Chang Y Y, Cronan J E
J Bacteriol. 1982 Sep;151(3):1279-89. doi: 10.1128/jb.151.3.1279-1289.1982.
Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from [1-14C]pyruvate by the method of Tabor et al. (J. Bacteriol. 128:485-486, 1976). One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well. To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon. These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from [1-14C]pyruvate. This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction. Calculations indicate that a similar enrichment should be expected for other genes. The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses. The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies. Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E. coli genetic map. The poxA locus is located at 94 min, close to the psd locus. The clockwise gene order is ampA, poxA, psd, purA. The poxA mutation is recessive and appears to be a regulatory gene.
通过采用塔博尔等人（《细菌学杂志》128:485 - 486, 1976年）的方法，从[1 - 14C]丙酮酸中筛选14CO2的产生情况，分离出了丙酮酸氧化酶缺陷的大肠杆菌K - 12突变体。其中一个损伤位点（命名为poxA）使丙酮酸氧化酶活性降至正常水平的10%至15%，但生长良好。为了定位这个非选择性突变，我们分离出了转座子Tn10插入到靠近poxA位点的染色体中的菌株，并对转座子进行了定位。这些插入是通过以下步骤分离得到的：（i）通过从λ::Tn10载体进行转座，制备转座子Tn10插入到两种不同Hfr菌株染色体中的文库；（ii）然后将这些携带Tn10的菌株与poxA受体菌株进行交配，并筛选出四环素抗性（Tetr）重组体；（iii）接着对Tetr重组体进行筛选，检测其从[1 - 14C]丙酮酸中产生14CO2的情况。与转导相比，该方法显示出在poxA基因附近Tn10插入的富集倍数大于40倍。计算表明，对于其他基因也应预期有类似的富集。这种富集是由于与转导杂交相比，在接合杂交中发现选择标记和未选择标记之间强连锁的图谱间隔要大得多。使用Hfr接合转移可以通过对数百个而非数千个菌落进行评分，分离出与非选择性基因紧密连锁的转座子插入。利用一个与poxA位点共转导率大于98%的Tn10插入，我们在大肠杆菌遗传图谱上定位了poxA基因。poxA位点位于94分钟处，靠近psd位点。顺时针方向的基因顺序是ampA、poxA、psd、purA。poxA突变是隐性的，似乎是一个调控基因。
