Analysis of RecA-independent recombination events between short direct repeats related to a genomic island and to a plasmid inEscherichia coliK12

RecA-independent recombination events between short direct repeats, leading to deletion of the intervening sequences, were found to occur in two genetic models in theEscherichia coliK12 background. The first model was a smallE. coligenomic island which had been shown to be mobile in its strain of origin and, when cloned, also in theE. coliK12 context. However, it did not encode a site-specific recombinase as mobile genomic islands usually do. It was then 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 inE. coliK12 mutants affected in a number of recombination functions, including the 16E. coliK12 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 fromSaccharomyces cerevisiae,which flanked thecatgene. 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 inE. coliK12. 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.

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

10.7287/peerj.preprints.2807v1 ◽

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.

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

10.7287/peerj.preprints.2807 ◽

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.

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Application of RtcB ligase to monitor self-cleaving ribozyme activity by RNA-seq

Biological Chemistry ◽

10.1515/hsz-2021-0408 ◽

2022 ◽

Vol 0 (0) ◽

Author(s):

V. Janett Olzog ◽

Lena I. Freist ◽

Robin Goldmann ◽

Jörg Fallmann ◽

Christina E. Weinberg

Keyword(s):

Rna Seq ◽

Site Specific ◽

Pyrococcus Horikoshii ◽

Total Rna ◽

E Coli ◽

Catalytic Rnas ◽

Adapter Ligation ◽

Domains Of Life ◽

Cyclic Phosphate ◽

A Site

Abstract Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

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Waterborne illness and injury: a feasibility study of a site-specific predictive model for beach water quality at Beachway Park in the City of Burlington

10.32920/ryerson.14668362 ◽

2021 ◽

Author(s):

Ramien Sereshk

Keyword(s):

Water Quality ◽

Predictive Model ◽

Real Time ◽

Site Specific ◽

E Coli ◽

Time Basis ◽

A Site ◽

Persistence Model ◽

Beach Water Quality ◽

Beach Water

It is commonly assumed that the persistence model, using day-old monitoring results, will provide accurate estimates of real-time bacteriological concentrations in beach water. However, the persistence model frequently provides incorrect results. This study: 1. develops a site-specific predictive model, based on factors significantly influencing water quality at Beachway Park; 2. determines the feasibility of the site-specific predictive model for use in accurately predicting near real-time E. coli levels. A site-specific predictive model, developed for Beachway Park, was evaluated and the results were compared to the persistence model. This critical performance evaluation helped to identify the inherent inaccuracy of the persistence model for Beachway Park, which renders it an unacceptable approach for safeguarding public health from recreational water-borne illnesses. The persistence model, supplemented with a site-specific predictive model, is recommended as a feasible method to accurately predict bacterial levels in water on a near real-time basis.

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Fbh1 Limits Rad51-Dependent Recombination at Blocked Replication Forks

Molecular and Cellular Biology ◽

10.1128/mcb.00471-09 ◽

2009 ◽

Vol 29 (17) ◽

pp. 4742-4756 ◽

Cited By ~ 44

Author(s):

Alexander Lorenz ◽

Fekret Osman ◽

Victoria Folkyte ◽

Sevil Sofueva ◽

Matthew C. Whitby

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Replication Fork ◽

Direct Repeat ◽

Dna Helicase ◽

Replication Forks ◽

Site Specific ◽

Replicative Stress ◽

Recombinant Formation ◽

A Site

ABSTRACT Controlling the loading of Rad51 onto DNA is important for governing when and how homologous recombination is used. Here we use a combination of genetic assays and indirect immunofluorescence to show that the F-box DNA helicase (Fbh1) functions in direct opposition to the Rad52 orthologue Rad22 to curb Rad51 loading onto DNA in fission yeast. Surprisingly, this activity is unnecessary for limiting spontaneous direct-repeat recombination. Instead it appears to play an important role in preventing recombination when replication forks are blocked and/or broken. When overexpressed, Fbh1 specifically reduces replication fork block-induced recombination, as well as the number of Rad51 nuclear foci that are induced by replicative stress. These abilities are dependent on its DNA helicase/translocase activity, suggesting that Fbh1 exerts its control on recombination by acting as a Rad51 disruptase. In accord with this, overexpression of Fbh1 also suppresses the high levels of recombinant formation and Rad51 accumulation at a site-specific replication fork barrier in a strain lacking the Rad51 disruptase Srs2. Similarly overexpression of Srs2 suppresses replication fork block-induced gene conversion events in an fbh1Δ mutant, although an inability to suppress deletion events suggests that Fbh1 has a distinct functionality, which is not readily substituted by Srs2.

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Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

Applied and Environmental Microbiology ◽

10.1128/aem.00901-12 ◽

2012 ◽

Vol 78 (19) ◽

pp. 6963-6974 ◽

Cited By ~ 10

Author(s):

Jose M. Quesada ◽

María Isabel Soriano ◽

Manuel Espinosa-Urgel

Keyword(s):

Pseudomonas Putida ◽

Genomic Island ◽

Mobile Element ◽

Direct Repeat ◽

Promoter Sequence ◽

Repeat Sequence ◽

Genomic Islands ◽

Pseudomonas Putida Kt2440 ◽

Bacterial Populations ◽

Content Type

ABSTRACTThe stability of seven genomic islands ofPseudomonas putidaKT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location,attsites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

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Local transposition of P elements in Drosophila melanogaster and recombination between duplicated elements using a site-specific recombinase.

Genetics ◽

10.1093/genetics/137.2.551 ◽

1994 ◽

Vol 137 (2) ◽

pp. 551-563 ◽

Cited By ~ 2

Author(s):

K G Golic

Keyword(s):

Drosophila Melanogaster ◽

Sister Chromatid ◽

P Element ◽

White Gene ◽

Direct Repeats ◽

Site Specific ◽

P Elements ◽

Dicentric Chromosomes ◽

A Site ◽

Different Levels

Abstract The transposase source delta 2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic white gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.

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The behavior of insertions near a site of mitotic gene conversion in yeast.

Genetics ◽

10.1093/genetics/119.3.535 ◽

1988 ◽

Vol 119 (3) ◽

pp. 535-540

Author(s):

J E Golin ◽

S C Falco

Keyword(s):

Gene Conversion ◽

High Frequency ◽

Direct Repeat ◽

Large Plasmid ◽

Direct Repeats ◽

Heteroduplex Dna ◽

Relative Stabilities ◽

Extensive Region ◽

Mitotic Gene Conversion ◽

A Site

Abstract In yeast, coincident gene conversion events involving the LEU1 and TRP5 loci (16 cM apart) occur at frequencies that are far greater than is expected for two independent acts of recombination. When a large plasmid (pJM53) is placed between these genes so that a direct repeat is produced, there is frequent loss of the insert among coincident convertants. Previous results strongly suggest that this is due to a separate, intrachromosomal exchange between the direct repeats rather than to excision from an extensive region of heteroduplex DNA. In this paper, we extend our genetic and molecular analysis to a plasmid insertion (pKSH) which replaces rather than duplicates the chromosomal material. The relative stabilities of pKSH and pJM53 are compared among coincident Leu+Trp+ convertants and convertants involving only one locus (LEU1). The pKSH insertion is significantly more stable in the latter which constitute a large majority of the selectable recombinants. In the former, both insertions are lost with high frequency. These results are used to argue that, while most mitotic conversion does not result from long intermediates, coincident convertants may arise from either multiple intermediates or extensive heteroduplex regions.

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Acquisition and Evolution of the exoU Locus in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.02000-05 ◽

2006 ◽

Vol 188 (11) ◽

pp. 4037-4050 ◽

Cited By ~ 79

Author(s):

Bridget R. Kulasekara ◽

Hemantha D. Kulasekara ◽

Matthew C. Wolfgang ◽

Lisa Stevens ◽

Dara W. Frank ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Type Iii Secretion ◽

Genomic Island ◽

Genomic Islands ◽

Targeted Mutagenesis ◽

Host Cells ◽

System A ◽

Transmissible Plasmid ◽

Genomic Regions ◽

Functional Analyses

ABSTRACT ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNALys gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of β- and γ-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNALys-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNALys gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

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