Laboratory for Nitrogen Fixation Research, Oregon State University, Corvallis, Oregon 97331.
Appl Environ Microbiol. 1983 Mar;45(3):892-7. doi: 10.1128/aem.45.3.892-897.1983.
A method has been developed for the rapid screening of Rhizobium 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 of R. 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 the R. japonicum hydrogen uptake system.
已经开发出一种基于呼吸抑制剂和氢气存在下还原亚甲基蓝能力的快速筛选根瘤菌（Rhizobium japonicum）菌落产氢酶活性的方法。通过用染料浸渍的滤纸圆盘的局部脱色，可以可视化具有吸氢活性的吸氢阳性（Hup）菌落。该方法适用于许多吸氢和野生型根瘤菌（Rhizobium japonicum）菌株以及吸氢突变体。根据与化能自养生长和其他遗传标记的存在的比较，在选定的菌株中进一步证实了其特异性。在含有克隆的 Hup 菌株 122DES DNA 片段的 Hup 突变株 PJ17nal 的 16000 个杂种二倍体衍生物中鉴定 Hup 菌落的方法的应用表明，它可作为根瘤菌（Rhizobium japonicum）吸氢系统遗传分析中的筛选程序。
