scholarly journals High efficiency generalized transduction in Escherichia coli O157:H7

F1000Research ◽  
2013 ◽  
Vol 2 ◽  
pp. 7 ◽  
Author(s):  
Martin G Marinus ◽  
Anthony R Poteete
Keyword(s):  
High Efficiency ◽  
Genetic Manipulation ◽  
Point Mutations ◽  
Ultra Violet ◽  
E Coli ◽  
Recombination System ◽  
Kanamycin Cassette ◽  
Uremic Syndrome ◽  
K 12 ◽  
Generalized Transduction

Genetic manipulation in enterohemorrhagicE. coliO157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changesinter aliainto the genome. Bacteriophage 933W is a prophage inE. coliO157:H7 strain EDL933, which encodes the genes (stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced thestx2ABgenes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers inE. coliK-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in bothE. coliK-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

scholarly journals Leveraging modern DNA assembly techniques for rapid, markerless genome modification

2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Ilya B Tikh ◽  
James C Samuelson
Keyword(s):  
High Efficiency ◽  
Point Mutations ◽  
Host Cells ◽  
Dna Assembly ◽  
E Coli ◽  
Recombination Efficiency ◽  
Genome Modification ◽  
Counter Selection ◽  
K 12 ◽  
Polymerase Chain Reaction Primers

Abstract The ability to alter the genomic material of a prokaryotic cell is necessary for experiments designed to define the biology of the organism. In addition, the production of biomolecules may be significantly improved by application of engineered prokaryotic host cells. Furthermore, in the age of synthetic biology, speed and efficiency are key factors when choosing a method for genome alteration. To address these needs, we have developed a method for modification of the Escherichia coli genome named FAST-GE for Fast Assembly-mediated Scarless Targeted Genome Editing. Traditional cloning steps such as plasmid transformation, propagation and isolation were eliminated. Instead, we developed a DNA assembly-based approach for generating scarless strain modifications, which may include point mutations, deletions and gene replacements, within 48 h after the receipt of polymerase chain reaction primers. The protocol uses established, but optimized, genome modification components such as I-SceI endonuclease to improve recombination efficiency and SacB as a counter-selection mechanism. All DNA-encoded components are assembled into a single allele-exchange vector named pDEL. We were able to rapidly modify the genomes of both E. coli B and K-12 strains with high efficiency. In principle, the method may be applied to other prokaryotic organisms capable of circular dsDNA uptake and homologous recombination.

Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress

1988 ◽  
Vol 34 (10) ◽  
pp. 1171-1176 ◽  
Author(s):  
Herb E. Schellhorn ◽  
Hosni M. Hassan
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Oxidant Stress ◽  
Relative Importance ◽  
E Coli ◽  
Redox Active ◽  
Chemical Oxidants ◽  
Dependent Growth ◽  
K 12 ◽  
Generalized Transduction

In Escherichia coli, the coordinate action of two antioxidant enzymes, superoxide dismutase and hydroperoxidase (catalase), protect the cell from the deleterious effects of oxyradicals generated during normal aerobic respiration. To evaluate the relative importance of these two classes of enzymes, strains of E. coli deficient in superoxide dismutase and (or) hydroperoxidase were constructed by generalized transduction and their physiological responses to oxygen and oxidant stress examined. Superoxide dismutase was found to be more important than hydroperoxidase in preventing oxygen-dependent growth inhibition and mutagenesis, and in reducing sensitivity to redox-active compounds known to generate the superoxide anion. However, both types of enzymes were required for an effective defense against chemical oxidants that generate superoxide radicals and hydrogen peroxide.

scholarly journals Molecular Basis of Commensalism in the Urinary Tract: Low Virulence or Virulence Attenuation?

2007 ◽  
Vol 76 (2) ◽  
pp. 695-703 ◽  
Author(s):  
Jaroslaw Zdziarski ◽  
Catharina Svanborg ◽  
Björn Wullt ◽  
Jörg Hacker ◽  
Ulrich Dobrindt
Keyword(s):  
Urinary Tract ◽  
Molecular Basis ◽  
Virulence Genes ◽  
Asymptomatic Bacteriuria ◽  
Point Mutations ◽  
General Mechanism ◽  
Comparative Genomic ◽  
E Coli ◽  
Virulence Attenuation ◽  
K 12

ABSTRACT In some patients, Escherichia coli strains establish significant bacteriuria without causing symptoms of urinary tract infection (UTI). These asymptomatic-bacteriuria (ABU) strains have been shown to express fewer virulence factors than the uropathogenic E. coli (UPEC) strains that cause severe, symptomatic UTI. Paradoxically, ABU strains carry many typical UPEC virulence genes, and the molecular basis of their low virulence therefore remains unclear. This study examined whether ABU strains might evolve from UPEC by genome loss and virulence gene attenuation. The presence of conserved E. coli K-12 genes was examined using an E. coli K-12 strain MG1655-specific DNA array and the distribution of UPEC virulence-related genes was examined with the E. coli pathoarray. Two groups of strains could be distinguished. Several ABU strains were shown by multilocus sequence typing and by comparative genomic analyses to be related to UPEC but to have smaller genome sizes. There were significant alterations in essential virulence genes, including reductive evolution by point mutations, DNA rearrangements, and deletions. Other strains were unrelated to UPEC and lacked most of the virulence-associated genes. The results suggest that some ABU strains arise from virulent strains by attenuation of virulence genes while others are nonvirulent and resemble commensal strains. We propose that virulence attenuation might constitute a general mechanism for mucosal pathogens to evolve toward commensalism.

Transduction of Escherichia coli in soil

1988 ◽  
Vol 34 (2) ◽  
pp. 190-193 ◽  
Author(s):  
James J. Germida ◽  
George G. Khachatourians
Keyword(s):  
Escherichia Coli ◽  
Silty Clay ◽  
Bacterial Populations ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Loam Soil ◽  
K 12 ◽  
Generalized Transduction ◽  
Potential Use

Bacteriophage P1-mediated generalized transduction of Escherichia coli K-12 was assessed in nonsterile soil. Auxotrophic recipient cells (thr−leu−thi−rpsL) were incubated in a sandy and a silty clay loam soil, and the transducing phage lysates from prototrophic strains carrying transposon 10 (Tn10) in either purE or aroL regions were added. At intervals, the bacterial populations derived from the soils were plated on selective-differential media to enumerate prototrophic (thr+, leu+, or Tcr) transductants. Of 100 bacterial isolates obtained on the selective-differential media, 58 (14 thr+; 11, leu+; 33 Tcr) were confirmed E. coli transductants. The frequency of transduction in soil was ca. 10−6. These data demonstrate the potential use of bacteriophage P1 to genetically manipulate E. coli in situ.

scholarly journals Use of Inducible Feedback-ResistantN-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically EngineeredEscherichia coli K-12 Strains

1998 ◽  
Vol 64 (5) ◽  
pp. 1805-1811 ◽  
Author(s):  
B. S. Rajagopal ◽  
Joseph DePonte ◽  
Mendel Tuchman ◽  
Michael H. Malamy
Keyword(s):  
Feedback Inhibition ◽  
Expression System ◽  
Point Mutations ◽  
Normal Growth ◽  
Growth Conditions ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
E Coli ◽  
Genes Encoding ◽  
K 12

ABSTRACT The goal of this work was to construct Escherichia colistrains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. colistrains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without the E. coli carABgenes encoding carbamyl phosphate synthetase and the argIgene for ornithine transcarbamylase. The free arginine production rates of “arg-derepressed” E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)argA. The expression system with fbr argAproduced 7- to 35-fold more arginine than a system overexpressingcarAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5α strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carABand argI genes. Plasmids containing wt or fbrargA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

scholarly journals Complete Bypass of Restriction Systems for Major Staphylococcus aureus Lineages

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Ian R. Monk ◽  
Jai J. Tree ◽  
Benjamin P. Howden ◽  
Timothy P. Stinear ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Plasmid Dna ◽  
High Efficiency ◽  
Recombinant Dna ◽  
Genetic Manipulation ◽  
Bacterial Pathogen ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Adenine Methylation

ABSTRACTStaphylococcus aureusis a prominent global nosocomial and community-acquired bacterial pathogen. A strong restriction barrier presents a major hurdle for the introduction of recombinant DNA into clinical isolates ofS. aureus. Here, we describe the construction and characterization of the IMXXB series ofEscherichia colistrains that mimic the type I adenine methylation profiles ofS. aureusclonal complexes 1, 8, 30, and ST93. The IMXXB strains enable direct, high-efficiency transformation and streamlined genetic manipulation of majorS. aureuslineages.IMPORTANCEThe genetic manipulation of clinicalS. aureusisolates has been hampered due to the presence of restriction modification barriers that detect and subsequently degrade inappropriately methylated DNA. Current methods allow the introduction of plasmid DNA into a limited subset ofS. aureusstrains at high efficiency after passage of plasmid DNA through the restriction-negative, modification-proficient strain RN4220. Here, we have constructed and validated a suite ofE. colistrains that mimic the adenine methylation profiles of different clonal complexes and show high-efficiency plasmid DNA transfer. The ability to bypass RN4220 will reduce the cost and time involved for plasmid transfer intoS. aureus. The IMXXB series ofE. colistrains should expedite the process of mutant construction in diverse genetic backgrounds and allow the application of new techniques to the genetic manipulation ofS. aureus.

scholarly journals Molecular Characterization of the Locus Encoding Biosynthesis of the Lipopolysaccharide O Antigen of Escherichia coliSerotype O113

1999 ◽  
Vol 67 (11) ◽  
pp. 5930-5937 ◽  
Author(s):  
Adrienne W. Paton ◽  
James C. Paton
Keyword(s):  
Immune Responses ◽  
Gastrointestinal Disease ◽  
Cosmid Library ◽  
E Coli ◽  
O Antigen ◽  
Uremic Syndrome ◽  
Human Immune ◽  
K 12

ABSTRACT Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, eae-positive STEC strains, particularly those belonging to serogroups O157 and O111, appear to have greater virulence for humans. However, in spite of beingeae negative, STEC strains belonging to serogroup O113 have frequently been associated with cases of severe STEC disease, including hemolytic-uremic syndrome (HUS). Western blot analysis with convalescent-phase serum from a patient with HUS caused by an O113:H21 STEC strain indicated that human immune responses were directed principally against lipopolysaccharide O antigen. Accordingly, the serum was used to isolate a clone expressing O113 O antigen from a cosmid library of O113:H21 DNA constructed in E. coli K-12. Sequence analysis indicated that the O113 O-antigen biosynthesis (rfb) locus contains a cluster of nine genes which may be cotranscribed. Comparison with sequence databases identified candidate genes for four glycosyl transferases, an O-acetyl transferase, an O-unit flippase, and an O-antigen polymerase, as well as copies of galE and gnd. Two additional, separately transcribed genes downstream of the O113 rfbregion were predicted to encode enzymes involved in synthesis of activated sugar precursors, one of which (designated wbnF) was essential for O113 O-antigen synthesis, and so is clearly a part of the O113 rfb locus. Interestingly, expression of O113 O antigen by E. coli K-12 significantly increased in vitro adherence to both HEp-2 and Henle 407 cells.

scholarly journals A minimal CRISPR-Cas3 system for genome engineering

2019 ◽  
Author(s):  
Bálint Csörgő ◽  
Lina M. León ◽  
Ilea J. Chau-Ly ◽  
Alejandro Vasquez-Rifo ◽  
Joel D. Berry ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Genome Engineering ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Point Mutations ◽  
Type I ◽  
Homology Directed Repair ◽  
Large Deletions ◽  
System Type ◽  
Genome Scale

AbstractCRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

scholarly journals Identification of Site-Specific Recombination Genesint and xis of the Rhizobium Temperate Phage 16-3

1999 ◽  
Vol 181 (14) ◽  
pp. 4185-4192 ◽  
Author(s):  
Szabolcs Semsey ◽  
IstvAn Papp ◽  
Zsuzsanna Buzas ◽  
Andras Patthy ◽  
Laszlo Orosz ◽  
...  
Keyword(s):  
Dna Sequences ◽  
High Efficiency ◽  
Rhizobium Meliloti ◽  
Temperate Phage ◽  
Host Chromosome ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
Tyrosine Recombinases ◽  
Recombination System

ABSTRACT Phage 16-3 is a temperate phage of Rhizobium meliloti 41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of attB and attP. Here we report the identification of two phage-encoded genes required for recombinations at these sites: int (phage integration) and xis(prophage excision). We concluded that Int protein of phage16-3 belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the 16-3 int xis att system is not active in Escherichia coli, probably due to requirements for host factors that differ in Rhizobium meliloti and E. coli. The application of the 16-3 site-specific recombination system in biotechnology is discussed.

A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli

2021 ◽  
Vol 53 (5) ◽  
pp. 620-627
Author(s):  
Qi Li ◽  
Bingbing Sun ◽  
Jun Chen ◽  
Yiwen Zhang ◽  
Yu Jiang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genome Editing ◽  
High Efficiency ◽  
Specific Sequence ◽  
E Coli ◽  
Strain Bl21 ◽  
Sacb Gene ◽  
Ccdb Gene ◽  
K 12 ◽  
Plasmid Elimination

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.

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