scholarly journals Cloning and Characterization of a Periplasmic Nuclease of Vibrio vulnificus and Its Role in Preventing Uptake of Foreign DNA

2001 ◽  
Vol 67 (1) ◽  
pp. 82-88 ◽  
Author(s):  
Shi-I Wu ◽  
Shi-Kan Lo ◽  
Chung-Ping Shao ◽  
Hsing-Wen Tsai ◽  
Lien-I Hor
Keyword(s):  
Parent Strain ◽  
Vibrio Vulnificus ◽  
Signal Sequence ◽  
Reduced Form ◽  
Wound Infections ◽  
Reading Frame ◽  
E Coli ◽  
Dna And Rna ◽  
Foreign Dna

ABSTRACT We have cloned a nuclease gene, vvn, from Vibrio vulnificus, an estuarine bacterium that causes wound infections and septicemia in humans and eels. The gene contained a 696-bp open reading frame encoding 232 amino acids (aa), including a signal sequence of 18 aa. The deduced amino acid sequence of the mature nuclease predicted a molecular mass of 25 kDa, which was confirmed by vital stain, and a pI of 8.6. Vvn was produced in the periplasm of either V. vulnificus or recombinant Escherichia coli strains and was active in the oxidized (but not the reduced) form. This nuclease was able to digest DNA and RNA, with differential thermostability in DNase and RNase activities. Expression of Vvn inE. coli DH5α reduced the frequencies of transformation with the divalent ion-treated cells and electroporation by about 6 and 2 logs, respectively. In addition, the transformation frequency of a Vvn-deficient V. vulnificus mutant (ND) was 10-fold higher than that of the parent strain. These data suggested that Vvn may be involved in preventing uptake of foreign DNA by transformation. However, Vvn expressed in the recipients had little effect on the conjugation frequency in either E. coli or V. vulnificus. Some other DNase(s) may be present in the periplasm and responsible for a residual DNase activity, which was about one-fourth of that of the parent strain, detected in the ND mutant. We also demonstrated that Vvn was not required for the virulence ofV. vulnificus mice.

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 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 Regulation of Metalloprotease Gene Expression in Vibrio vulnificus by a Vibrio harveyi LuxR Homologue

2001 ◽  
Vol 183 (4) ◽  
pp. 1369-1375 ◽  
Author(s):  
Chung-Ping Shao ◽  
Lien-I Hor
Keyword(s):  
Parent Strain ◽  
Vibrio Vulnificus ◽  
Vibrio Harveyi ◽  
Negative Regulation ◽  
Open Reading Frame ◽  
Protease Gene ◽  
Reading Frame ◽  
Allelic Exchange ◽  
Exchange Technique

ABSTRACT Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyiLuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene ofV. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificusvirulence in mice.

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 Rapid production and characterization of antimicrobial colicins using Escherichia coli-based cell-free protein synthesis

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Xing Jin ◽  
Weston Kightlinger ◽  
Yong-Chan Kwon ◽  
Seok Hoon Hong
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Host Cells ◽  
Persister Cells ◽  
Free Protein ◽  
E Coli ◽  
Dna And Rna ◽  
Rapid Production ◽  
Cell Free Protein Synthesis

Abstract Colicins are antimicrobial proteins produced by Escherichia coli, which, upon secretion from the host, kill non-host E. coli strains by forming pores in the inner membrane and degrading internal cellular components such as DNA and RNA. Due to their unique cell-killing activities, colicins are considered viable alternatives to conventional antibiotics. Recombinant production of colicins requires co-production of immunity proteins to protect host cells; otherwise, the colicins are lethal to the host. In this study, we used cell-free protein synthesis (CFPS) to produce active colicins without the need for protein purification and co-production of immunity proteins. Cell-free synthesized colicins were active in killing model E. coli cells with different modes of cytotoxicity. Pore-forming colicins E1 and nuclease colicin E2 killed actively growing cells in a nutrient-rich medium, but the cytotoxicity of colicin Ia was low compared to E1 and E2. Moreover, colicin E1 effectively killed cells in a nutrient-free solution, while the activity of E2 was decreased compared to nutrient-rich conditions. Both colicins E1 and E2 decreased the level of persister cells (metabolically dormant cell populations that are insensitive to antibiotics) by up to six orders of magnitude compared to that of the rifampin pretreated persister cells. This study finds that colicins can eradicate non-growing cells including persisters, and that CFPS is a promising platform for rapid production and characterization of toxic proteins.

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.

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.

scholarly journals Genetic Characterization of Vibrio vulnificus Strains from Tilapia Aquaculture in Bangladesh

2010 ◽  
Vol 76 (14) ◽  
pp. 4890-4895 ◽  
Author(s):  
Zahid H. Mahmud ◽  
Anita C. Wright ◽  
Shankar C. Mandal ◽  
Jianli Dai ◽  
Melissa K. Jones ◽  
...  
Keyword(s):  
Vibrio Vulnificus ◽  
Genetic Characterization ◽  
Phylogenetic Analyses ◽  
Wound Infections ◽  
Freshwater Aquaculture

ABSTRACT Outbreaks of Vibrio vulnificus wound infections in Israel were previously attributed to tilapia aquaculture. In this study, V. vulnificus was frequently isolated from coastal but not freshwater aquaculture in Bangladesh. Phylogenetic analyses showed that strains from Bangladesh differed remarkably from isolates commonly recovered elsewhere from fish or oysters and were more closely related to strains of clinical origin.

scholarly journals Characterization of an Endo-β-1,6-Galactanase from Streptomyces avermitilis NBRC14893

2008 ◽  
Vol 74 (8) ◽  
pp. 2379-2383 ◽  
Author(s):  
Hitomi Ichinose ◽  
Toshihisa Kotake ◽  
Yoichi Tsumuraya ◽  
Satoshi Kaneko
Keyword(s):  
Signal Sequence ◽  
Recombinant Enzyme ◽  
Streptomyces Avermitilis ◽  
Glycoside Hydrolase Family ◽  
Reading Frame ◽  
Secretion Signal ◽  
Acid Protein ◽  
Hydrolysis Of ◽  
Amino Acid Protein

ABSTRACT The putative endo-β-1,6-galactanase gene from Streptomyces avermitilis was cloned and expressed in Escherichia coli, and the enzymatic properties of the recombinant enzyme were characterized. The gene consisted of a 1,476-bp open reading frame and encoded a 491-amino-acid protein, comprising an N-terminal secretion signal sequence and glycoside hydrolase family 5 catalytic module. The recombinant enzyme, Sa1,6Gal5A, catalyzed the hydrolysis of β-1,6-linked galactosyl linkages of oligosaccharides and polysaccharides. The enzyme produced galactose and a range of β-1,6-linked galacto-oligosaccharides, predominantly β-1,6-galactobiose, from β-1,6-galactan chains. There was a synergistic effect between the enzyme and Sa1,3Gal43A in degrading tomato arabinogalactan proteins. These results suggest that Sa1,6Gal5A is the first identified endo-β-1,6-galactanase from a prokaryote.

scholarly journals Degradation of Substituted Phenylurea Herbicides byArthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene,puhA

2001 ◽  
Vol 67 (5) ◽  
pp. 2270-2275 ◽  
Author(s):  
Gillian A. Turnbull ◽  
Margaret Ousley ◽  
Allan Walker ◽  
Eve Shaw ◽  
J. Alun W. Morgan
Keyword(s):  
Protein Sequence ◽  
Sole Carbon Source ◽  
Selective Advantage ◽  
Arthrobacter Globiformis ◽  
Plasmid Curing ◽  
Phenylurea Herbicides ◽  
Reading Frame ◽  
E Coli ◽  
Degrading Bacteria

ABSTRACT Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kbEcoRI fragment of plasmid pHRIM620 was expressed inEscherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. colicoded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhAgene).

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