Harker A R, Lambert G R, Hanus F J, Evans H J
J Bacteriol. 1985 Oct;164(1):187-91. doi: 10.1128/jb.164.1.187-191.1985.
Eight strains of Rhizobium lacking hydrogenase uptake (Hup) activity and 17 transconjugant strains carrying the hup cosmids pHU1, pHU52, or pHU53 (G. R. Lambert, M. A. Cantrell, F. J. Hanus, S. A. Russell, K. R. Haddad, and H. J. Evans, Proc. Natl. Acad. Sci. USA, 82:3232-3236, 1985) were screened for Hup activity and the presence of immunologically detectable hydrogenase polypeptides. Crude extracts of these strains were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis with affinity-purified antibodies against the two subunits of purified hydrogenase (Mr 60,000 and 30,000). Derepressed transconjugants carrying the cosmid pHU52 were Hup+ and contained detectable levels of both hydrogenase subunit polypeptides. Non-derepressed strains, Hup- parent strains, and strains carrying cosmids other than pHU52 did not express Hup activity and contained no immunologically detectable protein. These data provide further evidence for the essential involvement of the smaller (Mr 30,000) subunit in the expression of hydrogenase activity in Rhizobium japonicum and suggest that the determinants for hydrogenase subunit synthesis are present on pHU52.
对8株缺乏吸氢酶（Hup）活性的根瘤菌菌株以及17株携带hup黏粒pHU1、pHU52或pHU53的转接合子菌株（G. R. 兰伯特、M. A. 坎特雷尔、F. J. 哈努斯、S. A. 拉塞尔、K. R. 哈达德和H. J. 埃文斯，《美国国家科学院院刊》，82:3232 - 3236，1985）进行了Hup活性筛选以及免疫可检测的氢化酶多肽存在情况的检测。对这些菌株的粗提取物进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，并使用针对纯化氢化酶的两个亚基（分子量60,000和30,000）的亲和纯化抗体进行蛋白质印迹分析。携带黏粒pHU52的去阻遏转接合子是Hup +，并且含有可检测水平的两种氢化酶亚基多肽。未去阻遏的菌株、Hup - 亲本菌株以及携带除pHU52以外黏粒的菌株均不表达Hup活性，且不含有免疫可检测的蛋白质。这些数据为较小（分子量30,000）亚基在日本根瘤菌氢化酶活性表达中的关键作用提供了进一步证据，并表明氢化酶亚基合成的决定因素存在于pHU52上。
