scholarly journals Heme A Synthase Enzyme Functions Dissected by Mutagenesis of Bacillus subtilis CtaA

2005 ◽  
Vol 187 (24) ◽  
pp. 8361-8369 ◽  
Author(s):  
Lars Hederstedt ◽  
Anna Lewin ◽  
Mimmi Throne-Holst
Keyword(s):  
Bacillus Subtilis ◽  
Catalytic Mechanism ◽  
Enzyme Reaction ◽  
Prosthetic Group ◽  
Histidine Residues ◽  
Functional Roles ◽  
Heme A Synthase ◽  
Respiratory Oxidases

ABSTRACT Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme O into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed anaffinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme O, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Exosomal S100A4 derived from highly metastatic hepatocellular carcinoma cells promotes metastasis by activating STAT3

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Haoting Sun ◽  
Chaoqun Wang ◽  
Beiyuan Hu ◽  
Xiaomei Gao ◽  
Tiantian Zou ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cancer Progression ◽  
Calcium Binding ◽  
Tumor Metastasis ◽  
Metastatic Potential ◽  
Functional Roles ◽  
Stat3 Phosphorylation ◽  
Hcc Cells

AbstractIntercellular cross-talk plays important roles in cancer progression and metastasis. Yet how these cancer cells interact with each other is still largely unknown. Exosomes released by tumor cells have been proved to be effective cell-to-cell signal mediators. We explored the functional roles of exosomes in metastasis and the potential prognostic values for hepatocellular carcinoma (HCC). Exosomes were extracted from HCC cells of different metastatic potentials. The metastatic effects of exosomes derived from highly metastatic HCC cells (HMH) were evaluated both in vitro and in vivo. Exosomal proteins were identified with iTRAQ mass spectrum and verified in cell lines, xenograft tumor samples, and functional analyses. Exosomes released by HMH significantly enhanced the in vitro invasion and in vivo metastasis of low metastatic HCC cells (LMH). S100 calcium-binding protein A4 (S100A4) was identified as a functional factor in exosomes derived from HMH. S100A4rich exosomes significantly promoted tumor metastasis both in vitro and in vivo compared with S100A4low exosomes or controls. Moreover, exosomal S100A4 could induce expression of osteopontin (OPN), along with other tumor metastasis/stemness-related genes. Exosomal S100A4 activated OPN transcription via STAT3 phosphorylation. HCC patients with high exosomal S100A4 in plasma also had a poorer prognosis. In conclusion, exosomes from HMH could promote the metastatic potential of LMH, and exosomal S100A4 is a key enhancer for HCC metastasis, activating STAT3 phosphorylation and up-regulating OPN expression. This suggested exosomal S100A4 to be a novel prognostic marker and therapeutic target for HCC metastasis.

scholarly journals In Bacillus subtilis, the Sirtuin Protein Deacetylase, Encoded by the srtN Gene (Formerly yhdZ), and Functions Encoded by the acuABC Genes Control the Activity of Acetyl Coenzyme A Synthetase

2009 ◽  
Vol 191 (6) ◽  
pp. 1749-1755 ◽  
Author(s):  
Jeffrey G. Gardner ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Bacillus Subtilis ◽  
Coenzyme A ◽  
Acetyl Coenzyme A ◽  
Acetyl Coenzyme ◽  
New Information ◽  
Low Concentrations ◽  
Dependent Protein ◽  
Protein Deacetylase

ABSTRACT This report provides in vivo evidence for the posttranslational control of the acetyl coenzyme A (Ac-CoA) synthetase (AcsA) enzyme of Bacillus subtilis by the acuA and acuC gene products. In addition, both in vivo and in vitro data presented support the conclusion that the yhdZ gene of B. subtilis encodes a NAD+-dependent protein deacetylase homologous to the yeast Sir2 protein (also known as sirtuin). On the basis of this new information, a change in gene nomenclature, from yhdZ to srtN (for sirtuin), is proposed to reflect the activity associated with the YdhZ protein. In vivo control of B. subtilis AcsA function required the combined activities of AcuC and SrtN. Inactivation of acuC or srtN resulted in slower growth and cell yield under low-acetate conditions than those of the wild-type strain, and the acuC srtN strain grew under low-acetate conditions as poorly as the acsA strain. Our interpretation of the latter result was that both deacetylases (AcuC and SrtN) are needed to maintain AcsA as active (i.e., deacetylated) so the cell can grow with low concentrations of acetate. Growth of an acuA acuC srtN strain on acetate was improved over that of the acuA + acuC srtN strain, indicating that the AcuA acetyltransferase enzyme modifies (i.e., inactivates) AcsA in vivo, a result consistent with previously reported in vitro evidence that AcsA is a substrate of AcuA.

scholarly journals A review on the bio-functional roles of phospholipids from marine resources

Food Research ◽  
2021 ◽  
Vol 5 (5) ◽  
pp. 1-16
Author(s):  
M. Haq ◽  
S. Suraiya
Keyword(s):  
Synergistic Effects ◽  
Chemical Properties ◽  
Functional Roles ◽  
Functional Activities ◽  
Marine Phospholipids ◽  
Liver Functions ◽  
Anti Inflammatory ◽  
Unique Chemical

Marine phospholipids (PLs) rich in ω-3 polyunsaturated fatty acids (ω-3 PUFAs) have drawn keen interest recently among researchers and consumers and could be assumed as a “miracle drug”. Substantial amount of EPA and DHA, amazing and unique chemical properties and super bio-functional activities of marine PLs make it superior compared to terrestrial PLs, which lack long chain ω-3 PUFAs. Many comparative studies revealed that marine PLs showed higher health beneficial activities compared to PLs obtained from land sources. Marine PLs are not only beneficial in containing a high amount of ω-3 PUFAs but also in absorbing and assimilating ω-3 PUFAs in different tissues. Synergistic effects of PL compounds and ω-3 PUFAs in marine PLs showed super bio-functional performances like anti-atherosis and cardioprotective, anti-inflammatory, neuroprotective, immunological, and liver functions. A number of in vivo and in vitro studies on the administration of marine PLs extracted from fishes, mollusks, crustaceans, echinoderms reduced triacylglycerol (TAG) level and enhanced cardioprotective functions, demonstrated anti-inflammatory activity, reduced cell proliferation and tumor, increased cognitive functions and memory, and prevented hepatic damages. Therefore, this review paper provides detailed accounts on the present research status of critical biological and nutritional functions of marine ω-3 PUFAs rich phospholipids focusing on the origin, animal models, treatment, and roles.

scholarly journals A hyperthermoactive-Cas9 editing tool reveals the role of a unique arsenite methyltransferase in the arsenic resistance system of Thermus thermophilus HB27

2021 ◽  
Author(s):  
Giovanni Gallo ◽  
Ioannis Mougiakos ◽  
Mauricio Bianco ◽  
Miriam Carbonaro ◽  
Andrea Carpentieri ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Fluorescent Protein ◽  
Efflux Pump ◽  
Thermus Thermophilus ◽  
Yellow Fluorescent Protein ◽  
Arsenic Resistance ◽  
Wide Range ◽  
Resistance System

Arsenic detoxification systems can be found in a wide range of organisms, from bacteria to man. In a previous study, we discovered an arsenic-responsive transcriptional regulator in the thermophilic bacterium Thermus thermophilus HB27 (TtSmtB). Here, we characterize the arsenic resistance system of T. thermophilus in more detail. We employed TtSmtB-based pull-down assays with protein extracts from cultures treated with arsenate and arsenite to obtain an S-adenosylmethionine (SAM)-dependent arsenite methyltransferase (TtArsM). In vivo and in vitro analyses were performed to shed light on this new component of the arsenic resistance network and its peculiar catalytic mechanism. Heterologous expression of TtarsM in Escherichia coli resulted in arsenite detoxification at mesophilic temperatures. Although TtArsM does not contain a canonical arsenite binding site, the purified protein does catalyse SAM-dependent arsenite methylation. In addition, in vitro analyses confirmed the unique interaction between TtArsM and TtSmtB. Next, a highly efficient ThermoCas9-based genome-editing tool was developed to delete the TtArsM-encoding gene on the T. thermophilus genome, and to confirm its involvement in the arsenite detoxification system. Finally, the TtarsX efflux pump gene in the T. thermophilus ΔTtarsM genome was substituted by a gene, encoding a stabilised yellow fluorescent protein (sYFP), to create a sensitive genome-based bioreporter system for the detection of arsenic ions.

scholarly journals In vivo and in vitro chemotactic methylation in Bacillus subtilis.

1981 ◽  
Vol 145 (2) ◽  
pp. 958-965 ◽  
Author(s):  
A H Ullah ◽  
G W Ordal
Keyword(s):  
Bacillus Subtilis

scholarly journals Mutagenesis and ultraviolet inactivation of transforming DNA of Haemophilus influenzae complexed with a Bacillus subtilis protein that alters DNA conformation.

1996 ◽  
Vol 43 (1) ◽  
pp. 107-114 ◽  
Author(s):  
J K Setlow ◽  
B C Setlow ◽  
P Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Haemophilus Influenzae ◽  
Dna Conformation ◽  
Erythromycin Resistance ◽  
Bacillus Subtilis Spore ◽  
Pyrimidine Dimers ◽  
Resistance Markers ◽  
Selection For

The wild-type Bacillus subtilis spore protein, SspCwt, binds to DNA in vitro and in vivo and changes the conformation of DNA from B to A. Synthesis of the cloned SspCwt gene in Escherichia coli also causes large increases in mutation frequency. Binding of SspCwt to transforming DNA from Haemophilus influenzae made the DNA resistant to ultraviolet (UV) radiation. The mutant protein, SspCala, which does not bind DNA, did not change the UV resistance. The UV sensitivity of the DNA/SspCwt complex was not increased when the recipients of the DNA were defective in excision of pyrimidine dimers. These data indicate that the H. influenzae excision mechanism does not operate on the spore photoproduct formed by UV irradiation of the complex. Selection for the streptomycin- or erythromycin-resistance markers on the transforming DNA evidenced significant mutations at loci closely linked to these, but not at other loci. SspCwt apparently entered the cell attached to the transforming DNA, and caused mutations in adjacent loci. The amount of such mutations decreased when the transforming DNA was UV irradiated, because UV unlinks linked markers.

scholarly journals Preliminary Characterization of the Probiotic Properties of a Bacterial Strain for Used in Monogastric Nutrition

2019 ◽  
Vol 76 (2) ◽  
pp. 102 ◽  
Author(s):  
Mihaela DUMITRU ◽  
Mihaela HĂBEANU ◽  
Cristina TABUC ◽  
Ștefana JURCOANE
Keyword(s):  
Bacillus Subtilis ◽  
Bile Salts ◽  
Good Growth ◽  
Probiotic Properties ◽  
Simulated Intestinal Fluid ◽  
Phenotypic Profile ◽  
Subtilis Atcc ◽  
Bacillus Subtilis Atcc

This study aimed to evaluate some probiotic properties of Bacillus subtilis ATCC 6051a. The phenotypic profile, resistance to pH by simulated gastric juice (pH 2 and 3), bile salts by simulated intestinal fluid, survivability (%), heat and antibiotics tolerance were investigated. The strain is a Gram-positive, rod-shaped bacteria, arranged in short chains or in small irregular pairs with the ability to produce spores. Good viability at pH 2 and 3, with a survival of more than ≥80%, was found. In the presence of bile salts 0.3%, over 4 h, the strain exhibited a survival ≥85%. At 80°C, for 120 min., the strain showed good growth (9.04 log CFU/ml). Results were sensitive to most antibiotics, with a highly susceptible (between 16 – 25 mm) to erythromycin, clindamycin, amoxicillin, chloramphenicol, ciprofloxacin, amikacin and kanamycin. The strain was found to be sensitive to vancomycin, gentamicin, and tetracycline. The present research demonstrated that Bacillus subtilis ATCC 6051a can survive under gastrointestinal conditions, which involves them to future in vitro and in vivo probiotic studies.

scholarly journals A note on the antibiotic properties of Bacillus subtilis against Colletotrichum trifolii

2005 ◽  
Vol 73 (1) ◽  
pp. 31-36 ◽  
Author(s):  
Y. Douville ◽  
J.G. Boland
Keyword(s):  
Bacillus Subtilis ◽  
Disease Incidence ◽  
Suppressive Effect ◽  
Mechanisms Of Action ◽  
Colletotrichum Trifolii ◽  
Growth Room ◽  
A Cell ◽  
Germ Tubes

The influence and mechanisms of action of Bacillus subtilis on Colletotrichum trifolii, a causal agent of anthracnose of alfalfa (Medicago sativa), were studied in vivo and in vitro. In growth room conditions, a cell-free culture filtrate of B. subtilis significantly reduced disease incidence and severity on alfalfa seedlings from 56% to 16% and from 2.0 to 1.2, respectively. Treatment of seedlings with washed cell suspensions of B. subtilis had no influence on disease. Applications of crude filtrate on alfalfa leaflets inoculated with C. trifolii were associated with reduced germination of conidia, lysis of conidia, and reduced formation of appressoria. Under in vitro conditions, crude filtrate reduced germination of conidia, and induced lysis of conidia and the formation of inflated germ tubes on germinating conidia. An antibiotic of the iturin family, iturin D, was tentatively identified as the active compound responsible for the suppressive effect of B. subtilis on C. trifolii.

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