title: Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.
abstract: Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.
authors.affiliation: Institute of Molecular Microbiology and Biotechnology, Westfalian Wilhelms University Münster, Münster, Germany.
authors.forename: Nuria
authors.initials: N
authors.lastname: Gómez-Santos
authors.forename: Anke
authors.initials: A
authors.lastname: Treuner-Lange
authors.forename: Aurelio
authors.initials: A
authors.lastname: Moraleda-Muñoz
authors.forename: Elena
authors.initials: E
authors.lastname: García-Bravo
authors.forename: Raquel
authors.initials: R
authors.lastname: García-Hernández
authors.forename: Marina
authors.initials: M
authors.lastname: Martínez-Cayuela
authors.forename: Juana
authors.initials: J
authors.lastname: Pérez
authors.forename: Lotte
authors.initials: L
authors.lastname: Søgaard-Andersen
authors.forename: José
authors.initials: J
authors.lastname: Muñoz-Dorado
mesh_terms: D003300:Copper / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D005827:Genetics, Microbial / Q000379:methods*; D008124:Locomotion; D008967:Molecular Biology / Q000379:methods*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D016941:Myxococcus xanthus / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D015533:Transcriptional Activation / Q000187:drug effects; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D003300:Copper; D001616:beta-Galactosidase