scholarly journals Viability of folA-null derivatives of wild-type (thyA+) Escherichia coli K-12.

1993 ◽  
Vol 175 (3) ◽  
pp. 909-911 ◽  
Author(s):  
B R Krishnan ◽  
D E Berg
Keyword(s):  
Escherichia Coli ◽  
Wild Type ◽  
K 12 ◽  
Derivatives Of

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

scholarly journals exrB: amalB-linked gene inEscherichia coliB involved in sensitivity to radiation and filament formation

1974 ◽  
Vol 23 (2) ◽  
pp. 175-184 ◽  
Author(s):  
Joseph Greenberg ◽  
Leonard J. Berends ◽  
John Donch ◽  
Michael H. L. Green
Keyword(s):  
Escherichia Coli ◽  
Normal Growth ◽  
Filament Formation ◽  
Wild Type ◽  
Sensitive Mutant ◽  
Γ Radiation ◽  
Linked Gene ◽  
Derivatives Of

SUMMARYPAM 26, a radiation-sensitive mutant ofEscherichia colistrain B, is described. Its properties are attributable to a mutation in a gene,exrB, which is cotransducible withmalB. It differs fromuvrA(alsomalB-linked) derivatives of strain B in being sensitive to 1-methyl-3-nitro-1-nitroso-guanidine and γ-radiation, and in being able to reactivate UV-irradiated phage T3. It differs fromexrA(alsomalB-linked) derivatives of strain B in forming filaments during the course of normal growth as well as after irradiation. WhenexrBwas transduced into a K12 (lon+) strain, filaments did not form spontaneously. Three-point transductions established the order of markers asmet A malB exrB. Based on an analysis of the frequency of wild-type recombinants in a reciprocal transduction betweenexrAandexrBstrains, it was inferred that they are not isogenic and that the order of markers ismalB exrA exrB.

scholarly journals First-Time Isolation and Characterization of a Bacteriophage Encoding the Shiga Toxin 2c Variant, Which Is Globally Spread in Strains of Escherichia coli O157

2004 ◽  
Vol 72 (12) ◽  
pp. 7030-7039 ◽  
Author(s):  
Eckhard Strauch ◽  
Christoph Schaudinn ◽  
Lothar Beutin
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Phage Lambda ◽  
Wild Type ◽  
Chromosomal Locus ◽  
O157 Strain ◽  
Diagnostic Pcr ◽  
E Coli ◽  
Isolation And Characterization ◽  
K 12

ABSTRACT A bacteriophage encoding the Shiga toxin 2c variant (Stx2c) was isolated from the human Escherichia coli O157 strain CB2851 and shown to form lysogens on the E. coli K-12 laboratory strains C600 and MG1655. Production of Stx2c was found in the wild-type E. coli O157 strain and the K-12 lysogens and was inducible by growing bacteria in the presence of ciprofloxacin. Phage 2851 is the first reported viable bacteriophage which carries an stx 2c gene. Electron micrographs of phage 2851 showed particles with elongated hexagonal heads and long flexible tails resembling phage lambda. Sequence analysis of an 8.4-kb region flanking the stx 2c gene and other genetic elements revealed a mosaic gene structure, as found in other Stx phages. Phage 2851 showed lysis of E. coli K-12 strains lysogenic for Stx phages encoding Stx1 (H19), Stx2 (933W), Stx (7888), and Stx1c (6220) but showed superinfection immunity with phage lambda, presumably originating from the similarity of the cI repressor proteins of both phages. Apparently, phage 2851 integrates at a different chromosomal locus than Stx2 phage 933W and Stx1 phage H19 in E. coli, explaining why Stx2c is often found in combination with Stx1 or Stx2 in E. coli O157 strains. Diagnostic PCR was performed to determine gene sequences specific for phage 2851 in wild-type E. coli O157 strains producing Stx2c. The phage 2851 q and o genes were frequently detected in Stx2c-producing E. coli O157 strains, indicating that phages related to 2851 are associated with Stx2c production in strains of E. coli O157 that were isolated in different locations and time periods.

scholarly journals An Epistasis Analysis of recA and recN in Escherichia coli K-12

Genetics ◽  
2020 ◽  
Vol 216 (2) ◽  
pp. 381-393
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Double Mutant ◽  
Strand Break ◽  
Wild Type ◽  
Epistasis Analysis ◽  
Chromatid Cohesion ◽  
K 12 ◽  
Structural Maintenance Of Chromosomes ◽  
Sos Protein

RecA is essential for double-strand-break repair (DSBR) and the SOS response in Escherichia coli K-12. RecN is an SOS protein and a member of the Structural Maintenance of Chromosomes family of proteins thought to play a role in sister chromatid cohesion/interactions during DSBR. Previous studies have shown that a plasmid-encoded recA4190 (Q300R) mutant had a phenotype similar to ∆recN (mitomycin C sensitive and UV resistant). It was hypothesized that RecN and RecA physically interact, and that recA4190 specifically eliminated this interaction. To test this model, an epistasis analysis between recA4190 and ∆recN was performed in wild-type and recBC sbcBC cells. To do this, recA4190 was first transferred to the chromosome. As single mutants, recA4190 and ∆recN were Rec+ as measured by transductional recombination, but were 3-fold and 10-fold decreased in their ability to do I-SceI-induced DSBR, respectively. In both cases, the double mutant had an additive phenotype relative to either single mutant. In the recBC sbcBC background, recA4190 and ∆recN cells were very UVS (sensitive), Rec−, had high basal levels of SOS expression and an altered distribution of RecA-GFP structures. In all cases, the double mutant had additive phenotypes. These data suggest that recA4190 (Q300R) and ∆recN remove functions in genetically distinct pathways important for DNA repair, and that RecA Q300 was not important for an interaction between RecN and RecA in vivo. recA4190 (Q300R) revealed modest phenotypes in a wild-type background and dramatic phenotypes in a recBC sbcBC strain, reflecting greater stringency of RecA’s role in that background.

scholarly journals Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa

2006 ◽  
Vol 74 (8) ◽  
pp. 4685-4693 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Hannah J. Knecht ◽  
Jie Li ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Clinical Manifestations ◽  
Rectal Mucosa ◽  
Wild Type ◽  
E Coli ◽  
Healthy Cattle ◽  
Life Threatening ◽  
K 12 ◽  
The Impact

ABSTRACT The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δtir, and Δeae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents

2016 ◽  
Vol 4 (2) ◽  
Author(s):  
Brian P. Anton ◽  
Alexey Fomenkov ◽  
Elisabeth A. Raleigh ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Complete Genome ◽  
Disulfide Bonds ◽  
Scientific Community ◽  
Genetically Engineered ◽  
Wild Type ◽  
E Coli ◽  
Biotech Industry ◽  
General Scientific ◽  
K 12

SHuffle strains are genetically engineered Escherichia coli strains that are capable of oxidizing cysteines within proteins to form disulfide bonds. Here we present the complete genome of both the K-12 and B versions of SHuffle strains along with their parental ancestors. These strains have been of significant use to both the general scientific community and the biotech industry, interested in producing novel disulfide-bonded proteins that were hitherto unable to be expressed in standard E. coli expression strains.

A new method for the isolation of methionyl transfer RNA synthetase mutants from Escherichia coli

1975 ◽  
Vol 21 (6) ◽  
pp. 754-758 ◽  
Author(s):  
John B. Armstrong ◽  
John A. Fairfield
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Transfer Rna ◽  
New Method ◽  
Wild Type ◽  
E Coli ◽  
Adenosyl Methionine ◽  
K 12 ◽  
Methionyl Trna Synthetase

Six methionine auxotrophs were isolated from an E. coli K-12 strain which required up to 100 times as much methionine for growth as a conventional auxotroph. In these mutants, the methionyl-tRNA synthetase had an increased Km for methionine. The Km value for the mutants ranged from 0.48 to 1.63 mM, compared to 0.078 mM for the wild type. The Km (methionine) for S-adenosyl methionine synthetase was not altered.

scholarly journals Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

2006 ◽  
Vol 74 (4) ◽  
pp. 2233-2244 ◽  
Author(s):  
Alison S. Low ◽  
Francis Dziva ◽  
Alfredo G. Torres ◽  
Jessenya L. Martinez ◽  
Tracy Rosser ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Enterohemorrhagic Escherichia Coli ◽  
Wild Type ◽  
Induced Expression ◽  
E Coli ◽  
Extracellular Matrix Components ◽  
K 12

ABSTRACT Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a sero- type O26:H- strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.

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