scholarly journals The Elemental Sulfur-Responsive Protein (SipA) from the Hyperthermophilic Archaeon Pyrococcus furiosus Is Regulated by Sulfide in an Iron-Dependent Manner

2010 ◽  
Vol 192 (21) ◽  
pp. 5841-5843 ◽  
Author(s):  
Sonya M. Clarkson ◽  
Elizabeth C. Newcomer ◽  
Everett G. Young ◽  
Michael W. W. Adams
Keyword(s):  
Elemental Sulfur ◽  
Iron Sulfide ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Dependent Manner ◽  
Intracellular Iron ◽  
Hyperthermophilic Archaeon

ABSTRACT The gene (sipA) encoding the sulfur-induced protein A (PF2025) is highly upregulated during growth of Pyrococcus furiosus on elemental sulfur (S0). Expression of sipA is regulated by sulfide, the product of S0 reduction, but in an iron-dependent manner. SipA is proposed to play a role in intracellular iron sulfide detoxification.

scholarly journals Key Role for Sulfur in Peptide Metabolism and in Regulation of Three Hydrogenases in the Hyperthermophilic ArchaeonPyrococcus furiosus

2001 ◽  
Vol 183 (2) ◽  
pp. 716-724 ◽  
Author(s):  
Michael W. W. Adams ◽  
James F. Holden ◽  
Angeli Lal Menon ◽  
Gerrit J. Schut ◽  
Amy M. Grunden ◽  
...  
Keyword(s):  
Growth Rates ◽  
Elemental Sulfur ◽  
Pyrococcus Furiosus ◽  
Growth Conditions ◽  
Growth Media ◽  
Hyperthermophilic Archaeon ◽  
Order Of Magnitude ◽  
Membrane Bound ◽  
Peptide Metabolism ◽  
Specific Activities

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosusgrows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S0). Growth rates were highest on media containing peptides and S0, with or without maltose. Growth did not occur on the peptide medium without S0. S0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S0 with or without maltose were the same, suggesting that S0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S0-reducing enzyme in this organism and the mechanism of the S0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.

scholarly journals Insights into the Metabolism of Elemental Sulfur by the Hyperthermophilic Archaeon Pyrococcus furiosus: Characterization of a Coenzyme A- Dependent NAD(P)H Sulfur Oxidoreductase

2007 ◽  
Vol 189 (12) ◽  
pp. 4431-4441 ◽  
Author(s):  
Gerrit J. Schut ◽  
Stephanie L. Bridger ◽  
Michael W. W. Adams
Keyword(s):  
Gene Cluster ◽  
Elemental Sulfur ◽  
Coenzyme A ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
Side Reaction ◽  
Primary Response ◽  
Secondary Response ◽  
Hyperthermophilic Archaeon ◽  
Membrane Bound

ABSTRACT The hyperthermophilic archaeon Pyrococcus furiosus uses carbohydrates as a carbon source and produces acetate, CO2, and H2 as end products. When S0 is added to a growing culture, within 10 min the rate of H2 production rapidly decreases and H2S is detected. After 1 hour cells contain high NADPH- and coenzyme A-dependent S0 reduction activity (0.7 units/mg, 85°C) located in the cytoplasm. The enzyme responsible for this activity was purified to electrophoretic homogeneity (specific activity, 100 units/mg) and is termed NAD(P)H elemental sulfur oxidoreductase (NSR). NSR is a homodimeric flavoprotein (M r, 100,000) and is encoded by PF1186. This designation was previously assigned to the gene encoding an enzyme that reduces coenzyme A disulfide, which is a side reaction of NSR. Whole-genome DNA microarray and quantitative PCR analyses showed that the expression of NSR is up-regulated up to sevenfold within 10 min of S0 addition. This primary response to S0 also involves the up-regulation (>16-fold) of a 13-gene cluster encoding a membrane-bound oxidoreductase (MBX). The cluster encoding MBX is proposed to replace the homologous 14-gene cluster that encodes the ferredoxin-oxidizing, H2-evolving membrane-bound hydrogenase (MBH), which is down-regulated >12-fold within 10 min of S0 addition. Although an activity for MBX could not be demonstrated, it is proposed to conserve energy by oxidizing ferredoxin and reducing NADP, which is used by NSR to reduce S0. A secondary response to S0 is observed 30 min after S0 addition and includes the up-regulation of genes encoding proteins involved in amino acid biosynthesis and iron metabolism, as well as two so-called sulfur-induced proteins termed SipA and SipB. This novel S0-reducing system involving NSR and MBX has been found so far only in the heterotrophic Thermococcales and is in contrast to the cytochrome- and quinone-based S0-reducing system in autotrophic archaea and bacteria.

scholarly journals Characterization of an Aminoacylase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2001 ◽  
Vol 183 (14) ◽  
pp. 4259-4268 ◽  
Author(s):  
Sherry V. Story ◽  
Amy M. Grunden ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Pyrococcus Furiosus ◽  
Protein A ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Protein Renaturation ◽  
Cell Extracts ◽  
Growth Substrates ◽  
Catalytically Active

ABSTRACT Aminoacylase was identified in cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by its ability to hydrolyze N-acetyl-l-methionine and was purified by multistep chromatography. The enzyme is a homotetramer (42.06 kDa per subunit) and, as purified, contains 1.0 ± 0.48 g-atoms of zinc per subunit. Treatment of the purified enzyme with EDTA resulted in complete loss of activity. This was restored to 86% of the original value (200 U/mg) by treatment with ZnCl2 (and to 74% by the addition of CoCl2). After reconstitution with ZnCl2, the enzyme contained 2.85 ± 0.48 g-atoms of zinc per subunit. Aminoacylase showed broad substrate specificity and hydrolyzed nonpolarN-acylated l amino acids (Met, Ala, Val, and Leu), as well as N-formyl-l-methionine. The high Km values for these compounds indicate that the enzyme plays a role in the metabolism of protein growth substrates rather than in the degradation of cellular proteins. Maximal aminoacylase activity withN-acetyl-l-methionine as the substrate occurred at pH 6.5 and a temperature of 100°C. The N-terminal amino acid sequence of the purified aminoacylase was used to identify, in theP. furiosus genome database, a gene that encodes 383 amino acids. The gene was cloned and expressed in Escherichia coli by using two approaches. One involved the T7lac promoter system, in which the recombinant protein was expressed as inclusion bodies. The second approach used the Trx fusion system, and this produced soluble but inactive recombinant protein. Renaturation and reconstitution experiments with Zn2+ ions failed to produce catalytically active protein. A survey of databases showed that, in general, organisms that contain a homolog of theP. furiosus aminoacylase (≥50% sequence identity) utilize peptide growth substrates, whereas those that do not contain the enzyme are not known to be proteolytic, suggesting a role for the enzyme in primary catabolism.

scholarly journals Genetic Analysis of Factors Affecting Susceptibility of Bacillus subtilis to Daptomycin

2009 ◽  
Vol 53 (4) ◽  
pp. 1598-1609 ◽  
Author(s):  
Anna-Barbara Hachmann ◽  
Esther R. Angert ◽  
John D. Helmann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Transcriptional Profiling ◽  
Protein A ◽  
Multidrug Resistant ◽  
Dependent Manner ◽  
Membrane Composition ◽  
Factors Affecting ◽  
Anionic Phospholipids ◽  
Cyclic Lipopeptide

ABSTRACT Daptomycin is the first of a new class of cyclic lipopeptide antibiotics used against multidrug-resistant, gram-positive pathogens. The proposed mechanism of action involves disruption of the functional integrity of the bacterial membrane in a Ca2+-dependent manner. We have used transcriptional profiling to demonstrate that treatment of Bacillus subtilis with daptomycin strongly induces the lia operon including the autoregulatory LiaRS two-component system (homologous to Staphylococcus aureus VraSR). The lia operon protects against daptomycin, and deletion of liaH, encoding a phage-shock protein A (PspA)-like protein, leads to threefold increased susceptibility. Since daptomycin interacts with the membrane, we tested mutants with altered membrane composition for effects on susceptibility. Deletion mutations of mprF (lacking lysyl-phosphatidylglycerol) or des (lipid desaturase) increased daptomycin susceptibility, whereas overexpression of MprF decreased susceptibility. Conversely, depletion of the cell for the anionic lipid phosphatidylglycerol led to increased resistance. Fluorescently labeled daptomycin localized to the septa and in a helical pattern around the cell envelope and was delocalized upon the depletion of phosphatidylglycerol. Together, these results indicate that the daptomycin-Ca2+ complex interacts preferentially with regions enriched in anionic phospholipids and leads to membrane stresses that can be ameliorated by PspA family proteins.

Oxygen modulates the differentiation of human fetal lung in vitro and its responsiveness to cAMP

1993 ◽  
Vol 264 (5) ◽  
pp. L465-L474 ◽  
Author(s):  
M. J. Acarregui ◽  
J. M. Snyder ◽  
C. R. Mendelson
Keyword(s):  
Gene Expression ◽  
Atmospheric Oxygen ◽  
Protein A ◽  
Morphological Differentiation ◽  
Fetal Lung ◽  
Morphological Development ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Human Fetal Lung

Previously, it was found that lung explants from mid-trimester human abortuses differentiate spontaneously in organ culture in serum-free defined medium in an atmosphere of 95% air-5% CO2. Dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) treatment of human fetal lung in culture increases the rate of morphological differentiation and enhances expression of the surfactant protein A (SP-A) gene. To begin to define the factors responsible for this accelerated in vitro differentiation, we analyzed the effects of atmospheric oxygen on the morphological and biochemical development of human fetal lung in culture and on responsiveness of the cultured tissue to DBcAMP. We found that when lung explants were maintained in an atmosphere containing 1% oxygen they failed to differentiate spontaneously and no induction of SP-A gene expression was apparent. Furthermore, at 1% oxygen, DBcAMP had no effect to stimulate morphological differentiation or SP-A gene expression. When lung tissues that had been maintained for 5 days in 1% oxygen were transferred to an environment containing 20% oxygen, there was rapid morphological development and induction of SP-A gene expression. The effects on morphological development were manifest within 24 h of transfer to the 20% oxygen environment; within 72 h, a marked stimulatory effect of DBcAMP on SP-A gene expression also was observed. Our findings further suggest that the effects of oxygen on the levels of SP-A and SP-A mRNA are concentration dependent. Interestingly, the inductive effects of DBcAMP on SP-A gene expression were apparent only at oxygen concentrations > or = 10%. Morphological differentiation of the cultured human fetal lung tissue also was influenced by oxygen in a concentration-dependent manner. These findings suggest that oxygen plays an important permissive role in the spontaneous differentiation of human fetal lung in vitro.

scholarly journals MicroRNA-132-3p suppresses type I IFN response through targeting IRF1 to facilitate H1N1 influenza A virus infection

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Fangyi Zhang ◽  
Xuefeng Lin ◽  
Xiaodong Yang ◽  
Guangjian Lu ◽  
Qunmei Zhang ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Peripheral Blood ◽  
Influenza A ◽  
Expression Profiles ◽  
Protein A ◽  
H1n1 Influenza ◽  
Type I ◽  
Dependent Manner ◽  
Type I Ifn

Abstract Increasing evidence has indicated that microRNAs (miRNAs) have essential roles in innate immune responses to various viral infections; however, the role of miRNAs in H1N1 influenza A virus (IAV) infection is still unclear. The present study aimed to elucidate the role and mechanism of miRNAs in IAV replication in vitro. Using a microarray assay, we analyzed the expression profiles of miRNAs in peripheral blood from IAV patients. It was found that miR-132-3p was significantly up-regulated in peripheral blood samples from IAV patients. It was also observed that IAV infection up-regulated the expression of miR-132-3p in a dose- and time-dependent manner. Subsequently, we investigated miR-132-3p function and found that up-regulation of miR-132-3p promoted IAV replication, whereas knockdown of miR-132-3p repressed replication. Meanwhile, overexpression of miR-132-3p could inhibit IAV triggered INF-α and INF-β production and IFN-stimulated gene (ISG) expression, including myxovirus protein A (MxA), 2′,5′-oligoadenylate synthetases (OAS), and double-stranded RNA-dependent protein kinase (PKR), while inhibition of miR-132-3p enhanced IAV triggered these effects. Of note, interferon regulatory factor 1 (IRF1), a well-known regulator of the type I IFN response, was identified as a direct target of miR-132-3p during HIN1 IAV infection. Furthermore, knockdown of IRF1 by si-IRF1 reversed the promoting effects of miR-132-3p inhibition on type I IFN response. Taken together, up-regulation of miR-132-3p promotes IAV replication by suppressing type I IFN response through its target gene IRF1, suggesting that miR-132-3p could represent a novel potential therapeutic target of IAV treatment.

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