Cloning and Characterization of a Carotenoid Cleavage Dioxygenase from Artemisia Annua L

2011 ◽  
Vol 108 ◽  
pp. 274-281
Author(s):  
Shuo Qian Liu ◽  
Na Tian ◽  
Zhong Hua Liu ◽  
Jia Nan Huang ◽  
Juan Li
Keyword(s):  
Amino Acid ◽  
Artemisia Annua ◽  
Amino Acid Identity ◽  
Carotenoid Cleavage Dioxygenase ◽  
E Coli ◽  
Acid Protein ◽  
Rapid Amplification ◽  
First Time

In order to discover the formation mechanism of carotenoid derived aroma, which has been wildly used on protection of crop against insect attacks, the full-length cDNA of an Artemisia annua carotenoid cleavage dioxygenase (AaCCD1) was cloned by rapid amplification of cDNA ends. The function of AaCCD1 was characterized by expression of AaCCD1 in a strain of E. coli accumulating carotenoids and enzyme assay in vitro. The completed open read frame of AaCCD1 was 1629 bp and it encoded a 542-amino acid protein with a 77% amino acid identity to Arabidopsis thaliana CCD1, a predicted molecular mass of 61.04 kDa and a pI of 5.8. AaCCD1 efficiently cleaves carotenoids and regulate the formation of terpenoid compounds. This is the first time to report the cloning and identification of carotenoid cleavage dioxygenase from Atemisia annua, which will play a great role on understanding the regulation of volatile compounds.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

scholarly journals Purification and Characterization of AsES Protein

2013 ◽  
Vol 288 (20) ◽  
pp. 14098-14113 ◽  
Author(s):  
Nadia R. Chalfoun ◽  
Carlos F. Grellet-Bournonville ◽  
Martín G. Martínez-Zamora ◽  
Araceli Díaz-Perales ◽  
Atilio P. Castagnaro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Foliar Spray ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Degenerate Primers ◽  
Acremonium Strictum ◽  
Rapid Amplification

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

scholarly journals New Plasmid-Mediated Quinolone Resistance Gene, qnrC, Found in a Clinical Isolate of Proteus mirabilis

2009 ◽  
Vol 53 (5) ◽  
pp. 1892-1897 ◽  
Author(s):  
Minghua Wang ◽  
Qinglan Guo ◽  
Xiaogang Xu ◽  
Xiaoying Wang ◽  
Xinyu Ye ◽  
...  
Keyword(s):  
Amino Acid ◽  
Resistance Gene ◽  
Clinical Isolate ◽  
Proteus Mirabilis ◽  
Amino Acid Identity ◽  
Quinolone Resistance ◽  
Clinical Strain ◽  
E Coli ◽  
Acid Protein ◽  
New Gene

ABSTRACT Since the discovery of qnrA in 1998, two additional qnr genes, qnrB and qnrS, have been described. These three plasmid-mediated genes contribute to quinolone resistance in gram-negative pathogens worldwide. A clinical strain of Proteus mirabilis was isolated from an outpatient with a urinary tract infection and was susceptible to most antimicrobials but resistant to ampicillin, sulfamethoxazole, and trimethoprim. Plasmid pHS10, harbored by this strain, was transferred to azide-resistant Escherichia coli J53 by conjugation. A transconjugant with pHS10 had low-level quinolone resistance but was negative by PCR for the known qnr genes, aac(6′)-Ib-cr and qepA. The ciprofloxacin MIC for the clinical strain and a J53/pHS10 transconjugant was 0.25 μg/ml, representing an increase of 32-fold relative to that for the recipient, J53. The plasmid was digested with HindIII, and a 4.4-kb DNA fragment containing the new gene was cloned into pUC18 and transformed into E. coli TOP10. Sequencing showed that the responsible 666-bp gene, designated qnrC, encoded a 221-amino-acid protein, QnrC, which shared 64%, 42%, 59%, and 43% amino acid identity with QnrA1, QnrB1, QnrS1, and QnrD, respectively. Upstream of qnrC there existed a new IS3 family insertion sequence, ISPmi1, which encoded a frameshifted transposase. qnrC could not be detected by PCR, however, in 2,020 strains of Enterobacteriaceae. A new quinolone resistance gene, qnrC, was thus characterized from plasmid pHS10 carried by a clinical isolate of P. mirabilis.

scholarly journals Molecular Characterization of OXA-20, a Novel Class D β-Lactamase, and Its Integron from Pseudomonas aeruginosa

1998 ◽  
Vol 42 (8) ◽  
pp. 2074-2083 ◽  
Author(s):  
Thierry Naas ◽  
Wladimir Sougakoff ◽  
Anne Casetta ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clavulanic Acid ◽  
Antibiotic Resistance Gene ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Class D ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum β-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188–2195, 1997). A second β-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 β-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D β-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar β-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) anintI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3′ conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3′ end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon.P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.

scholarly journals Characterization of VIM-2, a Carbapenem-Hydrolyzing Metallo-β-Lactamase and Its Plasmid- and Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in France

2000 ◽  
Vol 44 (4) ◽  
pp. 891-897 ◽  
Author(s):  
Laurent Poirel ◽  
Thierry Naas ◽  
Delphine Nicolas ◽  
Louis Collet ◽  
Samuel Bellais ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Variable Region ◽  
Gene Cassette ◽  
Amino Acid Identity ◽  
Resistance Pattern ◽  
Relative Molecular Mass ◽  
Class 1 Integron ◽  
E Coli

ABSTRACT Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to β-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing β-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B β-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, includingP. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 β-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 β-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-β-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 μM). VIM-2 conferred a resistance pattern to β-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime.bla VIM-2 was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. bla VIM-2 was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the bla VIM-1-containing integron. VIM-2 is the second carbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isolate outside Japan.

scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

Characterization of the small endogenous plasmid of Halobacterium strain SB3 and its use in transformation of H. halobium

1989 ◽  
Vol 35 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Neil R. Hackett ◽  
Shiladitya DasSarma
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Molecular Biology ◽  
Base Pair ◽  
Copy Number ◽  
Halobacterium Halobium ◽  
Acid Protein ◽  
Amino Acid Protein

To study the molecular biology of the halophilic archaebacterium Halobacterium halobium, the introduction of DNA engineered in vitro is desirable. As a first step in developing a cloning vector, the complete 1736 base pair nucleotide sequence of the natural, high copy number, Halobacterium plasmid pHSB1 has been determined. The plasmid was found to show homology to the small plasmids of Halobacterium strains GRB and GN101. Plasmid pHSB1 encodes a 317 amino acid protein of unknown function. The related halophile, H. halobium, could be transformed by pHSB1, demonstrating its utility as the basis of a cloning vector.Key words: archaebacteria, Halobacterium, plasmid.

scholarly journals Codon-Optimized Rhodotorula glutinis PAL Expressed in Escherichia coli With Enhanced Activities

2021 ◽  
Vol 8 ◽  
Author(s):  
Feiyan Xue ◽  
Zihui Liu ◽  
Yue Yu ◽  
Yangjie Wu ◽  
Yuxin Jin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Codon Optimization ◽  
Rhodotorula Glutinis ◽  
E Coli ◽  
Physical Conditions ◽  
Acid Protein ◽  
Heterogeneous Expression

PAL (phenylalanine ammonia lyase) is important for secondary metabolite production in plants and microorganisms. There is broad interest in engineering PAL for its biocatalytic applications in industry, agriculture, and medicine. The production of quantities of high-activity enzymes has been explored by gene cloning and heterogeneous expression of the corresponding protein. Here, we cloned the cDNA of Rhodotorula glutinis PAL (RgPAL) and introduced codon optimization to improve protein expression in Escherichia coli and enzyme activities in vitro. The RgPAL gene was cloned by reverse transcription and named pal-wt. It had a full-length of 2,121 bp and encoded a 706-amino-acid protein. The pal-wt was inefficiently expressed in E. coli, even when the expression host and physical conditions were optimized. Therefore, codon optimization was used to obtain the corresponding gene sequence, named pal-opt, in order to encode the same amino acid for the RgPAL protein. The recombinant protein encoded by pal-opt, named PAL-opt, was successfully expressed in E. coli and then purified to detect its enzymatic activity in vitro. Consequently, 55.33 ± 0.88 mg/L of PAL-opt protein with a specific activity of 1,219 ± 147 U/mg and Km value of 609 μM for substrate L-phenylalanine was easily obtained. The enzyme protein also displayed tyrosine ammonia lyase (TAL)–specific activity of 80 ± 2 U/mg and Km value of 13.3 μM for substrate L-tyrosine. The bifunctional enzyme RgPAL/TAL (PAL-opt) and its easy expression advantage will provide an important basis for further applications.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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