2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

ABSTRACT The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a Km of 152 μM. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R 2 = 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.

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Characterization of the Role of the FluG Protein in Asexual Development of Aspergillus nidulans

Genetics ◽

10.1093/genetics/158.3.1027 ◽

2001 ◽

Vol 158 (3) ◽

pp. 1027-1036 ◽

Cited By ~ 2

Author(s):

Cletus A D'Souza ◽

Bee Na Lee ◽

Thomas H Adams

Keyword(s):

Aspergillus Nidulans ◽

Negative Influence ◽

Asexual Development ◽

Wild Type ◽

Significant Similarity ◽

Developmental Defects ◽

Asexual Sporulation ◽

Type Strains

Abstract We showed previously that a ΔfluG mutation results in a block in Aspergillus nidulans asexual sporulation and that overexpression of fluG activates sporulation in liquid-submerged culture, a condition that does not normally support sporulation of wild-type strains. Here we demonstrate that the entire N-terminal region of FluG (∼400 amino acids) can be deleted without affecting sporulation, indicating that FluG activity resides in the C-terminal half of the protein, which bears significant similarity with GSI-type glutamine synthetases. While FluG has no apparent role in glutamine biosynthesis, we propose that it has an enzymatic role in sporulation factor production. We also describe the isolation of dominant suppressors of ΔfluG(dsg) that should identify components acting downstream of FluG and thereby define the function of FluG in sporulation. The dsgA1 mutation also suppresses the developmental defects resulting from ΔflbA and dominant activating fadA mutations, which both cause constitutive induction of the mycelial proliferation pathway. However, dsgA1 does not suppress the negative influence of these mutations on production of the aflatoxin precursor, sterigmatocystin, indicating that dsgA1 is specific for asexual development. Taken together, our studies define dsgA as a novel component of the asexual sporulation pathway.

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Immunomodulatory role of phagocyte-derived chloramines involving lymphocyte glutathione

Mediators of Inflammation ◽

10.1155/s0962935193000328 ◽

1993 ◽

Vol 2 (3) ◽

pp. 235-241 ◽

Cited By ~ 6

Author(s):

Véronique Witko-Sarsat ◽

Anh Thu Nguyen ◽

Béatrice Descamps-Latscha

Keyword(s):

Oxidative Stress ◽

Reduced Glutathione ◽

Human Lymphocytes ◽

Cell System ◽

Polymorphonuclear Neutrophils ◽

Myristate Acetate ◽

Defensive Role ◽

Free Model ◽

A Cell

This study shows that human lymphocytes markedly decrease chloramines (long-lived oxidants) generated by polymorphonuclear neutrophils (PMN) after stimulation by phorbol-myristate-acetate or opsonized zymosan. In a cell-free model, reduced glutathione (GSH) scavenged chloramines, giving rise to oxidized glutathione (GSSG). In the cell system, treatment of lymphocytes with autologous PMN-derived chloramines induced a profound decrease in their total and reduced glutathione (GSH) content and markedly inhibited their proliferate responses to concanavalin-A and, to a lesser extent, phytohaemagglutinin. It is concluded that (i) lymphocytes may play a defensive role against phagocyte-derived oxidative stress by scavenging chloramines, and (ii) as this effect which is mediated by GSH affects lymphocyte proliferative responses, it may help to elucidate the still obscure mechanisms of oxidative stress associated immunodeficiency.

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Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽

10.1182/blood.v93.12.4242.412k02_4242_4247 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4242-4247

Author(s):

T.A. Bayston ◽

A. Tripodi ◽

P.M. Mannucci ◽

E. Thompson ◽

H. Ireland ◽

...

Keyword(s):

Direct Sequencing ◽

Consensus Sequence ◽

Wild Type ◽

Antithrombin Deficiency ◽

Free System ◽

Heparin Interaction ◽

A Cell ◽

Variant Protein ◽

Very High

We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

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Recombinant Listeria monocytogenes Expressing a Cell Wall-Associated Listeriolysin O Is Weakly Virulent but Immunogenic

Infection and Immunity ◽

10.1128/iai.00419-09 ◽

2009 ◽

Vol 77 (10) ◽

pp. 4371-4382 ◽

Cited By ~ 5

Author(s):

Javier A. Carrero ◽

Boris Calderon ◽

Hector Vivanco-Cid ◽

Emil R. Unanue

Keyword(s):

Listeria Monocytogenes ◽

Listeriolysin O ◽

Wild Type ◽

Positive Bacterium ◽

Cell Responses ◽

A Cell ◽

Weakly Virulent

ABSTRACT Listeriolysin O (LLO) is an essential virulence factor for the gram-positive bacterium Listeria monocytogenes. Our goal was to determine if altering the topology of LLO would alter the virulence and toxicity of L. monocytogenes in vivo. A recombinant strain was generated that expressed a surface-associated LLO (sLLO) variant secreted at 40-fold-lower levels than the wild type. In culture, the sLLO strain grew in macrophages, translocated to the cytosol, and induced cell death. However, the sLLO strain showed decreased infectivity, reduced lymphocyte apoptosis, and decreased virulence despite a normal in vitro phenotype. Thus, the topology of LLO in L. monocytogenes was a factor in the pathogenesis of the infection and points to a role of LLO secretion during in vivo infection. The sLLO strain was cleared by severe combined immunodeficient (SCID) mice. Despite the attenuation of virulence, the sLLO strain was immunogenic and capable of eliciting protective T-cell responses.

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Evidence for a Fourth Hydrogenase in Desulfovibrio fructosovorans

Journal of Bacteriology ◽

10.1128/jb.184.3.853-856.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 853-856 ◽

Cited By ~ 11

Author(s):

Laurence Casalot ◽

Gilles De Luca ◽

Zorah Dermoun ◽

Marc Rousset ◽

Pascale de Philip

Keyword(s):

Methyl Viologen ◽

Wild Type ◽

Marker Exchange ◽

Reduction Activity ◽

Physiological Studies ◽

Type Strains ◽

Exchange Activity

ABSTRACT A strain devoid of the three hydrogenases characterized for Desulfovibrio fructosovorans was constructed using marker exchange mutagenesis. As expected, the H2-dependent methyl viologen reduction activity of the strain was null, but physiological studies showed no striking differences between the mutated and wild-type strains. The H+-D2 exchange activity measured in the mutated strain indicates the presence of a fourth hydrogenase in D. fructosovorans.

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The PAF1 complex cell-autonomously promotes oogenesis in Caenorhabditis elegans

10.1101/2021.08.18.456829 ◽

2021 ◽

Author(s):

Yukihiko Kubota ◽

Natsumi Ota ◽

Hisashi Takatsuka ◽

Takuma Unno ◽

Shuichi Onami ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Rna Polymerase Ii ◽

Oocyte Maturation ◽

Germ Line ◽

Biological Processes ◽

Wild Type ◽

Expression Of Genes ◽

A Cell ◽

Precise Role

The RNA polymerase II-associated factor 1 complex (PAF1C) is a protein complex that consists of LEO1, RTF1, PAF1, CDC73, and CTR9, and has been shown to be involved in Pol II-mediated transcriptional and chromatin regulation. Although it has been shown to regulate a variety of biological processes, the precise role of the PAF1C during germ line development has not been clarified. In this study, we found that reduction in the function of the PAF1C components, LEO-1, RTFO-1, PAFO-1, CDC-73, and CTR-9, in Caenorhabditis elegans affects cell volume expansion of oocytes. Defects in oogenesis were also confirmed using an oocyte maturation marker, OMA-1::GFP. While four to five OMA-1::GFP-positive oocytes were observed in wild-type animals, their numbers were significantly decreased in pafo-1 mutantand leo-1(RNAi), cdc-73(RNAi), and pafo-1(RNAi) animals. Expression of a functional PAFO-1::mCherry transgene in the germline significantly rescued the oogenesis-defective phenotype of the pafo-1 mutants, suggesting that expression of the PAF1C in germ cells is required for oogenesis. Notably, overexpression of OMA-1::GFP partially rescued the oogenesis defect in the pafo-1 mutants. Based on our findings, we propose that the PAF1C promotes oogenesis in a cell-autonomous manner by positively regulating the expression of genes involved in oocyte maturation.

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Familial Overexpression of β Antithrombin Caused by an Asn135Thr Substitution

Blood ◽

10.1182/blood.v93.12.4242 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4242-4247 ◽

Cited By ~ 9

Author(s):

T.A. Bayston ◽

A. Tripodi ◽

P.M. Mannucci ◽

E. Thompson ◽

H. Ireland ◽

...

Keyword(s):

Direct Sequencing ◽

Consensus Sequence ◽

Wild Type ◽

Antithrombin Deficiency ◽

Free System ◽

Heparin Interaction ◽

A Cell ◽

Variant Protein ◽

Very High

Abstract We have investigated the basis of antithrombin deficiency in an asymptomatic individual (and family) with borderline levels (≈70% antigen and activity) of antithrombin. Direct sequencing of amplified DNA showed a mutation in codon 135, AAC to ACC, predicting a heterozygous Asn135Thr substitution. This substitution alters the predicted consensus sequence for glycosylation, Asn-X-Ser, adjacent to the heparin interaction site of antithrombin. The antithrombin isolated from plasma of the proband by heparin-Sepharose chromatography contained amounts of β antithrombin (the very high affinity fraction) greatly increased (≈20% to 30% of total) above the trace levels found in normals. Expression of the residue 135 variant in both a cell-free system and COS-7 cells confirmed altered glycosylation arising as a consequence of the mutation. Wild-type and variant protein were translated and exported from COS-7 cells with apparently equal efficiency, in contrast to the reduced level of variant observed in plasma of the affected individual. This case represents a novel cause of antithrombin deficiency, removal of glycosylation concensus sequence, and highlights the potentially important role of β antithrombin in regulating coagulation.

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Role of CD8+ Lymphocytes in Control and Clearance of Measles Virus Infection of Rhesus Monkeys

Journal of Virology ◽

10.1128/jvi.77.7.4396-4400.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4396-4400 ◽

Cited By ~ 77

Author(s):

Sallie R. Permar ◽

Sherry A. Klumpp ◽

Keith G. Mansfield ◽

Woong-Ki Kim ◽

Darci A. Gorgone ◽

...

Keyword(s):

Rhesus Monkeys ◽

Measles Virus ◽

Measles Vaccine ◽

Virus Infections ◽

Wild Type ◽

Viral Loads ◽

Virus Clearance ◽

Cell Mediated Immune Response ◽

A Cell

ABSTRACT The creation of an improved vaccine for global measles control will require an understanding of the immune mechanisms of measles virus containment. To assess the role of CD8+ cytotoxic T lymphocytes in measles virus clearance, rhesus monkeys were depleted of CD8+ lymphocytes by monoclonal anti-CD8 antibody infusion and challenged with wild-type measles virus. The CD8+ lymphocyte-depleted animals exhibited a more extensive rash, higher viral loads at the peak of virus replication, and a longer duration of viremia than did the control antibody-treated animals. These findings indicate a central role for CD8+ lymphocytes in the control of measles virus infections and the importance of eliciting a cell-mediated immune response in new measles vaccine strategies.

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The Role of Ethylene Production in Virulence of Pseudomonas syringae pvs. glycinea and phaseolicola

Phytopathology ◽

10.1094/phyto.2001.91.5.511 ◽

2001 ◽

Vol 91 (5) ◽

pp. 511-518 ◽

Cited By ~ 60

Author(s):

Helge Weingart ◽

Henriette Ullrich ◽

Klaus Geider ◽

Beate Völksch

Keyword(s):

Pseudomonas Syringae ◽

Ethylene Production ◽

Type Strain ◽

Wild Type Strain ◽

Wild Type ◽

In Planta ◽

Population Sizes ◽

Soybean Plants ◽

Type Strains

The importance of ethylene production for virulence of Pseudomonas syringae pvs. glycinea and phaseolicola was assayed by comparing bacterial multiplication and symptom development in bean and soybean plants inoculated with ethylene-negative (efe) mutants and wild-type strains. The efe mutants of Pseudomonas syringae pv. glycinea were significantly reduced in their ability to grow in planta. However, the degree of reduction was strain-dependent. Population sizes of efe mutant 16/83-E1 that did not produce the phototoxin coronatine were 10- and 15-fold lower than those of the wild-type strain on soybean and on bean, and 16/83-E1 produced very weak symptoms compared with the wild-type strain. The coronatine-producing efe mutant 7a/90-E1 reached fourfold and twofold lower population sizes compared with the wild-type strain on soybean and bean, respectively, and caused disease symptoms typical of the wild-type strain. Experiments with ethylene-insensitive soybeans confirmed these results. The virulence of the wild-type strains was reduced to the same extent in ethylene-insensitive soybean plants as the virulence of the efe mutants in ethylene-susceptible soybeans. In contrast, the virulence of Pseudomonas syringae pv. phaseolicola was not affected by disruption of the efe gene.

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New Insights into the Potential Cytotoxic Role of Bacillus cytotoxicus Cytotoxin K-1

Toxins ◽

10.3390/toxins13100698 ◽

2021 ◽

Vol 13 (10) ◽

pp. 698

Author(s):

Klèma Marcel Koné ◽

Pauline Hinnekens ◽

Jelena Jovanovic ◽

Andreja Rajkovic ◽

Jacques Mahillon

Keyword(s):

Bacillus Cereus ◽

Tetrazolium Salt ◽

Wild Type ◽

Foodborne Outbreak ◽

Pathogenic Role ◽

Bacillus Cereus Group ◽

Mtt Method ◽

High Cytotoxicity ◽

Type Strains

The thermotolerant representative of the Bacillus cereus group, Bacillus cytotoxicus, reliably harbors the coding gene of cytotoxin K-1 (CytK-1). This protein is a highly cytotoxic variant of CytK toxin, initially recovered from a diarrheal foodborne outbreak that caused the death of three people. In recent years, the cytotoxicity of B. cytotoxicus has become controversial, with some strains displaying a high cytotoxicity while others show no cytotoxicity towards cell lines. In order to better circumscribe the potential pathogenic role of CytK-1, knockout (KO) mutants were constructed in two B. cytotoxicus strains, E8.1 and E28.3. The complementation of the cytK-1 KO mutation was implemented in a mutant strain lacking in the cytK-1 gene. Using the tetrazolium salt (MTT) method, cytotoxicity tests of the cytK-1 KO and complemented mutants, as well as those of their wild-type strains, were carried out on Caco-2 cells. The results showed that cytK-1 KO mutants were significantly less cytotoxic than the parental wild-type strains. However, the complemented mutant was as cytotoxic as the wild-type, suggesting that CytK-1 is the major cytotoxicity factor in B. cytotoxicus.

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