Summary
The structural gene sacB encoding B. subtilis levansucrase, a secreted enzyme, expresses in E. coli. E. coli hosts of the sacB gene are poisoned by sucrose. This property allowed a powerful selection of mutants affected in the cloned gene.
The plasmidic mutations were readily introduced in the B. subtilis chromosome. Using a collection of plasmids bearing various deletions extending in sacB we developed a technique of deletion mapping based on plasmid integration in the chromosome of B. subtilis. A generalization of this technique is discussed.
Résumé
Le gène de structure sacB d'une enzyme sécrétée de Bacillus subtilis, la lévane-saccharase, a été clonè dans Escherichia coli et s'y exprime (Gay et coll. 1983). Les souches d'E. coli qui synthétisent la lévane-saccharase sont intoxiquées par le saccharose. Cette propriété a permis la mise au point d'un crible sélectif d'isolement de mutants affectés dans le gène sacB cloné. Les mutations plasmidiques dans sacB peuvent aisément être introduites dans le chromosome de B. subtilis. Par ailleurs, une série de plasmides portant des fragments plus ou moins longs de sacB ont été construits, qui peuvent s'intégrer dans le chromosome de B. subtilis et être utilisés pour réaliser la cartographie par délétion de sacB. L'adapatation de cette technique à l'étude d'autres gènes ou groupes de gènes de B. subtili est brièvement discutée.
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Abbreviations
- AP:
-
ampicilline
- ApR/S :
-
résistant/sensible à l'ampicilline 150 μg/ml
- CM:
-
chloramphénicol
- CmR/S :
-
résistant/sensible au chloremphénicol (25 μg/ml pour E. coli, 5 μG/ml pour B. subtilis)
- His+/- :
-
prototrophie/auxotrophic pour l'histidine
- Lev+/- :
-
synthèse/absence de synthèse de lévanes
- Sac:
-
saccharose
- SacR/S :
-
résistant/sensible au saccharose (60 mM)
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Memoire présenté par K. Illmensee
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Steinmetz, M., Le Coq, D., Djemia, H.B. et al. Analyse génétique de sacB, gène de structure d'une enzyme secrétée, la lévane-saccharase de Bacillus subtilis Marburg. Molec Gen Genet 191, 138–144 (1983). https://doi.org/10.1007/BF00330901
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DOI: https://doi.org/10.1007/BF00330901