion under the control of the CIRCE operator intoEscherichia coli allowed induction of this fusion by heat shock, ethanol stress, and overproduction of GroESL substrates. The expression level of this hrcA-bgaB fusion inversely correlated with the amount of GroE machinery present in the cells. Therefore, all inducing conditions seem to lead to induction via titration of the GroE chaperonins by the increased level of nonnative proteins formed. Puromycin treatment failed to induce the ςB-dependent general stress regulon, indicating that nonnative proteins in general do not trigger this response. Reconstitution of HrcA-dependent heat shock regulation of B. subtilis in E. coli and complementation of E. coli groESL mutants by B. subtilis groESL indicate that the GroE chaperonin systems of the two bacterial species are functionally exchangeable.

scholarly journals Toward a Semisynthetic Stress Response System To Engineer Microbial Solvent Tolerance

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
Kyle A. Zingaro ◽  
Eleftherios Terry Papoutsakis
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Heat Shock ◽  
Heat Shock Proteins ◽  
Solvent Tolerance ◽  
Content Type ◽  
Response System ◽  
Wide Range ◽  
Stress Response System ◽  
Shock Proteins

ABSTRACT Strain tolerance to toxic metabolites is an important trait for many biotechnological applications, such as the production of solvents as biofuels or commodity chemicals. Engineering a complex cellular phenotype, such as solvent tolerance, requires the coordinated and tuned expression of several genes. Using combinations of heat shock proteins (HSPs), we engineered a semisynthetic stress response system in Escherichia coli capable of tolerating high levels of toxic solvents. Simultaneous overexpression of the HSPs GrpE and GroESL resulted in a 2-fold increase in viable cells (CFU) after exposure to 5% (vol/vol) ethanol for 24 h. Co-overexpression of GroESL and ClpB on coexisting plasmids resulted in 1,130%, 78%, and 25% increases in CFU after 24 h in 5% ethanol, 1% n-butanol, and 1% i-butanol, respectively. Co-overexpression of GrpE, GroESL, and ClpB on a single plasmid produced 200%, 390%, and 78% increases in CFU after 24 h in 7% ethanol, 1% n-butanol, or 25% 1,2,4-butanetriol, respectively. Overexpression of other autologous HSPs (DnaK, DnaJ, IbpA, and IbpB) alone or in combinations failed to improve tolerance. Expression levels of HSP genes, tuned through inducible promoters and the plasmid copy number, affected the effectiveness of the engineered stress response system. Taken together, these data demonstrate that tuned co-overexpression of GroES, GroEL, ClpB, and GrpE can be engaged to engineer a semisynthetic stress response system capable of greatly increasing the tolerance of E. coli to solvents and provides a starting platform for engineering customized tolerance to a wide variety of toxic chemicals. IMPORTANCE Microbial production of useful chemicals is often limited by the toxicity of desired products, feedstock impurities, and undesired side products. Improving tolerance is an essential step in the development of practical platform organisms for production of a wide range of chemicals. By overexpressing autologous heat shock proteins in Escherichia coli, we have developed a modular semisynthetic stress response system capable of improving tolerance to ethanol, n-butanol, and potentially other toxic solvents. Using this system, we demonstrate that a practical stress response system requires both tuning of individual gene components and a reliable framework for gene expression. This system can be used to seek out new interacting partners to improve the tolerance phenotype and can be used in the development of more robust solvent production strains.

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