Serina Stefania, Nozza Francesca, Nicastro Giovanna, Faggioni Federico, Mottl Harald, Dehò Gianni, Polissi Alessandra
Dipartimento di Scienze biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan, Italy.
Res Microbiol. 2004 Oct;155(8):692-701. doi: 10.1016/j.resmic.2004.05.006.
Analysis of the complete DNA sequences of many microbial genomes available reveals a fair number of putative ORFs without an identified function. A systematic scan of the Escherichia coli chromosome was achieved by random transposition with a newly developed Tn5 minitransposon derivative carrying the arabinose-inducible araP(BAD) promoter oriented outward at one end (Tn5-araP(BAD)). The transposon insertion mutants obtained were assayed for conditional lethal phenotypes (arabinose dependence or sensitivity), for growth at two temperatures (37 and 15 degrees C) and in different media (rich and minimal medium). The Tn5-araP(BAD)-tagged genes were identified by sequencing the transposon insertion points. In this way we found a new essential gene cluster (yhbN-yhbG), produced conditional lethal (arabinose-dependent) mutations in already known essential genes (folD, frr, plsC, thiL, serS, thrS, and trpS) and provided a new phenotype (cold sensitivity) to other known genes (holD, ahpC, and tolA). Moreover, we identified eight putative ORFs (kch, ycaM, ycbQ, yddA, yddB, ydeK, ydeX, and yliF) that appear to be required in optimum growth conditions (rich medium at 37 degrees C) but not in the cold and in minimal medium.
对现有许多微生物基因组的完整DNA序列进行分析后发现，有相当数量的推定开放阅读框（ORF）功能尚未明确。利用一种新开发的携带阿拉伯糖诱导型araP（BAD）启动子且该启动子一端向外的Tn5微型转座子衍生物（Tn5 - araP（BAD））进行随机转座，实现了对大肠杆菌染色体的系统扫描。对获得的转座子插入突变体进行条件致死表型（阿拉伯糖依赖性或敏感性）检测，以及在两种温度（37和15摄氏度）和不同培养基（丰富培养基和基本培养基）中的生长检测。通过对转座子插入点进行测序来鉴定Tn5 - araP（BAD）标记的基因。通过这种方式，我们发现了一个新的必需基因簇（yhbN - yhbG），在已知的必需基因（folD、frr、plsC、thiL、serS、thrS和trpS）中产生了条件致死（阿拉伯糖依赖性）突变，并为其他已知基因（holD、ahpC和tolA）赋予了一种新的表型（冷敏感性）。此外，我们鉴定出了八个推定的开放阅读框（kch、ycaM、ycbQ、yddA、yddB、ydeK、ydeX和yliF），它们似乎在最佳生长条件（37摄氏度的丰富培养基）下是必需的，但在低温和基本培养基中并非如此。
