scholarly journals Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58

2008 ◽  
Vol 191 (5) ◽  
pp. 1565-1573 ◽  
Author(s):  
Sang-Hwal Yoon ◽  
Tae Seok Moon ◽  
Pooya Iranpour ◽  
Amanda M. Lanza ◽  
Kristala Jones Prather
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Pseudomonas Syringae ◽  
Glucuronic Acid ◽  
Glucaric Acid ◽  
Michaelis Constant ◽  
Turnover Number ◽  
Reading Frame ◽  
E Coli ◽  
Uronate Dehydrogenase

ABSTRACT Uronate dehydrogenase has been cloned from Pseudomonas syringae pv. tomato strain DC3000, Pseudomonas putida KT2440, and Agrobacterium tumefaciens strain C58. The genes were identified by using a novel complementation assay employing an Escherichia coli mutant incapable of consuming glucuronate as the sole carbon source but capable of growth on glucarate. A shotgun library of P. syringae was screened in the mutant E. coli by growing transformed cells on minimal medium containing glucuronic acid. Colonies that survived were evaluated for uronate dehydrogenase, which is capable of converting glucuronic acid to glucaric acid. In this manner, a 0.8-kb open reading frame was identified and subsequently verified to be udh. Homologous enzymes in P. putida and A. tumefaciens were identified based on a similarity search of the sequenced genomes. Recombinant proteins from each of the three organisms expressed in E. coli were purified and characterized. For all three enzymes, the turnover number (k cat) with glucuronate as a substrate was higher than that with galacturonate; however, the Michaelis constant (K m ) for galacturonate was lower than that for glucuronate. The A. tumefaciens enzyme was found to have the highest rate constant (k cat = 1.9 × 102 s−1 on glucuronate), which was more than twofold higher than those of both of the pseudomonad enzymes.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Enhancing the thermostability and activity of uronate dehydrogenase from Agrobacterium tumefaciens LBA4404 by semi-rational engineering

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Hui-Hui Su ◽  
Fei Peng ◽  
Pei Xu ◽  
Xiao-Ling Wu ◽  
Min-Hua Zong ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Rational Design ◽  
Glucuronic Acid ◽  
Specific Activity ◽  
Value Added ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Glucaric Acid ◽  
Triple Mutant ◽  
Pharmaceutical Industries

Abstract Background Glucaric acid, one of the aldaric acids, has been declared a “top value-added chemical from biomass”, and is especially important in the food and pharmaceutical industries. Biocatalytic production of glucaric acid from glucuronic acid is more environmentally friendly, efficient and economical than chemical synthesis. Uronate dehydrogenases (UDHs) are the key enzymes for the preparation of glucaric acid in this way, but the poor thermostability and low activity of UDH limit its industrial application. Therefore, improving the thermostability and activity of UDH, for example by semi-rational design, is a major research goal. Results In the present work, three UDHs were obtained from different Agrobacterium tumefaciens strains. The three UDHs have an approximate molecular weight of 32 kDa and all contain typically conserved UDH motifs. All three UDHs showed optimal activity within a pH range of 6.0–8.5 and at a temperature of 30 °C, but the UDH from A. tumefaciens (At) LBA4404 had a better catalytic efficiency than the other two UDHs (800 vs 600 and 530 s−1 mM−1). To further boost the catalytic performance of the UDH from AtLBA4404, site-directed mutagenesis based on semi-rational design was carried out. An A39P/H99Y/H234K triple mutant showed a 400-fold improvement in half-life at 59 °C, a 5 °C improvement in $$ {\text{T}}_{ 5 0}^{ 1 0} $$ T 50 10 value and a 2.5-fold improvement in specific activity at 30 °C compared to wild-type UDH. Conclusions In this study, we successfully obtained a triple mutant (A39P/H99Y/H234K) with simultaneously enhanced activity and thermostability, which provides a novel alternative for the industrial production of glucaric acid from glucuronic acid.

scholarly journals Construction and Characterization of a proU-gfp Transcriptional Fusion That Measures Water Availability in a Microbial Habitat

2002 ◽  
Vol 68 (9) ◽  
pp. 4604-4612 ◽  
Author(s):  
Catherine A. Axtell ◽  
Gwyn A. Beattie
Keyword(s):  
Pseudomonas Syringae ◽  
Water Availability ◽  
Water Deprivation ◽  
Bacterial Species ◽  
Transcriptional Fusion ◽  
Bacterial Biosensor ◽  
E Coli ◽  
Quantitative Manner ◽  
Microbial Habitat

ABSTRACT We constructed and characterized a transcriptional fusion that measures the availability of water to a bacterial cell. This fusion between the proU promoter from Escherichia coli and the reporter gene gfp was introduced into strains of E. coli, Pantoea agglomerans, and Pseudomonas syringae. The proU-gfp fusion in these bacterial biosensor strains responded in a quantitative manner to water deprivation caused by the presence of NaCl, Na2SO4, KCl, or polyethylene glycol (molecular weight, 8000). The fusion was induced to a detectable level by NaCl concentrations of as low as 10 mM in all three bacterial species. Water deprivation induced proU-gfp expression in both planktonic and surface-associated cells; however, it induced a higher level of expression in the surface-associated cells. Following the introduction of P. agglomerans biosensor cells onto bean leaves, the cells detected a significant decrease in water availability within only 5 min. After 30 min, the populations were exposed, on average, to a water potential equivalent to that imposed by approximately 55 mM NaCl. These results demonstrate the effectiveness of a proU-gfp-based biosensor for evaluating water availability on leaves. Furthermore, the inducibility of proU-gfp in multiple bacterial species illustrates the potential for tailoring proU-gfp-based biosensors to specific habitats.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

scholarly journals Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

2008 ◽  
Vol 75 (3) ◽  
pp. 589-595 ◽  
Author(s):  
Tae Seok Moon ◽  
Sang-Hwal Yoon ◽  
Amanda M. Lanza ◽  
Joseph D. Roy-Mayhew ◽  
Kristala L. Jones Prather
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Glucuronic Acid ◽  
Value Added ◽  
Culture Broth ◽  
Glucaric Acid ◽  
Synthetic Pathway ◽  
Genes Encoding ◽  
Microbial System ◽  
Rate Limiting

ABSTRACT A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.

scholarly journals Characterization of the argA Gene Required for Arginine Biosynthesis and Syringomycin Production by Pseudomonas syringae pv. syringae

2003 ◽  
Vol 69 (12) ◽  
pp. 7273-7280 ◽  
Author(s):  
Shi-En Lu ◽  
Jonathan D. Soule ◽  
Dennis C. Gross
Keyword(s):  
Pseudomonas Syringae ◽  
Culture Media ◽  
Toxin Production ◽  
Arginine Biosynthesis ◽  
Reading Frame ◽  
High Conservation ◽  
Plant Pathogenicity ◽  
Arginine Deficiency ◽  
Dna Fragment

ABSTRACT Two types of necrosis-inducing lipodepsipeptide toxins, called syringomycin and syringopeptin, are major virulence factors of Pseudomonas syringae pv. syringae strain B301D. A previous study showed that a locus, called syrA, was required for both syringomycin production and plant pathogenicity, and the syrA locus was speculated to encode a regulator of toxin production. In this study, sequence analysis of the 8-kb genomic DNA fragment that complements the syrA phenotype revealed high conservation among a broad spectrum of fluorescent pseudomonads. The putative protein encoded by open reading frame 4 (ORF4) (1,299 bp) in the syrA locus region exhibited 85% identity to ArgA, which is involved in arginine biosynthesis in Pseudomonas aeruginosa. Growth of strain W4S2545, the syrA mutant, required supplementation of N minimal medium with arginine. Similarly, syringomycin production of syrA mutant W4S2545 was restored by the addition of arginine to culture media. Furthermore, the insertion of Tn5 in the genome of the syrA mutant W4S2545 was localized between nucleotides 146 and 147 in ORF4, and syringomycin production was complemented in trans with the wild-type DNA fragment containing intact ORF4. These results demonstrate that the syrA locus is the argA gene of P. syringae pv. syringae and that argA is directly involved in arginine biosynthesis and therefore indirectly affects syringomycin production because of arginine deficiency.

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

1999 ◽  
Vol 380 (12) ◽  
pp. 1455-1459 ◽  
Author(s):  
Eun Young Yun ◽  
Seok Woo Kang ◽  
Jae Sam Hwang ◽  
Tae Won Goo ◽  
Sang Hyun Kim ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cdna Sequence ◽  
The Self ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Defense System ◽  
Reading Frame ◽  
E Coli ◽  
Self Defense

Abstract We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

scholarly journals Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer

2002 ◽  
Vol 184 (7) ◽  
pp. 1932-1939 ◽  
Author(s):  
Karen C. Crasta ◽  
Kim-Lee Chua ◽  
Sumathi Subramaniam ◽  
Joachim Frey ◽  
Hilda Loh ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromosomal Dna ◽  
Pcr Cloning ◽  
Reading Frame ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Riemerella Anatipestifer ◽  
Hydrolysis Of

ABSTRACT Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam, from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp BamHI-BstXI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.

scholarly journals Knot Formation Caused by Pseudomonas syringae subsp. savastanoi on Olive Plants Is hrp-Dependent

2004 ◽  
Vol 94 (5) ◽  
pp. 484-489 ◽  
Author(s):  
A. Sisto ◽  
M. G. Cipriani ◽  
M. Morea
Keyword(s):  
Sequence Analysis ◽  
Pseudomonas Syringae ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Disease Symptoms ◽  
Phytopathogenic Bacteria ◽  
Reading Frame ◽  
Nonhost Plant ◽  
Induced Mutant

The virulence of Pseudomonas syringae subsp. savastanoi, which causes hyperplastic symptoms (knots) on olive plants, is associated with secreted phytohormones. We identified a Tn5-induced mutant of P. syringae subsp. savastanoi that did not cause disease symptoms on olive plants although it was still able to produce phytohormones. In addition, the mutant failed to elicit a hypersensitive response in a nonhost plant. Molecular characterization of the mutant revealed that a single Tn5 insertion occurred within an open reading frame encoding a protein 92% identical to the HrcC protein of P. syringae pv. syringae. Moreover, sequence analysis revealed that the gene encoding the HrcC protein in P. syringae subsp. savastanoi was part of an operon that included five genes arranged as in other phytopathogenic bacteria. These results imply that hrp/hrc genes are functional in P. syringae subsp. savastanoi and that they play a key role in the pathogenicity of this plant pathogen.

Rapid Gene Cloning, Overexpression and Characterization of a Thermophilic Catalase in E. coli

2011 ◽  
Vol 365 ◽  
pp. 367-374 ◽  
Author(s):  
Hui Luo ◽  
Yao Zhou ◽  
Yan Hong Chang ◽  
Lian Xiong ◽  
Lin Zhi Liu
Keyword(s):  
Prokaryotic Expression ◽  
Bacillus Stearothermophilus ◽  
Base Pairs ◽  
Reading Frame ◽  
E Coli ◽  
Metal Affinity ◽  
Culture Optimization ◽  
Dna Mixture ◽  
Encoding Gene

A thermophilic catalase-encoding gene was rapidly obtained by means a PCR-based protocol with the genomic DNA mixture from compost culture as the template. The open reading frame of this gene is composed of 2208 base pairs, sharing 92.5% homology with the reported Bacillus stearothermophilus gene (NCBI genbank accession No. AB020234. 1). A prokaryotic expression plasmid pET-CATHis was constructed for the gene expression, and two recombinant E. coli, BL21(DE3)/pET-CATHis and BL21(DE3)pLysS/pET-CATHis were finally obtained. After culture optimization, the highest activities for these two strains in shaking flask culture were 74.3 U/ml and 1055.3 U/ml, respectively. The 6 His-tagged recombinant catalase was then purified by using immobilized metal affinity chromatography, and the properties of the purified protein were finally characterized.

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