Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose.
Construction of TnphoA gene fusions in Rhodobacter sphaeroides: isolation and characterization of a respiratory mutant unable to utilize dimethyl sulfoxide as a terminal electron acceptor during anaerobic growth in the dark on glucose.
复制标题
球形红杆菌中 TnphoA 基因融合体的构建：在黑暗中葡萄糖厌氧生长期间无法利用二甲亚砜作为末端电子受体的呼吸突变体的分离和表征。
DOI：
10.1128/jb.171.8.4385-4394.1989
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发表时间：
1989
影响因子：
3.2
通讯作者：
Kaplan,S
中科院分区：
文献类型：
--
作者：
Moore,MD;Kaplan,S
我们已经构建了一个自杀载体，pU 1800，含有转座因子TnphoA（Tn 5 IS 50 L：：phoA），用于在大肠杆菌碱性磷酸酶（APase）和兼性光合异养菌，类球红细菌的蛋白质之间产生蛋白融合的目的。我们将TnphoA基因导入到R.在约1 × 10 ~（-6）的偶联共轭转座频率下，当将接合物接种在含有显色指示剂5-溴-4-氯-3-吲哚磷酸盐的生长培养基上时，通过蓝色菌落着色判断，在约1%的接合物中观察到引起AP酶表达的融合。通过这种方法已经产生了许多可区分的突变体表型，包括那些在无氧呼吸期间缺乏使用二甲基亚砜作为末端电子受体的能力的突变体表型，以及那些不能进行光合作用或色素合成改变的突变体表型，以及其他表达对氯酸盐的抗性的突变体表型。这些突变体中的几个的生长和光谱特性，以及在不同的生理条件下的亚细胞APase活性的定位和定量，已被检查。对于所分析的每个突变体，已证实TnphoA在宿主基因组中的存在，并且已鉴定和定位含有TnphoA的特异性标记的DNA片段;也已经分离出能够补充单个突变体的类球孢菌基因组DNA。这种方法在研究R. sphaeroides进行了讨论。
We have constructed a suicide vector, pU1800, containing the transposable element TnphoA (Tn5 IS50L::phoA), for the purpose of producing protein fusions in vivo between the Escherichia coli alkaline phosphatase (APase) and proteins of the facultative photoheterotroph, Rhodobacter sphaeroides. We introduced TnphoA into the genome of R. sphaeroides at a coupled conjugation-transposition frequency of approximately 1 x 10(-6). Fusions giving rise to APase expression, as judged by blue-colony pigmentation when exconjugants were plated on growth medium containing the chromogenic indicator 5-bromo-4-chloro-3-indolyl phosphate, were observed in about 1% of the exconjugants. Numerous, distinguishable mutant phenotypes have been generated by this method, including those which lack the ability to use dimethyl sulfoxide as a terminal electron acceptor during anaerobic respiration, as well as those which are photosynthetically incompetent or altered in pigment synthesis, and others that express resistance to chlorate. The growth and spectral characteristics of several of these mutants, as well as the localization and quantitation of subcellular APase activity under different physiological conditions, have been examined. The presence of TnphoA in the host genome has been confirmed for each mutant analyzed, and specifically tagged DNA fragments containing TnphoA have been identified and localized; cosmids containing R. sphaeroides genomic DNA capable of complementing individual mutants have also been isolated. The usefulness of this approach in studying gene activity in R. sphaeroides is discussed.