scholarly journals Characterization of a flavocytochrome that is induced during the anaerobic respiration of Fe3+ by Shewanella frigidimarina NCIMB400

1999 ◽  
Vol 342 (2) ◽  
pp. 439-448 ◽  
Author(s):  
Paul S. DOBBIN ◽  
Julea N. BUTT ◽  
Anne K. POWELL ◽  
Graeme A. REID ◽  
David J. RICHARDSON
Keyword(s):  
Reductase Activity ◽  
Anaerobic Respiration ◽  
Nucleotide Sequencing ◽  
Amino Acid Residues ◽  
Sequencing Data ◽  
Gene Encoding ◽  
Terminal Electron Acceptor ◽  
Reading Frame ◽  
Ferric Pyrophosphate ◽  
Reduction Potentials

A 63.9 kDa periplasmic tetrahaem flavocytochrome c3, designated Ifc3, was found to be expressed in Shewanellafrigidimarina NCIMB400 grown anaerobically with ferric citrate or ferric pyrophosphate as the sole terminal electron acceptor, but not in anaerobic cultures of the bacterium with other respiratory substrates. Ifc3 was purified to homogeneity and revealed by biochemical, spectroscopic and primary structure analyses to contain four low-spin bis-His-ligated c3-haems, with midpoint reduction potentials of -73, -141, -174 and -259 mV. A low-potential flavin was present in the form of non-covalently bound FAD; the protein possessed a unidirectional fumarate reductase activity. Disruption of the chromosomal gene encoding Ifc3, ifcA, did not lead to a significant change in the rate of Fe3+ reduction in batch culture. However, during such growth the Ifc3-deficient mutant produced both a 35 kDa periplasmic c-type cytochrome and a 45 kDa membrane-associated c-type cytochrome at markedly higher levels than did the parent strain. Nucleotide sequencing data from directly upstream of ifcA indicated the presence of an open reading frame encoding a putative outer-membrane β-barrel protein of 324 amino acid residues.

scholarly journals The Brucella abortus Cyclic β-1,2-Glucan Virulence Factor Is Substituted with O-Ester-Linked Succinyl Residues

2006 ◽  
Vol 188 (14) ◽  
pp. 5003-5013 ◽  
Author(s):  
Mara S. Roset ◽  
Andrés E. Ciocchini ◽  
Rodolfo A. Ugalde ◽  
Nora Iñón de Iannino
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Brucella Abortus ◽  
Brucella Melitensis ◽  
Open Reading Frame ◽  
Nucleotide Sequencing ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Spectrometry Analysis ◽  
Host Interaction

ABSTRACT Brucella periplasmic cyclic β-1,2-glucan plays an important role during bacterium-host interaction. Nuclear magnetic resonance spectrometry analysis, thin-layer chromatography, and DEAE-Sephadex chromatography were used to characterize Brucella abortus cyclic glucan. In the present study, we report that a fraction of B. abortus cyclic β-1,2-glucan is substituted with succinyl residues, which confer anionic character on the cyclic β-1,2-glucan. The oligosaccharide backbone is substituted at C-6 positions with an average of two succinyl residues per glucan molecule. This O-ester-linked succinyl residue is the only substituent of Brucella cyclic glucan. A B. abortus open reading frame (BAB1_1718) homologous to Rhodobacter sphaeroides glucan succinyltransferase (OpgC) was identified as the gene encoding the enzyme responsible for cyclic glucan modification. This gene was named cgm for cyclic glucan modifier and is highly conserved in Brucella melitensis and Brucella suis. Nucleotide sequencing revealed that B. abortus cgm consists of a 1,182-bp open reading frame coding for a predicted membrane protein of 393 amino acid residues (42.7 kDa) 39% identical to Rhodobacter sphaeroides succinyltransferase. cgm null mutants in B. abortus strains 2308 and S19 produced neutral glucans without succinyl residues, confirming the identity of this protein as the cyclic-glucan succinyltransferase enzyme. In this study, we demonstrate that succinyl substituents of cyclic β-1,2-glucan of B. abortus are necessary for hypo-osmotic adaptation. On the other hand, intracellular multiplication and mouse spleen colonization are not affected in cgm mutants, indicating that cyclic-β-1,2-glucan succinylation is not required for virulence and suggesting that no low-osmotic stress conditions must be overcome during infection.

Gene Cloning, Nucleotide Sequencing, and Purification and Characterization of the Low-Specificityl-Threonine Aldolase from Pseudomonas sp. Strain NCIMB 10558

1998 ◽  
Vol 64 (2) ◽  
pp. 549-554 ◽  
Author(s):  
Ji-Quan Liu ◽  
Saeko Ito ◽  
Tohru Dairi ◽  
Nobuya Itoh ◽  
Michihiko Kataoka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Significant Loss ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequencing ◽  
Predicted Amino Acid Sequence ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Reading Frame ◽  
Threonine Aldolase

ABSTRACT A low-specificity l-threonine aldolase (l-TA) gene from Pseudomonas sp. strain NCIMB 10558 was cloned and sequenced. The gene contains an open reading frame consisting of 1,041 nucleotides corresponding to 346 amino acid residues. The gene was overexpressed in Escherichia colicells, and the recombinant enzyme was purified and characterized. The enzyme, requiring pyridoxal 5′-phosphate as a coenzyme, is strictlyl specific at the α position, whereas it cannot distinguish between threo and erythro forms at the β position. In addition to threonine, the enzyme also acts on various other l-β-hydroxy-α-amino acids, includingl-β-3,4-dihydroxyphenylserine,l-β-3,4-methylenedioxyphenylserine, andl-β-phenylserine. The predicted amino acid sequence displayed less than 20% identity with those of low-specificityl-TA from Saccharomyces cerevisiae,l-allo-threonine aldolase from Aeromonas jandaei, and four relevant hypothetical proteins from other microorganisms. However, lysine 207 of low-specificity l-TA from Pseudomonas sp. strain NCIMB 10558 was found to be completely conserved in these proteins. Site-directed mutagenesis experiments showed that substitution of Lys207 with Ala or Arg resulted in a significant loss of enzyme activity, with the corresponding disappearance of the absorption maximum at 420 nm. Thus, Lys207 of thel-TA probably functions as an essential catalytic residue, forming an internal Schiff base with the pyridoxal 5′-phosphate of the enzyme to catalyze the reversible aldol reaction.

Cloning and Expression of a Novel Protease with Fibrinolytic Activity from Arenicola cristata

2014 ◽  
Vol 998-999 ◽  
pp. 210-213
Author(s):  
Chun Ling Zhao ◽  
Wen Jing Yu ◽  
Ji Yu Ju
Keyword(s):  
Amino Acid ◽  
Recombinant Protein ◽  
Active Form ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Arenicola Cristata ◽  
Fibrin Plate ◽  
Serine Protease Family

cDNA of a novel protease, designated as AFEI, was cloned from digestive tract of Arenicola cristata by RACE. The cDNA of AFEIcomprised 897bp and an open reading frame that encoded polypeptides of 264 amino acid residues. AFEIshowed similarity to serine protease family and contained the conserved catalytic amino acid residues. The gene encoding the active form of AFEIwas expressed in E.coli and the purified recombinant protein could dissolve an artificial fibrin plate with plasminogen, which indicated the recombinant protein might be a plasminogen activator for thrombosis therapy.

Molecular Cloning and Characterization of cDNA Encoding Fibrinolytic Enzyme-3 from Earthworm Eisenia foetida

2004 ◽  
Vol 36 (4) ◽  
pp. 303-308 ◽  
Author(s):  
Guo-Qing Dong ◽  
Xiao-Ling Yuan ◽  
Ya-Jun Shan ◽  
Zhen-Hu Zhao ◽  
Jia-Pei Chen ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Fibrinolytic Enzyme ◽  
Eisenia Foetida ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Native Form ◽  
Reading Frame ◽  
Fibrin Plate ◽  
Earthworm Fibrinolytic Enzyme ◽  
High Degree

Abstract The earthworm fibrinolytic enzyme-3 (EFE-3, GenBank accession No: AY438622), from the earthworm Eisenia foetida, is a component of earthworm fibrinolytic enzymes. In this study, cDNA encoding the EFE-3 was cloned by RT-PCR. The cDNA contained an open reading frame of 741 nucleotides, which encoded a deduced protein of 247 amino acid residues, including signal sequences. EFE-3 showed a high degree of homology to earthworm (Lumbricus rebullus) proteases F-III-1, F-III-2, and bovine trypsin. The recombinant EFE-3 was expressed in E. coli as inclusion bodies, and the gene encoding the native form of EFE-3 was expressed in COS-7 cells in the medium. Both the refolding product of inclusion bodies and the secreted protease could dissolve the artificial fibrin plate.

scholarly journals Modulation of Anaerobic Energy Metabolism of Bacillus subtilis by arfM(ywiD)

2001 ◽  
Vol 183 (23) ◽  
pp. 6815-6821 ◽  
Author(s):  
Marco Marino ◽  
Hugo Cruz Ramos ◽  
Tamara Hoffmann ◽  
Philippe Glaser ◽  
Dieter Jahn
Keyword(s):  
Bacillus Subtilis ◽  
Nitrate Reductase ◽  
Reductase Activity ◽  
Transcriptional Start Site ◽  
Anaerobic Respiration ◽  
Regulatory System ◽  
Reading Frame ◽  
Regulatory Cascade ◽  
Anaerobic Induction ◽  
Respiratory Nitrate Reductase

ABSTRACT Bacillus subtilis grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame ywiDlocated upstream of the respiratory nitrate reductase operonnarGHJI resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of nasDE expression was found to be significantly dependent on intact ywiD, while anaerobicnarGHJI expression was ywiD independent. Anaerobic transcription of hmp, encoding a flavohemoglobin-like protein, and of the fermentative operonslctEP and alsSD, responsible for lactate and acetoin formation, was partially dependent on ywiD. Expression of pta, encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced byywiD. Transcription of the ywiD gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of ywiDby resDE was found to be indirect viaresDE-dependent activation of fnr. TheywiD gene is subject to autorepression and nitrite repression. These results suggest a ResDE → Fnr → YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of B. subtilis. Therefore,ywiD was renamed arfM for anaerobic respiration and fermentation modulator.

scholarly journals Isolation and characterization of a gene encoding farnesyl diphosphate synthase from \(\textit{Panax vietnamensis}\) Ha et Grushv

2021 ◽  
Vol 43 (4) ◽  
pp. 119-128
Author(s):  
Nguyen Van Giang ◽  
Luu Han Ly ◽  
Pham Le Bich Hang ◽  
Le Thi Thu Hien
Keyword(s):  
Farnesyl Diphosphate ◽  
Farnesyl Diphosphate Synthase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
Ginsenoside Biosynthesis ◽  
Isolation And Characterization ◽  
Key Enzyme ◽  
As Stress ◽  
Root Tissues

Panax vietnamensis Ha et Grushv. is a species of the genus Panax native to Central Vietnam, containing a family of triterpene saponins named ginsenosides. This group of biomolecules possesses valuable therapeutic properties against cancer, hepatitis, diabetes, inflammation as well as stress and anxiety. Farnesyl diphosphate synthase (FPS) is a key enzyme participating in the ginsenoside biosynthesis pathway. In this study, a FPS gene from P. vietnamensis (PvFPS) was isolated and characterized. The PvFPS cDNA contained an open reading frame of 1032 bp, encoding a polypeptide chain of 342 amino acid residues. Nucleotide sequence comparison showed that FPS was highly conserved among most species, with two Aspartate-rich motifs responsible for product chain length determination strongly sustained. PvFPS was closely related to those of the same genera and order and differed from those from other kingdoms. PvFPS expression was detected at a greater level in root tissues than in leaves in all ages. Our findings provided information concerning the properties of a crucial gene in the ginsenoside biosynthesis, thus enhancing our understanding of this important pathway.

scholarly journals Molecular cloning, expression and characterization of Bmserpin-2 gene from Bombyx mori.

2009 ◽  
Vol 56 (4) ◽  
Author(s):  
Ye Pan ◽  
Hengchuan Xia ◽  
Peng Lü ◽  
KePing Chen ◽  
Qin Yao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Developmental Stages ◽  
Pcr Analysis ◽  
Post Inoculation ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Real Time Quantitative Pcr ◽  
Localization Analysis

Serpins are a broadly distributed family of protease inhibitors. In this study, the gene encoding Bombyx mori serpin-2 (Bmserpin-2) was cloned and expressed in E. coli. The Bmserpin-2 cDNA contains a 1125 bp open reading frame (ORF). The deduced protein has 374 amino-acid residues, contains a conserved SERPIN domain and shares extensive homology with other invertebrate serpins. RT-PCR analysis showed that Bmserpin-2 was expressed in all developmental stages of B. mori larvae and various larval tissues. Subcellular localization analysis indicated that Bmserpin-2 protein was located in the cytoplasm. Interestingly, real-time quantitative PCR revealed that the expression of Bmserpin-2 in the midgut of susceptible B. mori strain 306 significantly increased at 72 hours post inoculation (hpi) when infected with BmNPV. However, there was no significant increase of the Bmserpin-2 expression in resistant strain NB infected with BmNPV. Thus, our data indicates that Bmserpin-2 may be involved in B. mori antiviral response.

scholarly journals Escherichia coli RNA Polymerase Is the Target of the Cyclopeptide Antibiotic Microcin J25

2001 ◽  
Vol 183 (15) ◽  
pp. 4543-4550 ◽  
Author(s):  
Mónica A. Delgado ◽  
Marı́a R. Rintoul ◽  
Ricardo N. Farı́as ◽  
Raúl A. Salomón
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Cyclic Peptide ◽  
Nucleotide Sequencing ◽  
Peptide Antibiotic ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Microcin J25

ABSTRACT Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in therpoC gene, encoding the β′ subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-typerpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoCgene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

scholarly journals Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

1998 ◽  
Vol 64 (12) ◽  
pp. 4883-4890 ◽  
Author(s):  
Belén Floriano ◽  
José L. Ruiz-Barba ◽  
Rufino Jiménez-Díaz
Keyword(s):  
Enterococcus Faecium ◽  
Pathogenic Bacteria ◽  
Specific Activity ◽  
Sequence Similarity ◽  
Fold Increase ◽  
Leader Peptide ◽  
Nucleotide Sequencing ◽  
Gene Encoding ◽  
Reading Frame ◽  
Fast Flow

ABSTRACT Enterocin I (ENTI) is a novel bacteriocin produced byEnterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI.entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1.

scholarly journals Gene Cloning and Nucleotide Sequencing and Properties of a Cocaine Esterase from Rhodococcus sp. Strain MB1

2000 ◽  
Vol 66 (3) ◽  
pp. 904-908 ◽  
Author(s):  
Matthew M. Bresler ◽  
Susan J. Rosser ◽  
Amrik Basran ◽  
Neil C. Bruce
Keyword(s):  
Rhodococcus Erythropolis ◽  
Sole Source ◽  
Tropane Alkaloid ◽  
Nucleotide Sequencing ◽  
Serine Esterase ◽  
Gene Encoding ◽  
Genomic Libraries ◽  
Reading Frame ◽  
Apparent Homogeneity ◽  
Cocaine Esterase

ABSTRACT A strain of Rhodococcus designated MB1, which was capable of utilizing cocaine as a sole source of carbon and nitrogen for growth, was isolated from rhizosphere soil of the tropane alkaloid-producing plant Erythroxylum coca. A cocaine esterase was found to initiate degradation of cocaine, which was hydrolyzed to ecgonine methyl ester and benzoate; both of these esterolytic products were further metabolized byRhodococcus sp. strain MB1. The structural gene encoding a cocaine esterase, designated cocE, was cloned fromRhodococcus sp. strain MB1 genomic libraries by screening recombinant strains of Rhodococcus erythropolis CW25 for growth on cocaine. The nucleotide sequence of cocEcorresponded to an open reading frame of 1,724 bp that codes for a protein of 574 amino acids. The amino acid sequence of cocaine esterase has a region of similarity with the active serine consensus of X-prolyl dipeptidyl aminopeptidases, suggesting that the cocaine esterase is a serine esterase. The cocE coding sequence was subcloned into the pCFX1 expression plasmid and expressed in Escherichia coli. The recombinant cocaine esterase was purified to apparent homogeneity and was found to be monomeric, with anM r of approximately 65,000. The apparentKm of the enzyme (mean ± standard deviation) for cocaine was measured as 1.33 ± 0.085 mM. These findings are of potential use in the development of a linked assay for the detection of illicit cocaine.

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