Jenks P J, Chevalier C, Ecobichon C, Labigne A
Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris, France.
Res Microbiol. 2001 Oct;152(8):725-34. doi: 10.1016/s0923-2508(01)01253-0.
Analysis of the published genome sequences of Helicobacter pylori revealed that approximately 40% of the predicted open reading frames (ORFs) were of unknown function. We have developed the random mutagenesis and loop amplification (RMLA) strategy, and used this approach both to characterize individual virulence factors and to collectively screen comparatively large numbers of H. pylori mutants to identify genes that are not essential for viability in vitro. The mini-Tn3-Km transposon was used to generate a random mutant library in H. pylori strain G27. By screening the library of mutants we were able to demonstrate that the transposon integrated randomly into the chromosome of H. pylori and that RMLA was able to identify mutants in known virulence genes (urease and catalase). To test whether this strategy could be used as a high-throughput approach for the simultaneous identification of a series of nonessential genes of H. pylori, the transposon-chromosomal junctions of a pool of mutants were amplified by inverse PCR using circular fragments of genomic DNA obtained after chromosomal DNA extracted from the pool of mutants had been digested with HindIII and self-ligated. The amplification products were radioactively labelled and hybridized to a high density macroarray membrane containing a duplicated target sequence for every gene of H. pylori strain 26695. For the positive ORFs the precise site of transposon insertion was confirmed by PCR mapping. In total 78 H. pylori genes were unambiguously identified as nonessential for viability in vitro, including 20 with orthologues of unknown function in other species and 21 which were H. pylori-specific.
对已公布的幽门螺杆菌基因组序列分析表明，大约40%的预测开放阅读框（ORF）功能未知。我们开发了随机诱变和环扩增（RMLA）策略，并使用该方法来表征单个毒力因子，以及集体筛选相对大量的幽门螺杆菌突变体，以鉴定体外生存所非必需的基因。mini-Tn3-Km转座子用于在幽门螺杆菌G27菌株中生成随机突变体文库。通过筛选突变体文库，我们能够证明转座子随机整合到幽门螺杆菌染色体中，并且RMLA能够鉴定已知毒力基因（脲酶和过氧化氢酶）中的突变体。为了测试该策略是否可用作高通量方法来同时鉴定幽门螺杆菌的一系列非必需基因，使用从突变体库中提取的染色体DNA经HindIII消化并自身连接后获得的基因组DNA环状片段，通过反向PCR扩增一组突变体的转座子-染色体连接点。扩增产物经放射性标记后与高密度宏阵列膜杂交，该膜包含幽门螺杆菌26695菌株每个基因的重复靶序列。对于阳性ORF，通过PCR定位确认转座子插入的精确位点。总共明确鉴定出78个幽门螺杆菌基因在体外生存是非必需的，其中包括20个在其他物种中具有功能未知直向同源物的基因和21个幽门螺杆菌特异性基因。
