Expression of chIL-6 gene encoding chicken interleukin-6 in E. coli BL21(DE3)

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

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The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

Microbiology ◽

10.1099/mic.0.27476-0 ◽

2005 ◽

Vol 151 (5) ◽

pp. 1421-1431 ◽

Cited By ~ 34

Author(s):

Patrice Bruscella ◽

Laure Cassagnaud ◽

Jeanine Ratouchniak ◽

Gaël Brasseur ◽

Elisabeth Lojou ◽

...

Keyword(s):

Escherichia Coli ◽

Acidithiobacillus Ferrooxidans ◽

Biochemical Properties ◽

Gene Encoding ◽

Iron Sulfur Cluster ◽

E Coli ◽

Iron Sulfur ◽

Sulfur Protein ◽

Tat System ◽

Micro Organisms

The gene encoding a putative high-potential iron–sulfur protein (HiPIP) from the strictly acidophilic and chemolithoautotrophic Acidithiobacillus ferrooxidans ATCC 33020 has been cloned and sequenced. This potential HiPIP was overproduced in the periplasm of the neutrophile and heterotroph Escherichia coli. As shown by optical and EPR spectra and by electrochemical studies, the recombinant protein has all the biochemical properties of a HiPIP, indicating that the iron–sulfur cluster was correctly inserted. Translocation of this protein in the periplasm of E. coli was not detected in a ΔtatC mutant, indicating that it is dependent on the Tat system. The genetic organization of the iro locus in strains ATCC 23270 and ATCC 33020 is different from that found in strains Fe-1 and BRGM. Indeed, in A. ferrooxidans ATCC 33020 and ATCC 23270 (the type strain), iro was not located downstream from purA but was instead downstream from petC2, encoding cytochrome c 1 from the second A. ferrooxidans cytochrome bc 1 complex. These findings underline the genotypic heterogeneity within the A. ferrooxidans species. The results suggest that Iro transfers electrons from a cytochrome bc 1 complex to a terminal oxidase, as proposed for the HiPIP in photosynthetic bacteria.

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A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

Applied and Environmental Microbiology ◽

10.1128/aem.66.9.3945-3950.2000 ◽

2000 ◽

Vol 66 (9) ◽

pp. 3945-3950 ◽

Cited By ~ 6

Author(s):

Harald J. Ruijssenaars ◽

Sybe Hartmans ◽

Jan C. Verdoes

Keyword(s):

Signal Sequence ◽

Fold Increase ◽

Side Chain ◽

Cloning And Sequencing ◽

Gene Encoding ◽

E Coli ◽

Mature Enzyme ◽

Locust Bean ◽

Encoding Gene ◽

Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

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Phosphorylation of Nucleosides by the Mutated Acid Phosphatase from Morganella morganii

Applied and Environmental Microbiology ◽

10.1128/aem.66.7.2811-2816.2000 ◽

2000 ◽

Vol 66 (7) ◽

pp. 2811-2816 ◽

Cited By ~ 44

Author(s):

Yasuhiro Mihara ◽

Takashi Utagawa ◽

Hideaki Yamada ◽

Yasuhisa Asano

Keyword(s):

Acid Phosphatase ◽

Random Mutagenesis ◽

Reaction Yield ◽

Mutant Library ◽

Morganella Morganii ◽

Gene Encoding ◽

E Coli ◽

Acid Phosphatase Gene ◽

Phosphate Source

ABSTRACT A novel nucleoside phosphorylation process using the food additive pyrophosphate as the phosphate source was investigated. TheMorganella morganii gene encoding a selective nucleoside pyrophosphate phosphotransferase was cloned. It was identical to theM. morganii PhoC acid phosphatase gene. Sequential in vitro random mutagenesis was performed on the gene by error-prone PCR to construct a mutant library. The mutant library was introduced intoEscherichia coli, and the transformants were screened for the production of 5′-IMP. One mutated acid phosphatase with an increased phosphotransferase reaction yield was obtained. With E. coli overproducing the mutated acid phosphatase, 101 g of 5′-IMP per liter (192 mM) was synthesized from inosine in an 88% molar yield. This improvement was achieved with two mutations, Gly to Asp at position 92 and Ile to Thr at position 171. A decreasedKm value for inosine was responsible for the increased productivity.

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Synthesis of the novel transporter YdhC, is regulated by the YdhB transcription factor controlling adenosine and adenine uptake

10.1101/2020.05.03.074617 ◽

2020 ◽

Cited By ~ 1

Author(s):

Irina A. Rodionova ◽

Ye Gao ◽

Anand Sastry ◽

Reo Yoo ◽

Dmitry A. Rodionov ◽

...

Keyword(s):

Nitrogen Sources ◽

Gene Arrangement ◽

The Novel ◽

Rna Seq ◽

Gene Encoding ◽

E Coli ◽

Binding Sequence ◽

Predicted Binding Site ◽

Lysr Family ◽

Mg1655 Strain

AbstractThe YdhB transcriptional factor, re-named here AdnB, homologous to the allantoin regulator, AllS, was shown to regulate ydhC gene expression in Escherichia coli, which is divergently transcribed from adnB, and this gene arrangement is conserved in many Protreobacteria. The predicted consensus DNA binding sequence for YdhB is also conserved in Entrobacterial genomes. RNA-seq data confirmed the activation predicted due to the binding of AdnB as shown by Chip-Exo results. Fluorescent polarization experiments revealed binding of YdhB to the predicted binding site upstream of ydhC in the presence of 0.35 mM adenine, but not in its absence. The E. coli MG1655, strain lacking the ydhB gene, showed a lower level of ydhC mRNA in cells grown in M9-glucose supplemented with 2 mM adenosine. Adenosine and adenine are products of purine metabolism and provide sources of ammonium for many organisms. They are utilized under nitrogen starvation conditions as single nitrogen sources. Deletion of either the ydhC or the ydhB gene leads to a substantially decreased growth rate for E. coli in minimal M9 medium with glycerol as the carbon source and adenosine or adenine as the single nitrogen source. The ydhC mutant showed increased resistance to Paromomycine, Sulfathiazole and Sulfamethohazole using Biolog plates. We provide evidence that YdhB, (a novel LysR family regulator) activates expression of the ydhC gene, encoding a novel adenosine/adenine transporter in E. coli. The YdhB binding consensus for different groups of Enterobacteria was predicted.

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EXPLORATION OF BROTH CHICKEN GUT AS GROWTH MEDIA OF Escherichia coli BL21 pET-Endo FOR ENDO-Β-1,4-D-XYLANASE PRODUCTION

ALCHEMY Jurnal Penelitian Kimia ◽

10.20961/alchemy.13.2.4356.191-204 ◽

2017 ◽

Vol 13 (2) ◽

pp. 191

Author(s):

Anak Agung Istri Ratnadewi ◽

Moch. Yoris Alidion ◽

Agung Budi Santoso ◽

Ika Oktavianawatia

Keyword(s):

Large Scale ◽

Incubation Temperature ◽

Specific Activity ◽

Hydrolytic Enzyme ◽

Optimal Growth ◽

Good Alternative ◽

Growth Media ◽

Gene Encoding ◽

Xylanase Production ◽

E Coli

<p>Endo-β-1,4-D-xylanase is a hydrolytic enzyme that breakdown the 1.4 chain of xylan polysaccharide. We have succes to transform the plasmid pET-Endo gene encoding endo-1,4-β-D-xylanase from Bacillus sp. originally from termites abdominal to E. coli BL21. The clone was ready for large scale of enzyme production. To reduce production cost, we look for subtitute media for the expensive Luria Berthani broth. Chicken guts broth is good alternative while rich of protein and very cheap. The content of N dissolved chicken guts broth reaches 87 % of LB broth. Growth of E. Coli BL21 in Chicken guts broth and LB broth (as control) was observed by Optical Density (OD) using spectrofotometer. Concentration of glucose added in broth and incubation temperature was varied. The result showed that optimal growth was in addition of 1.5 % glucose and incubated at 37 <sup>o</sup>C for 16 h. This optimal condition was used to grow E. coli BL21 pET-Endo for xylanase production. Enzyme purification was done by Ni-NTA affinity chromatography. Highest protein yield was 0.076 mg/mL obtained in 100 mM imidazole elucidation. The activity and specific activity of xylanase were estimated as 0.042 U/mL and 0.556 U/µg, respectively. The purification factor was 3.16 time and the molecular weight of enzyme was ± 30, 000 Dalton</p>

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Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.7555 ◽

2015 ◽

Vol 10 (2) ◽

Author(s):

Wayan T. Artama ◽

Yulia Sari ◽

Didik Tulus Subekti ◽

Soenarwan Hery Poerwanto ◽

Jarot Subandono

Keyword(s):

Toxoplasma Gondii ◽

Recombinant Plasmid ◽

Messenger Rna ◽

Secretory Protein ◽

Gene Encoding ◽

Isolation System ◽

Complementary Dna ◽

E Coli ◽

Local Isolate ◽

Cell Target

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2

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PCR and Blood Culture for Detection ofEscherichia coli Bacteremia in Rats

Journal of Clinical Microbiology ◽

10.1128/jcm.37.8.2479-2482.1999 ◽

1999 ◽

Vol 37 (8) ◽

pp. 2479-2482 ◽

Cited By ~ 55

Author(s):

Alexandra Heininger ◽

Marlies Binder ◽

Sibylle Schmidt ◽

Klaus Unertl ◽

Konrad Botzenhart ◽

...

Keyword(s):

Blood Culture ◽

Detection Rate ◽

False Negative ◽

Diagnostic Procedures ◽

Microbial Pathogens ◽

Gene Encoding ◽

E Coli ◽

Negative Results ◽

False Negative Results ◽

Male Wistar Rats

Critically ill patients often develop symptoms of sepsis and therefore require microbiological tests for bacteremia that use conventional blood culture (BC) techniques. However, since these patients frequently receive early empirical antibiotic therapy before diagnostic procedures are completed, examination by BC can return false-negative results. We therefore hypothesized that PCR could improve the rate of detection of microbial pathogens over that of BC. To test this hypothesis, male Wistar rats were challenged intravenously with 106 CFU of Escherichia coli. Blood was then taken at several time points for detection of E. coliby BC and by PCR with E. coli-specific primers derived from the uidA gene, encoding β-glucuronidase. In further experiments, cefotaxime (100 or 50 mg/kg of body weight) was administered intravenously to rats 10 min after E. colichallenge. Without this chemotherapy, the E. coli detection rate decreased at 15 min and at 210 min after challenge from 100% to 62% of the animals with PCR and from 100% to 54% of the animals with BC (P, >0.05). Chemotherapy decreased the E. coli detection rate at 25 min and at 55 min after challenge from 100% to 50% with PCR and from 100% to 0% with BC (P, <0.05). Thus, at clinically relevant serum antibiotic levels, PCR affords a significantly higher detection rate than BC in this rat model. The results suggest that PCR could be a useful adjunct tool supplementing conventional BC techniques in diagnosing bacteremia.

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Functional characterization of the Haemophilus influenzae 4.5S RNA

Canadian Journal of Microbiology ◽

10.1139/w97-124 ◽

1998 ◽

Vol 44 (1) ◽

pp. 91-94

Author(s):

G Scott Jenkins ◽

Mark S Chandler ◽

Pamela S Fink

Keyword(s):

Haemophilus Influenzae ◽

Functional Characterization ◽

Coli Strain ◽

Northern Analysis ◽

Gene Encoding ◽

E Coli ◽

Functional Homolog ◽

Polymerase Chain ◽

4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

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