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Transposition of pGh9:ISS1 is random and efficient in Streptococcus thermophilus CNRZ368

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Abstract:

Streptococcus thermophilus bacteria are used as a starter in the fermentation of yogurts and many cheeses. To construct mutants of S. thermophilus CNRZ368, the use of the plasmid pGh9:ISS1 was considered. This plasmid is known to be a good tool for insertional mutagenesis in gram-positive bacteria, owing to its ability to integrate in the genome by a mechanism of replicative transposition. However, the presence of three endogenous ISS1 copies in the genome of S. thermophilus CNRZ368 and the possible occurrence of homologous recombination could reduce the efficiency of pGh9:ISS1 as a tool for generating mutants. To address this question, the ability of pGh9:ISS1 to transpose randomly in the genome of strain CNRZ368 was investigated. The results of our experiments indicated that: (i) the frequency of transposition of ISS1 was high, approximately 2 × 10–2, in S. thermophilus CNRZ368; (ii) the integration of multiple tandem copies of the plasmid was frequent; (iii) homologous recombination events between ISS1 were not predominant; and (iv) plasmid pGh9:ISS1 transposed randomly around the S. thermophilus CNRZ368 chromosome. In addition, we describe the strategy used to localize the pGh9:ISS1 insertion locus on the physical map of strain CNRZ368 and the method used to clone the regions flanking this insertion site, especially when multiple copies of the plasmid were integrated in tandem.Key words: insertional mutagenesis, random transposition, Streptococcus thermophilus, homologue recombination, ISS1.

Les bactéries de l'espèce Streptococcus thermophilus sont utilisées pour la fermentation des yaourts et de certains fromages. Afin de construire des mutants de la souche S. thermophilus CNRZ368, l'utilisation du plasmide pGh9:ISS1 a été envisagée. Ce plasmide est employé en tant qu'agent de mutagenèse de bactéries à Gram positives pour sa capacité à intégrer le chromosome par un mécanisme de transposition réplicative. Toutefois, la présence de trois copies endogènes de l'élément ISS1 et la possibilité d'événements de recombinaison homologue entre les copies endogènes et la copie portée par le plasmide pourraient réduire l'efficacité de pGh9:ISS1 en tant qu'outil de mutagenèse. C'est pourquoi, la capacité de pGh9:ISS1 à transposer aléatoirement dans le génome de la souche CNRZ368 a été testée. Les résultats de nos expériences indiquent que : (i) la fréquence de transposition de l'élément ISS1 chez S. thermophilus CNRZ368 est forte et atteint 2 × 10–2 ; (ii) l'intégration en tandem de multiples copies du plasmide est fréquente ; (iii) les événements de recombinaison homologue ne sont pas prédominants; et (iv) la transposition du plasmide est aléatoire. De plus, dans cet article est décrite la stratégie utilisée pour la localisation du locus d'insertion de pGh9:ISS1 sur la carte physique du chromosome de la souche CNRZ368, ainsi que celle utilisée lors du clonage des régions flanquant ce site y compris lorsque de multiples copies du plasmide sont intégrées en tandem.Mots clés : mutagenèse insertionnelle, transposition aléatoire, Streptococcus thermophilus, recombinaison homologue, ISS1.

Keywords: ISS1; Streptococcus thermophilus; homologue recombination; insertional mutagenesis; mutagenèse insertionnelle; random transposition; recombinaison homologue; transposition aléatoire

Document Type: Research Article

Publication date: May 1, 2002

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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