Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.
Complementation of an Escherichia coli proC mutation by a gene cloned from Treponema pallidum.
复制标题
梅毒螺旋体克隆基因对大肠杆菌 proC 突变的补充。
DOI：
10.1128/jb.172.6.2996-3002.1990
复制
发表时间：
1990
影响因子：
3.2
通讯作者：
BassfordJr,PJ
中科院分区：
文献类型：
--
作者：
Gherardini,FC;Hobbs,MM;Stamm,LV;BassfordJr,PJ
关于梅毒病原体梅毒螺旋体的生物合成和代谢能力知之甚少，因为无法在体外连续培养该生物体。为了解决培养的问题，研究人员利用重组DNA技术在大肠杆菌中表达密螺旋体蛋白抗原。然而，除了少数值得注意的例外，这些克隆的密螺旋体蛋白的特定细胞作用尚未确定。本研究利用T.通过在体内重新包装感染性λ噬菌体颗粒构建并扩增苍白球基因组DNA。能够互补E. coliproC基因编码1-吡咯啉-5-羧酸（P5 C）还原酶（EC 1.5.1.2）。互补活性最终定位于2.3-内切酶BglII-HindIII片段，该片段与T.苍白球DNA两个蛋白质的41和27千道尔顿（kDa）的编码由该片段，确定由maxicell分析。虽然只有41-kDa的蛋白质可以特异性沉淀实验梅毒兔抗血清，这是27-kDa的蛋白质，负责前C-互补活性。重组P5 C还原酶不同于天然E.大肠杆菌酶的一些生化特性。T. pallidum基因编码P5 C还原酶，强烈表明该病原体具有合成脯氨酸和可能的其他氨基酸的能力。
Little is known concerning the biosynthetic and metabolic capabilities of the syphilis agent, Treponema pallidum, because of the inability to cultivate continuously the organism in vitro. To circumvent the problem of cultivation, researchers have used recombinant DNA technology to express treponemal protein antigens in Escherichia coli. However, with a few notable exceptions, the specific cellular roles of these cloned treponemal proteins have not been determined. In this study, a cosmid library of T. pallidum genomic DNA was constructed and amplified by repackaging infective lambda bacteriophage particles in vivo. Recombinant clones capable of complementing a null mutation in the E. coli proC gene encoding 1-pyrroline-5-carboxylate (P5C) reductase (EC 1.5.1.2) were subsequently identified. The complementing activity was eventually localized to a 2.3-kilobase BglII-HindIII fragment that hybridized to the same-size fragment of a BglII-HindIII digest of T. pallidum DNA. Two proteins of 41 and 27 kilodaltons (kDa) were encoded by this fragment, as determined by maxicell analysis. Although only the 41-kDa protein could be specifically precipitated by experimental syphilitic rabbit antisera, it was the 27-kDa protein that was responsible for the proC-complementing activity. The recombinant P5C reductase differed from the native E. coli enzyme by a number of biochemical properties. The cloning of a T. pallidum gene encoding P5C reductase strongly suggests that this pathogen has the ability to synthesize proline and possibly other amino acids.