scholarly journals Context effects in the formation of deletions in Escherichia coli.

Genetics ◽  
1990 ◽  
Vol 126 (1) ◽  
pp. 17-24 ◽  
Author(s):  
T Kazic ◽  
D E Berg
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Copy Number ◽  
Context Effects ◽  
Direct Repeat ◽  
Single Copy ◽  
Multicopy Plasmid ◽  
Direct Repeats ◽  
Specific Manner ◽  
Allele Specific

Abstract We have examined the frequency with which identical deletions are formed in different chromosomal contexts. A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac. Deletion of the palindromes and one of the direct repeats results in reversion to Ampr. The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome. The magnitude of the declines varies in an allele-specific and location-specific manner. Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.

Reversion of the Tyrosine Ochre Strain Escherichia coli WU3610 under Starvation Conditions Depends on a New Gene tas

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1627-1635 ◽  
Author(s):  
Andrew R Timms ◽  
Bryn A Bridges
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Spontaneous Mutation ◽  
Single Copy ◽  
Chorismate Mutase ◽  
Multicopy Plasmid ◽  
Reading Frame ◽  
Prephenate Dehydrogenase ◽  
Slow Growing ◽  
Starvation Conditions

Abstract When 3 × 108 bacteria of the Escherichia coli tyrA14(oc) leu308(am) strain WU3610 are plated on glucose salts agar supplemented with leucine only, colonies of slow-growing Tyr+ suppressor mutants begin to appear after about a week and increase in numbers roughly linearly with time thereafter (stationary phase or starvation-associated mutation). From a library constructed from two of these mutants, a clone was obtained that suppressed the tyrosine requirement of WU3610 when present on a multicopy plasmid. The activity was identified to an open reading frame we call tas, the sequence for which has homology with a variety of known genes with aldo-keto reductase activity. The activity of tas complements the prephenate dehydrogenase dysfunction of tyrA14 (the chorismate mutase activity of tyrA possibly being still functional). A strain deleted for tas showed no spontaneous mutation under starvation conditions. Whereas neither tas+ nor tas bacteria showed any increase in viable or total count when plated under conditions of tyrosine starvation at 3 × 108 cells per plate, at lower density (~107 per plate) tas+ but not tas bacteria showed considerable residual growth. We suggest that the single copy of tas present in WU3610 allows cryptic cell or DNA turnover under conditions of tyrosine starvation and that this is an essential prerequisite for starvation-associated mutation in this system. The target gene for mutation is not tas, although an increase in the expression of this gene, for example, resulting from a suppressor mutation affecting supercoiling, could be responsible for the slow-growing Tyr+ phenotype.

scholarly journals IS103, a new insertion element in Escherichia coli: characterization and distribution in natural populations.

Genetics ◽  
1989 ◽  
Vol 121 (3) ◽  
pp. 423-431 ◽  
Author(s):  
B G Hall ◽  
L L Parker ◽  
P W Betts ◽  
R F DuBose ◽  
S A Sawyer ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Insertion Sequence ◽  
Natural Populations ◽  
Single Copy ◽  
Target Sequence ◽  
Wild Type ◽  
Insertion Element ◽  
E Coli ◽  
Natural Isolates

Abstract IS103 is a previously unknown insertion sequence found in Escherichia coli K12. We have sequenced IS103 and find that it is a 1441-bp element that consists of a 1395-bp core flanked by imperfect 23-bp inverted repeats. IS103 causes a 6-bp duplication of the target sequence into which it inserts. There is a single copy of IS103 present in wild-type E. coli K12 strain HfrC. In strain X342 and its descendents there are two additional copies, one of which is located within the bglF gene. IS103 is capable of excising from within bglF and restoring function of that gene. IS103 exhibits 44% sequence identity with IS3, suggesting that the two insertion sequences are probably derived from a common ancestor. We have examined the distribution of IS103 in the chromosomes and plasmids of the ECOR collection of natural isolates of E. coli. IS103 is found in 36 of the 71 strains examined, and it strongly tends to inhabit plasmids rather than chromosomes. Comparison of the observed distribution of IS103 with distributions predicted by nine different models for the regulation of transposition according to copy number and of the effects of copy number on fitness suggest that transposition of IS103 is strongly regulated and that it has only minor effects on fitness. The strong clustering of IS103 within one phylogenetic subgroup of the E. coli population despite its presence on plasmids suggests that plasmids tend to remain within closely related strains and that transfer to distantly related strains is inhibited.

scholarly journals The role of FIS in the Rcd checkpoint and stable maintenance of plasmid ColE1

Microbiology ◽  
2009 ◽  
Vol 155 (8) ◽  
pp. 2676-2682 ◽  
Author(s):  
I. K. Blaby ◽  
D. K. Summers
Keyword(s):  
Copy Number ◽  
Bacterial Population ◽  
Multicopy Plasmid ◽  
Concerted Action ◽  
Global Regulator ◽  
Synaptic Complex ◽  
Daughter Cells ◽  
Specific Manner ◽  
Stable Maintenance

Escherichia coli plasmid ColE1 lacks active partitioning, and copies are distributed randomly to daughter cells at division. The plasmid is maintained stably in the bacterial population as long as its copy number remains high. The accumulation of plasmid dimers and higher multimers depresses copy number, and is an important cause of multicopy plasmid instability. ColE1 dimers are restored to the monomeric state by site-specific recombination, which requires the host-encoded proteins XerCD, ArgR and PepA acting at the plasmid cer site. In addition, a 70 nt RNA expressed from the cer site of plasmid dimers delays the division of dimer-containing cells. Here, we report that the global regulator FIS binds to cer in a sequence-specific manner, close to the Rcd promoter (P cer ). FIS is not required for plasmid dimer resolution, but is essential for repression of P cer in plasmid monomers. Repression also requires the XerCD recombinase, but not ArgR or PepA. We propose a model for monomer–dimer control of P cer in which the promoter is repressed in plasmid monomers by the concerted action of FIS and XerCD. Rcd transcription is triggered in plasmid dimers by the lifting of XerCD-mediated repression in the synaptic complex.

scholarly journals Exchange of spacer regions between rRNA operons in Escherichia coli.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 683-690 ◽  
Author(s):  
S Harvey ◽  
C W Hill
Keyword(s):  
Escherichia Coli ◽  
Gene Conversion ◽  
Copy Number ◽  
Single Copy ◽  
The Other ◽  
Gene Conversion Event ◽  
E Coli ◽  
Coding Regions ◽  
Sister Chromatids ◽  
Rrn Operons

Abstract The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala1B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala1B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids.

scholarly journals Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

2017 ◽  
Author(s):  
María F Azpiroz ◽  
Magela D Laviña
Keyword(s):  
Escherichia Coli ◽  
Genomic Island ◽  
Direct Repeat ◽  
Host Cells ◽  
Dna Repeats ◽  
Direct Repeats ◽  
Site Specific ◽  
E Coli ◽  
A Site ◽  
New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

scholarly journals Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid in Escherichia coli K12

2017 ◽  
Author(s):  
María F Azpiroz ◽  
Magela D Laviña
Keyword(s):  
Escherichia Coli ◽  
Genomic Island ◽  
Direct Repeat ◽  
Host Cells ◽  
Dna Repeats ◽  
Direct Repeats ◽  
Site Specific ◽  
E Coli ◽  
A Site ◽  
New Evidence

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in the Escherichia coli K12 background. The first model was a small E. coli genomic island which had been shown to be mobile in its strain of origin and, when cloned, in the E. coli K12 context too. However, it did not encode a site-specific recombinase as mobile genomic island usually do. Then, it was deduced that the host cells should provide the recombination function. This latter was searched for by means of a PCR approach to detect the island excision in E. coli K12 mutants affected in a number of recombination functions, including the 16 E. coli K12 site-specific recombinases, the RecET system, and multiple proteins that participate in the RecA-dependent pathways of homologous recombination. None of these appeared to be involved in the island excision. The second model, analyzed in a RecA deficient context, was a plasmid construction containing a short direct repeat proceeding from Saccharomyces cerevisiae, which flanked the cat gene. The excision of this gene by recombination of the DNA repeats was confirmed by PCR and through the detection, recovery and characterization of the plasmid deleted form. In sum, we present new evidence on the occurrence of RecA-independent recombination events in E. coli K12. Although the mechanism underlying these processes is still unknown, their existence suggests that RecA-independent recombination may confer mobility to other genetic elements, thus contributing to genome plasticity.

Local DNA Sequence Control of Deletion Formation in Escherichia coli Plasmid pBR322

Genetics ◽  
1987 ◽  
Vol 115 (1) ◽  
pp. 41-49
Author(s):  
Ujjala DasGupta ◽  
Kathleen Weston-Hafer ◽  
Douglas E Berg
Keyword(s):  
Escherichia Coli ◽  
Dna Sequence ◽  
Specific Interactions ◽  
Plasmid Pbr322 ◽  
Direct Repeats ◽  
Ampicillin Resistance ◽  
Deletion Formation ◽  
Coli Plasmid ◽  
Insertion Mutations ◽  
Dna Processing

ABSTRACT The specificity of deletion formation was studied using tests involving reversion of palindromic insertion mutations. Insertions of a Tn5-related transposon at 13 sites in the ampicillin-resistance (amp) gene of plasmid pBR322 were shortened to a nested set of perfect palindromes, 22, 32 and 90 bp long. We monitored frequencies of reversion to Ampr, which is the result of deletion of the palindrome plus one copy of the flanking 9 bp direct repeats (which had been formed by transposition). Revertant frequencies were found to depend on the location and the sequence of the palindromic insert. Changing a 45-kb interrupted palindrome to a 22-bp perfect palindrome stimulated deletion formation by factors of from fourfold to 545-fold among the 13 sites, while elongation of the perfect palindrome from 22 to 90 bp stimulated deletion formation by factors of from eight- to 18,000-fold. We conclude that deletion formation is strongly affected by subtle features of DNA sequence or conformation, both inside and outside the deleted segment, and that these effects may reflect specific interactions of DNA processing proteins with template DNAs.

scholarly journals Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells

2017 ◽  
Vol 133 ◽  
pp. 46-51 ◽  
Author(s):  
Whitney N. Wood ◽  
Kyle D. Smith ◽  
Jennifer A. Ream ◽  
L. Kevin Lewis
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Single Copy ◽  
Plasmid Dnas ◽  
Copy Number Plasmid ◽  
Escherichia Coli Cells

scholarly journals Roles of the membrane-reentrant β-hairpin-like loop of RseP protease in selective substrate cleavage

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Koichiro Akiyama ◽  
Shinya Mizuno ◽  
Yohei Hizukuri ◽  
Hiroyuki Mori ◽  
Terukazu Nogi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteolytic Activity ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Substrate Recognition ◽  
Membrane Domain ◽  
Transmembrane Segments ◽  
Specific Manner ◽  
Allele Specific ◽  
Substrate Cleavage

Molecular mechanisms underlying substrate recognition and cleavage by Escherichia coli RseP, which belongs to S2P family of intramembrane-cleaving proteases, remain unclear. We examined the function of a conserved region looped into the membrane domain of RseP to form a β-hairpin-like structure near its active site in substrate recognition and cleavage. We observed that mutations disturbing the possible β-strand conformation of the loop impaired RseP proteolytic activity and that some of these mutations resulted in the differential cleavage of different substrates. Co-immunoprecipitation and crosslinking experiments suggest that the loop directly interacts with the transmembrane segments of substrates. Helix-destabilising mutations in the transmembrane segments of substrates suppressed the effect of loop mutations in an allele-specific manner. These results suggest that the loop promotes substrate cleavage by selectively recognising the transmembrane segments of substrates in an extended conformation and by presenting them to the proteolytic active site, which contributes to substrate discrimination.

scholarly journals An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function

1984 ◽  
Vol 221 (1) ◽  
pp. 43-51 ◽  
Author(s):  
D A Jans ◽  
A L Fimmel ◽  
L Hatch ◽  
F Gibson ◽  
G B Cox
Keyword(s):  
Escherichia Coli ◽  
Oxidative Phosphorylation ◽  
Atpase Activity ◽  
Dna Sequence ◽  
Mutant Allele ◽  
Adenosine Triphosphatase ◽  
The Other ◽  
Multicopy Plasmid ◽  
B Subunit ◽  
And Function

Glycine at position 9 is replaced by aspartic acid in the mutant b-subunit of Escherichia coli F1F0-ATPase coded for by the uncF476 allele. The mutant b-subunit is not assembled into the membrane in haploid strains carrying the uncF476 allele, but, if the mutant allele is incorporated into a multicopy plasmid, then some assembly of the mutant b-subunit occurs. Two revertant strains were characterized, one of which (AN2030) was a full revertant, the other (AN1953) a partial revertant. DNA sequencing indicated that in strain AN2030 the uncF476 mutation had reverted to give the sequence found in the normal uncF gene. The partial-revertant strain AN1953, however, retained the DNA sequence of the uncF476 allele, and complementation analysis indicated that the second mutation may be in the uncA gene. Membranes prepared from the partial-revertant strain carried out oxidative phosphorylation, although the membranes appeared to be impermeable to protons, and the ATPase activity was sensitive to the inhibitor dicyclohexylcarbodi-imide.

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