scholarly journals The Escherichia coli motA Flagellar Gene as a Potential Integration Site for Large Synthetic DNA

2019 ◽  
Vol 48 (1) ◽  
pp. 81-91
Author(s):  
Chee-Hoo Yip ◽  
Orr Yarkoni ◽  
Mario Juhas ◽  
James Ajioka ◽  
Kiew-Lian Wan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Integration Site ◽  
Flagellar Gene ◽  
Synthetic Dna ◽  
Potential Integration

O6-methylguanine-DNA methyltransferase in wild-type and ada mutants of Escherichia coli

1982 ◽  
Vol 152 (1) ◽  
pp. 534-537
Author(s):  
S Mitra ◽  
B C Pal ◽  
R S Foote
Keyword(s):  
Escherichia Coli ◽  
Dna Methyltransferase ◽  
Wild Type ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Synthetic Dna ◽  
In The Wild ◽  
Low Levels ◽  
Enzyme Molecules ◽  
Dna Substrate

O(6)-Methylguanine-DNA methyltransferase is induced in Escherichia coli during growth in low levels of N-methyl-N'-nitro-N-nitrosoguanidine. We have developed a sensitive assay for quantitating low levels of this activity with a synthetic DNA substrate containing 3H-labeled O(6)-methylguanine as the only modified base. Although both wild-type and adaptation-deficient (ada) mutants of E. coli contained low but comparable numbers (from 13 to 60) of the enzyme molecules per cell, adaptation treatment caused a significant increase of the enzyme in the wild type but not in the ada mutants, suggesting that the ada mutation is in a regulatory locus and not in the structural gene for the methyltransferase.

scholarly journals Bacteriophage Lambda: a Paradigm Revisited

2010 ◽  
Vol 84 (13) ◽  
pp. 6876-6879 ◽  
Author(s):  
Paul C. M. Fogg ◽  
Heather E. Allison ◽  
Jon R. Saunders ◽  
Alan J. McCarthy
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
High Frequency ◽  
Southern Hybridization ◽  
Integration Site ◽  
Bacteriophage Lambda ◽  
Rare Event ◽  
Low Frequency ◽  
E Coli ◽  
Very Low Frequency

ABSTRACT Bacteriophage lambda has an archetypal immunity system, which prevents the superinfection of its Escherichia coli lysogens. It is now known that superinfection can occur with toxigenic lambda-like phages at a high frequency, and here we demonstrate that the superinfection of a lambda lysogen can lead to the acquisition of additional lambda genomes, which was confirmed by Southern hybridization and quantitative PCR. As many as eight integration events were observed but at a very low frequency (6.4 × 10−4) and always as multiple insertions at the established primary integration site in E. coli. Sequence analysis of the complete immunity region demonstrated that these multiply infected lysogens were not immunity mutants. In conclusion, although lambda superinfection immunity can be confounded, it is a rare event.

A method for the specific Inhibition of poly[d(A-T)] synthesis using the A-T specific quinoxaline antibiotic TANDEM

1982 ◽  
Vol 60 (2) ◽  
pp. 131-136 ◽  
Author(s):  
D. H. Evans ◽  
J. S. Lee ◽  
A. R. Morgan ◽  
R. K. Olsen
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Thermal Denaturation ◽  
Strong Preference ◽  
Specific Inhibition ◽  
Dna Polymerase I ◽  
Synthetic Dna ◽  
Polymerase I

A serious problem in the replication of repeating-sequence DNA polymers using Escherichia coli DNA polymerase I arises from the fact that this polymerase has a very strong preference for the replication of poly[d(A-T)]. Thus reactions primed with DNA containing small amounts of contaminating poly[d(A-T)] will eventually result in complete domination of the synthesis by poly[d(A-T)]. This problem can be overcome by the addition to the reaction mixture of the synthetic quinoxaline antibiotic TANDEM which binds specifically to poly[d(A-T)] completely inhibiting its replication. Using thermal denaturation experiments it can be shown that TANDEM does not bind to most other synthetic DNA polymers (e.g., poly(dA)∙poly(dT) and poly[d(A-T-C)]∙poly[d(G-A-T)]) and therefore their replication is not inhibited. The only exception we have encountered is poly[d(T-A-C)]∙poly[d(G-T-A)] which does bind TANDEM and therefore the drug cannot be used during the synthesis of this polymer. The fact that poly[d(T-A-C)]-poly[d(G-T-A)] does bind TANDEM while poly [d(A-T-C)]-poly[d(G-A-T)] does not, suggests that the drug recognizes T-A rather than A-T sequences.

scholarly journals Recycling of Shiga Toxin 2 Genes in Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:NM

2007 ◽  
Vol 74 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Alexander Mellmann ◽  
Shan Lu ◽  
Helge Karch ◽  
Jian-guo Xu ◽  
Dag Harmsen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Integration Site ◽  
Hot Spot ◽  
Blot Hybridization ◽  
Enterohemorrhagic Escherichia Coli ◽  
Biologically Active ◽  
E Coli ◽  
Shiga Toxin 2

ABSTRACT Using colony blot hybridization with stx 2 and eae probes and agglutination in anti-O157 lipopolysaccharide serum, we isolated stx 2-positive and eae-positive sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:NM (nonmotile) strains from initial stool specimens and stx-negative and eae-positive SF E. coli O157:NM strains from follow-up specimens (collected 3 to 8 days later) from three children. The stx-negative isolates from each patient shared with the corresponding stx 2-positive isolates fliC H7, non-stx virulence traits, and multilocus sequence types, which indicates that they arose from the stx 2-positive strains by loss of stx 2 during infection. Analysis of the integrity of the yecE gene, a possible stx phage integration site in EHEC O157, in the consecutive stx 2-positive and stx-negative isolates demonstrated that yecE was occupied in stx 2-positive but intact in stx-negative strains. It was possible to infect and lysogenize the stx-negative E. coli O157 strains in vitro using an stx 2-harboring bacteriophage from one of the SF EHEC O157:NM isolates. The acquisition of the stx 2-containing phage resulted in the occupation of yecE and production of biologically active Shiga toxin 2. We conclude that the yecE gene in SF E. coli O157:NM is a hot spot for excision and integration of Shiga toxin 2-encoding bacteriophages. SF EHEC O157:NM strains and their stx-negative derivatives thus represent a highly dynamic system that can convert in both directions by the loss and gain of stx 2-harboring phages. The ability to recycle stx 2, a critical virulence trait, makes SF E. coli O157:NM strains ephemeral EHEC that can exist as stx-negative variants during certain phases of their life cycle.

scholarly journals Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli

2021 ◽  
Vol 12 ◽  
Author(s):  
Silje N. Ramstad ◽  
Yngvild Wasteson ◽  
Bjørn-Arne Lindstedt ◽  
Arne M. Taxt ◽  
Jørgen V. Bjørnholt ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Integration Site ◽  
Severe Disease ◽  
High Sequence Identity ◽  
Shiga Toxins ◽  
Sequence Identity ◽  
Diarrhea Case ◽  
The Usa ◽  
Uremic Syndrome

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
Dylan C. Alexander ◽  
David J. Devlin ◽  
Duane D. Hewitt ◽  
Ann C. Horan ◽  
Thomas J. Hosted
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Integration Site ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Temperate Bacteriophage ◽  
Site Specific ◽  
Sequence Identity ◽  
Site Specific Integration ◽  
Chromosome Sequence

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

scholarly journals The role of PI3K signaling in enteropathogenic escherichia coli induced apoptosis in epithelial cells

2021 ◽  
Author(s):  
Heather Park
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Integration Site ◽  
Pi3k Signaling ◽  
Enteropathogenic Escherichia Coli ◽  
Apoptotic Signaling ◽  
Signaling Protein ◽  
Infected Cells ◽  
Induced Apoptosis

Enteropathogenic Escherichia coli (EPEC) is a diarrheagic pathogen that has been the cause of severe and persistent infant diarrhea worldwide. EPEC invades the gastrointestinal tract where it hijacks host cell signaling and evades immune response long enough to cause the disease. This study was done to establish the role of PI3K signaling in EPEC induced apoptosis in epithelial cells. This report demonstrates that EPEC infected cells activate the anti-apoptotic signaling protein Akt via phosphorylation along with another anti-apoptotic signaling protein, Bcl-2. At the same time during EPEC infection the pro-aptoptotic protein Bax is inhibited. The activation of Akt was also observed with the addition of lipopolysaccharide (LPS) in the presence of serum. This thesis suggests that there are likely separate sensing mechanisms for EPEC, LPS and serum that are independent but synergistic and that Akt is the integration site of these signaling cascades.

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