Open AccessRapid Colony Screening Method for Identifying Hydrogenase Activity in Rhizobium japonicum
Author(s) -
Richard A. Haugland,
F. J. Hanus,
Michael A. Cantrell,
Harold J. Evans
Publication year - 1983
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.45.3.892-897.1983
Subject(s) - hydrogenase , strain (injury) , mutant , biology , methylene blue , rhizobiaceae , microbiology and biotechnology , rhizobium , bacteria , gene , biochemistry , enzyme , genetics , catalysis , symbiosis , anatomy , photocatalysis
A method has been developed for the rapid screening ofRhizobium japonicum colonies for hydrogenase activity based on their ability to reduce methylene blue in the presence of respiratory inhibitors and hydrogen. Hydrogen uptake-positive (Hup+ ) colonies derepressed for hydrogenase activity were visualized by their localized decolorization of filter paper disks impregnated with the dye. Appropriate responses were seen with a number of Hup+ and Hup− wild-type strains ofR. japonicum as well as Hup− mutants. Its specificity was further confirmed in selected strains on the basis of comparisons with chemolithotrophic growth and the presence of other genetic markers. Utilization of the method in identifying Hup+ colonies among 16,000 merodiploid derivatives of the Hup− mutant strain PJ17nal containing cloned DNA fragments of the Hup+ strain 122 DES has demonstrated its applicability as a screening procedure in the genetic analysis of theR. japonicum hydrogen uptake system.
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