Construction of hermes shuttle vectors: a versatile system useful for genetic complementation of transformable and non-transformableNeisseria mutants |
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Authors: | Eva-Marià Kupsch Dominique Aubel Carol P Gibbs Andreas F Kahrs Thomas Rudel and Thomas F Meyer |
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Institution: | (1) Abteilung Infektionsbiologie, Max-Planck-Institut für Biologie, Spemannstr. 34, D-72076 Tübingen, Germany;(2) Present address: Dow Europe SA, CH-8810 Horgen, Switzerland;(3) Present address: Immuno AG, A-2304 Orth a.d. Donau, Austria;(4) Present address: Evotec GmbH, Grandweg 64, D-22529 Hamburg, Germany |
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Abstract: | A versatile shuttle system has been developed for genetic complementation with cloned genes of transformable and non-transformableNeisseria mutants. By random insertion of a selectable marker into the conjugativeNeisseria plasmidptetM25.2, a site within this plasmid was identified that is compatible with plasmid replication and with conjugative transfer of plasmid. Regions flanking the permissive insertion site of ptetM25.2 were cloned inEscherichia coli and served as a basis for the construction of the Hermes vectors. Hermes vectors are composed of anE. coli replicon that does not support autonomous replication inNeisseria, e.g. ColE1, p15A, orori
fd, fused with a shuttle consisting of a selectable marker and a multiple cloning site flanked by the integration region of ptetM25.2. Complementation of a non-transformableNeisseria strain involves a three-step process: (i) insertion of the desired gene into a Hermes vector; (ii) transformation of Hermes into aNeisseria strain containing ptetM25.2 to create a hybrid ptetM25.2 via gene replacement by the Hermes shuttle cassette; and (iii) conjugative transfer of the hybrid ptetM25.2 into the finalNeisseria recipient. Several applications for the genetic manipulation of pathogenicNeisseriae are described. |
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Keywords: | Plasmid vector Conjugation Generalized mutagenesis Homologous recombination Natural transformation competence |
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