scholarly journals Characterization of Ten Heterotetrameric NDP-Dependent Acyl-CoA Synthetases of the Hyperthermophilic ArchaeonPyrococcus furiosus

Archaea ◽  
2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Joseph W. Scott ◽  
Farris L. Poole ◽  
Michael W. W. Adams
Keyword(s):  
Amino Acids ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Pyrococcus Furiosus ◽  
Hyperthermophilic Archaea ◽  
E Coli ◽  
Proteomic Data ◽  
Genes Encoding ◽  
Derivatives Of ◽  
Dependent Processes

The hyperthermophilic archaeonPyrococcus furiosusgrows by fermenting peptides and carbohydrates to organic acids. In the terminal step, acyl-CoA synthetase (ACS) isoenzymes convert acyl-CoA derivatives to the corresponding acid and conserve energy in the form of ATP. ACS1 and ACS2 were previously purified fromP. furiosusand haveα2β2structures but the genome contains genes encoding three additionalα-subunits. The ten possible combinations ofαandβgenes were expressed inE. coliand each resulted in stable and activeα2β2isoenzymes. Theα-subunit of each isoenzyme determined CoA-based substrate specificity and between them they accounted for the CoA derivatives of fourteen amino acids. Theβ-subunit determined preference for adenine or guanine nucleotides. The GTP-generating isoenzymes are proposed to play a role in gluconeogenesis by producing GTP for GTP-dependent phosphoenolpyruvate carboxykinase and for other GTP-dependent processes. Transcriptional and proteomic data showed that all ten isoenzymes are constitutively expressed indicating that both ATP and GTP are generated from the metabolism of most of the amino acids. A phylogenetic analysis showed that the ACSs ofP. furiosusand other members of the Thermococcales are evolutionarily distinct from those found throughout the rest of biology, including those of other hyperthermophilic archaea.

scholarly journals Molecular characterization of clinical carbapenem-resistant Enterobacterales from Qatar

2021 ◽  
Author(s):  
Fatma Ben Abid ◽  
Clement K. M. Tsui ◽  
Yohei Doi ◽  
Anand Deshmukh ◽  
Christi L. McElheny ◽  
...  
Keyword(s):  
Common Species ◽  
Clinical Samples ◽  
Whole Genome ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Genes Encoding ◽  
Encoding Genes ◽  
Sequence Types ◽  
Common Sequence

AbstractOne hundred forty-nine carbapenem-resistant Enterobacterales from clinical samples obtained between April 2014 and November 2017 were subjected to whole genome sequencing and multi-locus sequence typing. Klebsiella pneumoniae (81, 54.4%) and Escherichia coli (38, 25.5%) were the most common species. Genes encoding metallo-β-lactamases were detected in 68 (45.8%) isolates, and OXA-48-like enzymes in 60 (40.3%). blaNDM-1 (45; 30.2%) and blaOXA-48 (29; 19.5%) were the most frequent. KPC-encoding genes were identified in 5 (3.6%) isolates. Most common sequence types were E. coli ST410 (8; 21.1%) and ST38 (7; 18.4%), and K. pneumoniae ST147 (13; 16%) and ST231 (7; 8.6%).

scholarly journals Identification of the Tyrosine- and Phenylalanine-Derived Soluble Metabolomes of Sorghum

2021 ◽  
Vol 12 ◽  
Author(s):  
Jeffrey P. Simpson ◽  
Jacob Olson ◽  
Brian Dilkes ◽  
Clint Chapple
Keyword(s):  
Amino Acids ◽  
Cinnamic Acid ◽  
Aromatic Amino Acids ◽  
Flavonoid Glycosides ◽  
Coumaric Acid ◽  
Biotic And Abiotic Stress ◽  
Proportional Contribution ◽  
Negative Ionization ◽  
Derivatives Of

The synthesis of small organic molecules, known as specialized or secondary metabolites, is one mechanism by which plants resist and tolerate biotic and abiotic stress. Many specialized metabolites are derived from the aromatic amino acids phenylalanine (Phe) and tyrosine (Tyr). In addition, the improved characterization of compounds derived from these amino acids could inform strategies for developing crops with greater resilience and improved traits for the biorefinery. Sorghum and other grasses possess phenylalanine ammonia-lyase (PAL) enzymes that generate cinnamic acid from Phe and bifunctional phenylalanine/tyrosine ammonia-lyase (PTAL) enzymes that generate cinnamic acid and p-coumaric acid from Phe and Tyr, respectively. Cinnamic acid can, in turn, be converted into p-coumaric acid by cinnamate 4-hydroxylase. Thus, Phe and Tyr are both precursors of common downstream products. Not all derivatives of Phe and Tyr are shared, however, and each can act as a precursor for unique metabolites. In this study, 13C isotopic-labeled precursors and the recently developed Precursor of Origin Determination in Untargeted Metabolomics (PODIUM) mass spectrometry (MS) analytical pipeline were used to identify over 600 MS features derived from Phe and Tyr in sorghum. These features comprised 20% of the MS signal collected by reverse-phase chromatography and detected through negative-ionization. Ninety percent of the labeled mass features were derived from both Phe and Tyr, although the proportional contribution of each precursor varied. In addition, the relative incorporation of Phe and Tyr varied between metabolites and tissues, suggesting the existence of multiple pools of p-coumaric acid that are fed by the two amino acids. Furthermore, Phe incorporation was greater for many known hydroxycinnamate esters and flavonoid glycosides. In contrast, mass features derived exclusively from Tyr were the most abundant in every tissue. The Phe- and Tyr-derived metabolite library was also utilized to retrospectively annotate soluble MS features in two brown midrib mutants (bmr6 and bmr12) identifying several MS features that change significantly in each mutant.

scholarly journals Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals The characterization of ESBL genes in Escherichia coli and Klebsiella pneumoniae causing nosocomial infections in Vietnam

2013 ◽  
Vol 7 (12) ◽  
pp. 922-928 ◽  
Author(s):  
Nguyen Hoang Thu Trang ◽  
Tran Vu Thieu Nga ◽  
James I Campbell ◽  
Nguyen Trong Hiep ◽  
Jeremy Farrar ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Klebsiella Pneumoniae ◽  
Nosocomial Infections ◽  
Enteric Bacteria ◽  
E Coli ◽  
Plasmid Analysis ◽  
Resistance Plasmids ◽  
Genes Encoding ◽  
Third Generation Cephalosporins

Background: Extended-spectrum β-lactamases (ESBLs) are enzymes capable of hydrolyzing oxyimino-β-lactams and inducing resistance to third generation cephalosporins. The genes encoding ESBLs are widespread and generally located on highly transmissible resistance plasmids. We aimed to investigate the complement of ESBL genes in E. coli and Klebsiella pneumoniae causing nosocomial infections in hospitals in Ho Chi Minh City, Vietnam. Methodology: Thirty-two non-duplicate isolates of E. coli and Klebsiella pneumoniae causing nosocomial infections, isolated between March and June 2010, were subjected to antimicrobial susceptibility testing. All isolates were PCR-amplified to detect the blaSHV, blaTEM and blaCTX-M ESBL genes and subjected to plasmid analysis. Results: We found that co-resistance to multiple antimicrobials was highly prevalent, and we report the predominance of the blaCTX-M-15 and blaCTX-M-27 genes, located on highly transmissible plasmids ranging from 50 to 170 kb in size. Conclusions: Our study represents a snap shot of ESBL-producing enteric bacteria causing nosocomial infections in this setting. We suggest that antimicrobial resistance in nosocomial E. coli and Klebsiella pneumoniae is rampant in Vietnam and ESBL organisms are widespread. In view of these data and the dramatic levels of antimicrobial resistance reported in Vietnam we advocate an urgent review of antimicrobial use in the Vietnamese healthcare system.

Biochemical and functional characterization of Mycobacterium tuberculosis ketol-acid reductoisomerase

Microbiology ◽  
2021 ◽  
Vol 167 (9) ◽  
Author(s):  
Nirbhay Singh ◽  
Anu Chauhan ◽  
Ram Kumar ◽  
Sudheer Kumar Singh
Keyword(s):  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Type Species ◽  
Low Ph ◽  
Stress Conditions ◽  
Starvation Stress ◽  
Content Type ◽  
E Coli ◽  
Link Type

Branched-chain amino acids (BCAAs) are essential amino acids, but their biosynthetic pathway is absent in mammals. Ketol-acid reductoisomerase (IlvC) is a BCAA biosynthetic enzyme that is coded by Rv3001c in Mycobacterium tuberculosis H37Rv (Mtb-Rv) and MRA_3031 in M. tuberculosis H37Ra (Mtb-Ra). IlvCs are essential in Mtb-Rv as well as in Escherichia coli . Compared to wild-type and IlvC-complemented Mtb-Ra strains, IlvC knockdown strain showed reduced survival at low pH and under low pH+starvation stress conditions. Further, increased expression of IlvC was observed under low pH and starvation stress conditions. Confirmation of a role for IlvC in pH and starvation stress was achieved by developing E. coli BL21(DE3) IlvC knockout, which was defective for growth in M9 minimal medium, but growth could be rescued by isoleucine and valine supplementation. Growth was also restored by complementing with over-expressing constructs of Mtb-Ra and E. coli IlvCs. The E. coli knockout also had a survival deficit at pH=5.5 and 4.5 and was more susceptible to killing at pH=3.0. The biochemical characterization of Mtb-Ra and E. coli IlvCs confirmed that both have NADPH-dependent activity. In conclusion, this study demonstrates the functional complementation of E. coli IlvC by Mtb-Ra IlvC and also suggests that IlvC has a role in tolerance to low pH and starvation stress.

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

2006 ◽  
Vol 26 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Si Sun ◽  
Jo Han Gan ◽  
Jennifer J. Paynter ◽  
Stephen J. Tucker
Keyword(s):  
Functional Properties ◽  
Functional Characterization ◽  
Unicellular Eukaryote ◽  
Channel Blockers ◽  
E Coli ◽  
Inwardly Rectifying Potassium Channels ◽  
Genes Encoding ◽  
Functionally Diverse ◽  
Inwardly Rectifying

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

ISOLATION AND CHARACTERIZATION OF RIBONUCLEIC ACID FROM RAT LENS

1962 ◽  
Vol 40 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Anima Devi
Keyword(s):  
Amino Acids ◽  
The Other ◽  
Ocular Lens ◽  
E Coli ◽  
Isolation And Characterization ◽  
Coiled Structure ◽  
Calf Thymus ◽  
Original Procedure ◽  
Polynucleotide Chain

RNA from rat ocular lens has been isolated by a method based on Kirby's original procedure (7), but greatly modified so as to avoid any degradation of RNA by RNase during the process of its extraction from lenses. The absorption at 260 mμ of a 1.0% solution of this purified material in a 1-cm cell is 1.95. Its N/P ratio is 1.58. It has 20 to 25% activity of that of yeast-soluble RNA in accepting activated amino acids. When this RNA (like all other RNA's) is heated and cooled the polynucleotide chain can again form loops, thus suggesting a randomly coiled structure for this RNA. On the other hand, DNA preparations from calf thymus, rat liver, and E. coli showed irreversible changes when heated and cooled.

Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

1990 ◽  
Vol 45 (5) ◽  
pp. 538-543 ◽  
Author(s):  
D. Friedberg ◽  
J. Seijffers
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Characterization ◽  
Amino Acid Level ◽  
Acetolactate Synthase ◽  
Mutant Gene ◽  
Wild Type ◽  
E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

scholarly journals Postgenomic Scan of Metallo-β-Lactamase Homologues in Rhizobacteria: Identification and Characterization of BJP-1, a Subclass B3 Ortholog from Bradyrhizobium japonicum

2006 ◽  
Vol 50 (6) ◽  
pp. 1973-1981 ◽  
Author(s):  
Magdalena Stoczko ◽  
Jean-Marie Frère ◽  
Gian Maria Rossolini ◽  
Jean-Denis Docquier
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Catalytic Efficiency ◽  
Open Reading Frames ◽  
Human Pathogens ◽  
Microbial Genomes ◽  
E Coli ◽  
Genes Encoding ◽  
And Function ◽  
Identification And Characterization

ABSTRACT The diffusion of metallo-β-lactamases (MBLs) among clinically important human pathogens represents a therapeutic issue of increasing importance. However, the origin of these resistance determinants is largely unknown, although an important number of proteins belonging to the MBL superfamily have been identified in microbial genomes. In this work, we analyzed the distribution and function of genes encoding MBL-like proteins in the class Rhizobiales. Among 12 released complete genomes of members of the class Rhizobiales, a total of 57 open reading frames (ORFs) were found to have the MBL conserved motif and identity scores with MBLs ranging from 8 to 40%. On the basis of the best identity scores with known MBLs, four ORFs were cloned into Escherichia coli for heterologous expression. Among their products, one (blr6230) encoded by the Bradyrhizobium japonicum USDA110 genome, named BJP-1, hydrolyzed β-lactams when expressed in E. coli. BJP-1 enzyme is most closely related to the CAU-1 enzyme from Caulobacter vibrioides (40% amino acid sequence identity), a member of subclass B3 MBLs. A kinetic analysis revealed that BJP-1 efficiently hydrolyzed most β-lactam substrates, except aztreonam, ticarcillin, and temocillin, with the highest catalytic efficiency measured with meropenem. Compared to other MBLs, BJP-1 was less sensitive to inactivation by chelating agents.

Primary structure, expression and localization of two intermediate subunit lectins ofEntamoeba disparthat contain multiple CXXC motifs

Parasitology ◽  
2007 ◽  
Vol 134 (14) ◽  
pp. 1989-1999 ◽  
Author(s):  
H. TACHIBANA ◽  
X.-J. CHENG ◽  
S. KOBAYASHI ◽  
Y. OKADA ◽  
J. ITOH ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Confocal Microscopy ◽  
Phenotypic Expression ◽  
Surface Proteins ◽  
Rt Pcr ◽  
Entamoeba Dispar ◽  
Genes Encoding ◽  
Sequence Identities

SUMMARYWe have recently identified 2 surface proteins inEntamoeba histolyticaas intermediate subunits of galactose- andN-acetyl-D-galactosamine-inhibitable lectin (EhIgl1 and EhIgl2); these proteins both contain multiple CXXC motifs. Here, we report the molecular characterization of the corresponding proteins inEntamoeba dispar, which is neither pathogenic nor invasive. TwoIglgenes encoding 1110 and 1106 amino acids (EdIgl1 and EdIgl2) were cloned from 2 strains ofE. dispar. The amino acid sequence identities were 79% between EdIgl1 and EdIgl2, 75–76% between EdIgl1 and EhIgl1, and 73–74% between EdIgl2 and EhIgl2. However, all the CXXC motifs were conserved in the EdIgl proteins, suggesting that the fold conferred by this motif is important for function. Comparison of the expression level of theIglgenes by real-time RT-PCR showed 3–5 times higher expression ofEdIgl1compared toEdIgl2. Most EdIgl1 and EdIgl2 proteins were co-localized on the surface and in the cytoplasm of trophozoites, based on confocal microscopy. However, a different localization of EdIgl1 and EdIgl2 in intracellular vacuoles and a different level of phenotypic expression of the two Igls were also observed. These results demonstrate that Igls are important proteins even in non-pathogenic amoeba and that Igl1 and Igl2 may possess different functions.

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