title: Activity-Based Screening of Metagenomic Libraries for Hydrogenase Enzymes.
abstract: Here we outline how to identify hydrogenase enzymes from metagenomic libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis (ΔhyaB) via triparental mating. If a fosmid exhibits hydrogen uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. This new method enables screening of 48 metagenomic fosmid clones in parallel.
authors.affiliation: Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Adam
authors.affiliation: Molecular Biology of Microbial Consortia, Biocenter Klein Flottbek, University of Hamburg, Ohnhorststr. 18, 22609, Hamburg, Germany. mirjam.perner@uni-hamburg.de.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Perner
mesh_terms: D004789:Enzyme Activation; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics* / Q000378:metabolism*; D054892:Metagenome; D056186:Metagenomics / Q000379:methods
chemical_list: D006859:Hydrogen; D006864:Hydrogenase
keywords: Function-based screen; Hydrogen uptake; Hydrogenase; Metagenome