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首页> 外文期刊>Journal of bacteriology >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.
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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.

机译：球形球形红细菌中TnphoA基因融合体的构建：在黑暗中葡萄糖无氧生长过程中无法利用二甲基亚砜作为末端电子受体的呼吸突变体的分离和鉴定。

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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.
机译：我们构建了一个自杀载体pU1800，该载体包含转座因子TnphoA（Tn5 IS50L :: phoA），目的是在体内产生大肠杆菌碱性磷酸酶（APase）与兼性光异养细菌球形红球菌蛋白质之间的蛋白质融合。我们以大约1 x 10（-6）的偶联共轭转座频率将TnphoA引入到球形红球菌的基因组中。当将准接合剂涂在含有生色指示剂5-溴-4-氯-3-吲哚基磷酸的生长培养基上时，通过蓝殖民地色素沉着判断，融合物会引起APase表达，在大约1％的准接合剂中观察到。通过这种方法已经产生了许多可区分的突变表型，包括在厌氧呼吸过程中缺乏使用二甲基亚砜作为末端电子受体的能力，以及在色素合成中光合无能或改变的那些，以及其他表现出抗性的表型。去氯酸盐。研究了这些突变体中几种突变体的生长和光谱特征，以及在不同生理条件下亚细胞APase活性的定位和定量。对于每个分析的突变体，已经证实宿主基因组中存在TnphoA，并且已经鉴定并定位了包含TnphoA的特定标记DNA片段。还已经分离出含有能够互补单个突变体的球形红球菌基因组DNA的粘粒。讨论了该方法在研究球形红球菌基因活性中的有用性。

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