A new approach using multiplex long accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing.

Sorokin, Alexeï; Lapidus, Alla; Capuano, Véronique; Galleron, Nathalie; Pujic, Petar; Ehrlich, S. Dusko

doi:10.1101/gr.6.5.448

Cited by 37 publications

(31 citation statements)

References 16 publications

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“…The amplifications were performed with 0.1 g of E. coli chromosomal DNA, prepared as described (Te Riele et al, 1986). Reactions and cycling conditions were as described by Sorokin et al (1996). PCR products were purified by using the Wizard PCR Preps kit (Promega) or agarose gel electrophoresis.…”

Section: Plasmidsmentioning

confidence: 99%

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Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

Bierne

Vilette

Ehrlich

et al. 1997

Molecular Microbiology

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SummaryThe mechanism of recombination of tandem repeats in the chromosome of Escherichia coli was investigated by genetic means. Tandem repeats 624 bp long were introduced into the lacZ gene of E. coli and the efficiency of deletion of one repeat was compared in different recombination mutants. No effects of the recA, recBC, recF, ruvA or ruvA recG mutations were detected. Hence, tandem repeat deletion appears to not proceed via the RecBCD or RecF homologous recombination pathways. A new mutant in which RecA-independent recombination is increased 15-fold was isolated. The mutation lies in the dnaE gene coding for the alpha subunit of polymerase III: it is a Gly to Asp change at codon 133. Another dnaE mutation, dnaE486, was tested and also shown to stimulate RecA-independent recombination. It is proposed that tandem-repeat recombination occurs by a replication slippage mechanism. RecA-independent recombination is also enhanced in a rep mutant, in which chromosomal replication is slowed down by the absence of the Rep helicase, suggesting that replication pausing may facilitate slippage.

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Section: Plasmidsmentioning

confidence: 99%

“…PCR products used for sequencing were prepared as described above and in Sorokin et al (1996). Oligonucleotides were synthesized with a Beckman DNA Oligo 1000 synthesizer.…”

Section: Dna Sequence Analysismentioning

confidence: 99%

Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

Bierne

Vilette

Ehrlich

et al. 1997

Molecular Microbiology

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“…The length of the PCR products, 1422 bp for all parental clones and 798 bp for all recombinant clones, was verified by gel electrophoresis. PCR products used for sequencing were prepared as described by Sorokin et al (1996). Oligonucleotides were synthesized with a Beckman DNA synthesizer Oligo 1000.…”

Section: Analysis Of Parental and Deletion Productsmentioning

confidence: 99%

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

Bierne

Seigneur

et al. 1997

Molecular Microbiology

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SummaryIt has previously been shown that recombination between tandem repeats is not significantly affected by a recA mutation in Escherichia coli. Here, we describe the activation of a RecA-dependent recombination pathway in a hyper-recombination mutant. In order to analyse how tandem repeat deletion may proceed, we searched for mutants that affect this process. Three hyper-recombination clones were characterized and shown to be mutated in the uvrD gene. Two of the mutations were identified as opal mutations at codons 130 and 438. A uvrD ::Tn5 mutation was used to investigate the mechanism of deletion formation in these mutants. The uvrD-mediated stimulation of deletion was abolished by a lexAind3 mutation or by inactivation of either the recA, recF, recQ or ruvA genes. We conclude that (i) this stimulation requires SOS induction and (ii) tandem repeat recombination in uvrD mutants occurs via the RecF pathway. In uvrD þ cells, constitutive expression of SOS genes is not sufficient to stimulate deletion formation. This suggests that the RecF recombination pathway activated by SOS induction is antagonized by the UvrD protein. Paradoxically, we observed that the overproduction of UvrD from a plasmid also stimulates tandem repeat deletion. However, this stimulation is RecA independent, as is deletion in a wild-type strain. We propose that the presence of an excess of the UvrD helicase favours replication slippage. This work suggests that the UvrD helicase controls a balance between different routes of tandem repeat deletion.

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“…La séquence géno-mique a été obtenue en utilisant une straté-gie de séquençage par shot-gun suivie de PCRs longue portée (Long range PCR) et de PCR combinatoires (Multiplex PCR [18] Les données de séquence nous seront très utiles pour la caractérisation des protéines de stress visibles sur les gels bidimensionnels. Une analyse préliminaire de 40 spots d'intensité variable sur le gel de référence à pH 6, nous a permis d'identifier 36 protéines en couplant la spectrométrie de masse aux données de séquence.…”

Section: Projet De Séquençage De L Bulgaricusunclassified

Article

Guillouard

Lim²,

Guchte

et al. 2004

Lait

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-Tolerance and adaptative acid stress response of Lactobacillus delbrueckii ssp. bulgaricus. Using several approaches, we studied the acid stress response of different strains of Lactobacillus bulgaricus. We showed that after an acid shock, the cell survival varied considerably between strains and that all the studied strains were able to develop an acid adaptive response. For one of these strains, we characterized, using two-dimensional electrophoresis, the modifications in the protein pattern induced by acid adaptation. Out of the 50 proteins up-regulated under the adaptive conditions, 21 were identified after N-terminal sequencing. They belong to different functional categories including stress response and general metabolism. To allow a larger genetic exploration of L. bulgaricus, the genome of the type strain ATCC 11842 was sequenced in collaboration with the Genoscope. All together, these genomic and post-genomic data should lead to a better understanding of L. bulgaricus physiology and its environmental adaptability.Lactobacillus bulgaricus / acid stress / proteome / genome sequence Résumé -En utilisant plusieurs approches, nous avons étudié le comportement de différentes souches de Lactobacillus bulgaricus face à un stress acide. Nous avons observé que la survie des cellules à un choc acide variait considérablement entre les souches et que toutes les souches étudiées étaient capables d'initier une réponse adaptative à l'acidité. Pour l'une des souches, nous avons caractérisé, par électrophorèse bidimensionnelle, les changements protéiques intervenant dans la réponse adaptative à l'acidité. Parmi les 50 protéines induites pendant l'adaptation, 21 ont pu être identifiées après séquençage N-terminal. Elles appartiennent à différentes catégories fonctionnelles dont celles des réponses aux stress et du métabolisme général. Afin de permettre une exploration génétique plus large de L. bulgaricus, le génome complet de la souche type ATCC 11842 a été séquencé en collaboration avec le Génoscope. Toutes ces données de génomique et de postgénomique nous conduiront à une meilleure compréhension de la physiologie de L. bulgaricus et notamment de son adaptabilité environnementale.Lactobacillus bulgaricus / stress acide / protéome / séquence génomique * Auteur correspondant : maguin@jouy.inra.fr 2 I. Guillouard et al.

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A new approach using multiplex long accurate PCR and yeast artificial chromosomes for bacterial chromosome mapping and sequencing.

Cited by 37 publications

References 16 publications

Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

Isolation of a dnaE mutation which enhances RecA‐independent homologous recombination in the Escherichia coli chromosome

uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway

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