scholarly journals Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

2020 ◽  
Author(s):  
Beltrán Ortolá ◽  
Teresa Cordero ◽  
Xu Hu ◽  
José-Antonio Daròs
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Rna Stability ◽  
Diabrotica Virgifera Virgifera ◽  
Septate Junction ◽  
Circular Rnas ◽  
Double Stranded Rna ◽  
E Coli ◽  
Group I ◽  
Insecticide Activity

ABSTRACTRNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

scholarly journals Engineering a Circular Riboregulator in Escherichia coli

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
William Rostain ◽  
Shensi Shen ◽  
Teresa Cordero ◽  
Guillermo Rodrigo ◽  
Alfonso Jaramillo
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Messenger Rna ◽  
Noncoding Rna ◽  
Circular Rna ◽  
Circular Rnas ◽  
Fluorescent Reporter ◽  
Small Noncoding Rna ◽  
E Coli ◽  
Group I

RNAs of different shapes and sizes, natural or synthetic, can regulate gene expression in prokaryotes and eukaryotes. Circular RNAs have recently appeared to be more widespread than previously thought, but their role in prokaryotes remains elusive. Here, by inserting a riboregulatory sequence within a group I permuted intron-exon ribozyme, we created a small noncoding RNA that self-splices to produce a circular riboregulator in Escherichia coli. We showed that the resulting riboregulator can trans-activate gene expression by interacting with a cis-repressed messenger RNA. We characterized the system with a fluorescent reporter and with an antibiotic resistance marker, and we modeled this novel posttranscriptional mechanism. This first reported example of a circular RNA regulating gene expression in E. coli adds to an increasing repertoire of RNA synthetic biology parts, and it highlights that topological molecules can play a role in the case of prokaryotic regulation.

scholarly journals Iron Acquisition of Urinary Tract Infection Escherichia coli Involves Pathogenicity in Caenorhabditis elegans

2021 ◽  
Vol 9 (2) ◽  
pp. 310
Author(s):  
Masayuki Hashimoto ◽  
Yi-Fen Ma ◽  
Sin-Tian Wang ◽  
Chang-Shi Chen ◽  
Ching-Hao Teng
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Caenorhabditis Elegans ◽  
Large Scale ◽  
Iron Acquisition ◽  
Infection Model ◽  
High Throughput Analysis ◽  
E Coli ◽  
C Elegans ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.

scholarly journals Dinucleoside polyphosphates act as 5’-RNA caps in Escherichia coli

2019 ◽  
Author(s):  
Oldřich Hudeček ◽  
Roberto Benoni ◽  
Martin Culka ◽  
Martin Hubálek ◽  
Lubomír Rulíšek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Stability ◽  
E Coli ◽  
Chemical Structures ◽  
Additional Layer ◽  
Similar Chemical ◽  
Decapping Enzyme ◽  
Almost All ◽  
Dinucleoside Polyphosphates ◽  
Rna Caps

Dinucleoside polyphosphates (NpnNs), discovered more than 50 years ago,1 are pleiotropic molecules present in almost all types of cells.2 It has been shown that their intracellular concentration can under stress conditions increase from the µM to mM range 2,3. However, the cellular roles and mechanisms of action of NpnNs are still speculative4,5. They have never been considered as part of the RNA, even though they have similar chemical structures as already known RNA caps, such as the nicotinamide adenine dinucleotide (NAD)6-8 and 7-methylguanylate cap9. Here, we show that both methylated and non-methylated Npn Ns serve as RNA caps in Escherichia coli (E. coli). NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAP) and efficiently initiate transcription. Further, we demonstrate that the E. coli decapping enzyme RNA 5’ pyrophosphohydrolase (RppH) is able to remove the NpnNs-cap from the RNA. RppH was, however, not able to cleave the methylated forms of the NpnN-caps, suggesting that the methylation adds an additional layer to the RNA stability regulation. Our work introduces an original perspective on the chemical structure of RNA in prokaryotes and the function of RNA caps. This is the first evidence that small molecules like NpnNs can act in cells via their incorporation into RNA and influence the cellular metabolism.

scholarly journals Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing

1993 ◽  
Vol 294 (2) ◽  
pp. 521-527 ◽  
Author(s):  
S A Morris ◽  
W P Revill ◽  
J Staunton ◽  
P F Leadlay
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Acyl Carrier Protein ◽  
Saccharopolyspora Erythraea ◽  
Carrier Protein ◽  
Freezing And Thawing ◽  
Acyl Carrier Proteins ◽  
Expression Plasmid ◽  
E Coli ◽  
Protein Dimer

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

scholarly journals Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments

2010 ◽  
Vol 76 (17) ◽  
pp. 5659-5668 ◽  
Author(s):  
G. M. Luna ◽  
C. Vignaroli ◽  
C. Rinaldi ◽  
A. Pusceddu ◽  
L. Nicoletti ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Marine Sediments ◽  
Large Scale ◽  
Microbiological Quality ◽  
Genotypic Diversity ◽  
Coastal Sediments ◽  
Fecal Coliforms ◽  
Biochemical Tests ◽  
Term Survival ◽  
E Coli

ABSTRACT Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx 1, stx 2, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.

Growth of Escherichia coli O157:H7 and Listeria monocytogenes in Packaged Fresh-Cut Romaine Mix at Fluctuating Temperatures during Commercial Transport, Retail Storage, and Display

2014 ◽  
Vol 77 (2) ◽  
pp. 197-206 ◽  
Author(s):  
WENTING ZENG ◽  
KEITH VORST ◽  
WYATT BROWN ◽  
BRADLEY P. MARKS ◽  
SANGHYUP JEONG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Large Scale ◽  
Microbial Growth ◽  
Escherichia Coli O157 ◽  
Temperature Profiles ◽  
E Coli ◽  
Leafy Greens ◽  
Retail Display ◽  
Fluctuating Temperatures

Temperature abuse during commercial transport and retail sale of leafy greens negatively impacts both microbial safety and product quality. Consequently, the effect of fluctuating temperatures on Escherichia coli O157:H7 and Listeria monocytogenes growth in commercially-bagged salad greens was assessed during transport, retail storage, and display. Over a 16-month period, a series of time-temperature profiles for bagged salads were obtained from five transportation routes covering four geographic regions (432 profiles), as well as during retail storage (4,867 profiles) and display (3,799 profiles). Five different time-temperature profiles collected during 2 to 3 days of transport, 1 and 3 days of retail storage, and 3 days of retail display were then duplicated in a programmable incubator to assess E. coli O157:H7 and L. monocytogenes growth in commercial bags of romaine lettuce mix. Microbial growth predictions using the Koseki-Isobe and McKellar-Delaquis models were validated by comparing the root mean square error (RMSE), bias, and the acceptable prediction zone between the laboratory growth data and model predictions. Monte Carlo simulations were performed to calculate the probability distribution of microbial growth from 8,122,127,472 scenarios during transport, cold room storage, and retail display. Using inoculated bags of retail salad, E. coli O157:H7 and L. monocytogenes populations increased a maximum of 3.1 and 3.0 log CFU/g at retail storage. Both models yielded acceptable RMSEs and biases within the acceptable prediction zone for E. coli O157:H7. Based on the simulation, both pathogens generally increased <2 log CFU/g during transport, storage, and display. However, retail storage duration can significantly impact pathogen growth. This large-scale U.S. study—the first using commercial time/temperature profiles to assess the microbial risk of leafy greens—should be useful in filling some of the data gaps in current risk assessments for leafy greens.

scholarly journals Diversity of Escherichia coli from the enteric microbiota in patients with Escherichia coli bacteraemia

2019 ◽  
Author(s):  
Mia Mosavie ◽  
Oliver Blandy ◽  
Elita Jauneikaite ◽  
Isabel Caldas ◽  
Matthew J. Ellington ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Bloodstream Infections ◽  
Sample Type ◽  
E Coli ◽  
Rapd Pcr ◽  
Hospitalised Patients ◽  
Rapd Pattern ◽  
Chromogenic Agar ◽  
The Relationship

ABSTRACTObjectiveThe increase in Escherichia coli bloodstream infections mandates better characterisation of the relationship between commensal and invasive isolates. This study established a simple approach to characterize diversity of E. coli in the gut reservoir from patients with either E. coli bacteraemia, other Gram-negative bacteraemia, or patients without bacteraemia not receiving antibiotics. Stool or rectal swabs from patients in the three groups were cultured on selective chromogenic agar. Genetic diversity of E. coli in gut microbiota was estimated by RAPD-PCR.ResultsEnteric samples from E. coli bacteraemia patients yielded a median of one E. coli RAPD pattern (range 1-4) compared with two (range 1-5) from groups without E. coli bacteraemia. Of relevance to large-scale clinical studies, observed diversity of E. coli among E. coli bacteraemia patients was not significantly altered by sample type (rectal swab or stool), nor by increasing the colonies tested beyond ten. Overall, hospitalised patients demonstrated an apparently limited diversity of E. coli in the enteric microbiota and this was further reduced in those with E. coli bacteraemia. The reduced diversity of E. coli within the gut during E. coli bacteraemia raises the possibility that dominant strains may outcompete other lineages in patients with bloodstream infection.

scholarly journals Media Optimization for Production of Recombinant Carrier Protein (CRM197) in Escherichia coli

2021 ◽  
Vol 13 (1) ◽  
pp. 283-297
Author(s):  
S. Shukla ◽  
D. Mishra
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Luria Bertani ◽  
Lag Phase ◽  
Carrier Protein ◽  
Scale Production ◽  
E Coli ◽  
Defined Media

Since the advent of vaccines, the mankind has benefited from the same and has been able to curb the mortality rate around the globe. Amongst different types of available vaccines, polysaccharide based vaccines are very widely used against various infectious diseases. The polysaccharide vaccines need to be conjugated with a carrier protein to make the vaccine more immunogenic. Recombinant Escherichia coli cells are the organism of choice for large scale production of a carrier protein because of its widely studied scientific aspects. In the present study, for proof of concept, the recombinant E. coli cells were cultured in Luria-Bertani media to check the expression of rCRM197. At 80L scale, it was observed that when recombinant E. coli cells were grown in a chemically defined media, it resulted in inconsistent growth and a long lag phase. When the defined media was supplemented with yeast extract, the lag phase of the culture was substantially reduced and the maximum growth of the culture was achieved. Protein expression was checked using SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) and Western blot technique. The optimized media resulted in a robust fermentation process to achieve high cell density and maximum biomass for the production of recombinant protein.

scholarly journals Growth of cell wall-defective variants of Escherichia coli: comparison of aerobic and anaerobic induction frequencies

1977 ◽  
Vol 6 (2) ◽  
pp. 166-171
Author(s):  
T W Huber ◽  
A W Brinkley
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
High Frequency ◽  
Anaerobic Incubation ◽  
Anaerobic Conditions ◽  
Group Iii ◽  
E Coli ◽  
Induction Frequency ◽  
Group I ◽  
Aerobic And Anaerobic

A method for quantitating the conversion of Escherichia coli to colony-forming, cell wall-defective (CWD) bacteria has been developed. The induction frequency, i.e., the percentage of the population recovered as CWD colonies was determined for 20 randomly selected clinical isolates of E. coli under aerobic and anaerobic incubation conditions. Penicillin (1,000 U/ML) was the inducing agent. The 20 strains segregated into three groups. Group I organisms produced CWD colonies with high frequency both aerobically and anaerobically. Grout II organisms showed a much higher induction frequency anaerobically than aerobically. Group III organisms were poor inducers. Thirty percent of the strains were group I, 50% were group II, and 20% were group III organisms. These data indicate that anaerobic conditions enhance the induction and growth of CWD E. coli in the research laboratory and suggest that anaerobic incubation may be important in recovery of medically significant CWD bacteria.

scholarly journals PENGARUH PEMBERIAN Lactobacillus TERHADAP GAMBARAN MIKROSKOPIS MUKOSA USUS HALUS TIKUS WISTAR (Rattus norvegicus) YANG DIINFEKSI DENGAN Escherichia coli

2013 ◽  
Vol 1 (2) ◽  
Author(s):  
Steviany Towoliu
Keyword(s):  
Escherichia Coli ◽  
Wistar Rats ◽  
Pathogenic Bacteria ◽  
Control Group ◽  
Group Iii ◽  
Treatment Groups ◽  
E Coli ◽  
Group 4 ◽  
Group I ◽  
Microscopic Features

Abstract: E. coli is part of the normal flora of the human and animal intestine and is commonly non pathogenic. However, one of the serotypes of this bacteria, which is enteropathogenic E. coli (EPEC), can cause primary infection on the intestine such diarrhea.  The growth of pathogenic bacteria in diarrheal patients can be inhibited by Lactobacillus.  Lactobacillus can function as probiotic which can affect the immune system of the digestive canals.  In addition, Lactobacillus also produce mucus  which can act as barrier to the pathogens.  The objective of this study was to reveal the effects of the administration of Lactobacillus on the microscopic features of the mucosa of the intestine of wistar rats infected by Escherichia coli.  This study was a laboratory experimental research employing 16 wistar rats divided into the control group (4 rats) and three treatment groups (12 rats) consisting of 4 rats each.  Results showed that; in group I the histological features were normal, in group II part of the epithelium of the mucosa showed erosion, dilatation of the capillary vessels, and many lymphosites were observed, in group III the epithelium of the mucosa was intact and the number of lymphosites was liitle, and in group IV the surface of the epithelium was intact, the presence of cell regeneration indicated by the increase number of goblet cells and a small number of  lymphocyes. Conclusions: The administration of after infection by E. coli has benefial effects indicated by the improvement of epithelial cells and the absence of denudation of the epithelium of the intestine. Keywords: E.coli, Lactobacillus, intestinal mucosa.   Abstrak: E.coli merupakan flora normal usus halus manusia dan hewan umumnya tidak menyebabkan penyakit.  Namun salah satu serotipe E.coli yaitu E.coli Enteropatogenik (EPEK) bersifat patogen dan dapat menyebabkan infeksi primer pada usus misalnya diare.  Pertumbuhan bakteri patogen pada pasien diare dapat dihambat oleh Lactobacillus. Lactobacillus merupakan probiotik yang akan mempengaruhi sistem imun saluran cerna serta memproduksi mukus yang berfungsi sebagai penghalang saluran cerna terhadap bakteri patogen.  Penelitian ini bertujuan untuk melihat efek pemberian Lactobacillus terhadap gambaran mikroskopis mukosa usus halus tikus wistar yang diinfeksi dengan Escherichia coli.  Penelitian ini merupakan penelitian eksperimental laboratorik.  Subjek penelitian terdiri dari 16 ekor tikur wistar yang dibagi dalam kelompok kontrol (4 ekor) dan kelompok  perlakuan (12 ekor) dibagi dalam 3 kelompok masing-masing 4 ekor.  Hasil penelitian kelompok I dengan gambaran histologik jaringan usus normal, kelompok II sebagian epitel mukosa usus halus terlihat erosi,  ada pelebaran pembuluh darah kapiler, dan banyak limfosit, kelompok III dengan permukaan epitel mukosa yang utuh dan jumlah limfosit sedikit, dan kelompok IV dengan permukaan epitel yang utuh, adanya regenerasi sel ditandai dengan bertambahnya sel goblet, dan sedikit limfosit.  Simpulan: Pemberian Lactobacillus setelah diberikan E.coli memberi efek yang baik berupa perbaikan sel epitel permukaan dan tidak terlihat denudasi epitel permukaan usus halus. Kata Kunci: E.coli, Lactobacillus, mukosa usus halus.

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