de Jonge Ramon, Bakker Dennis, van Vliet Arnoud H M, Kuipers Ernst J, Vandenbroucke-Grauls Christina M J E, Kusters Johannes G
Department of Gastroenterology and Hepatology, Erasmus MC-University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands.
J Microbiol Methods. 2003 Jan;52(1):93-100. doi: 10.1016/s0167-7012(02)00136-7.
Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.
随机插入诱变是一种广泛用于鉴定细菌毒力基因的技术。大多数随机诱变策略涉及在大肠杆菌中克隆以传代质粒或进行表型选择。由于克隆DNA的限制或不稳定性，或编码产物的毒性，这可能导致有偏向性的选择。因此，我们使用一种简单、直接的诱变技术，在人类病原体幽门螺杆菌中创建了两个突变体文库，该技术不需要大肠杆菌作为中间宿主。幽门螺杆菌的总DNA被消化、环化，然后用一种切割频率高的限制性内切酶再次消化，所得片段与卡那霉素抗生素抗性盒连接。随后，将连接混合物转化到亲本幽门螺杆菌菌株1061中。通过双同源重组将卡那霉素盒插入幽门螺杆菌1061的基因组中，产生了大约2500个卡那霉素抗性菌落。通过Southern印迹法证实了卡那霉素盒插入的异质性。从该文库中分离出两个脲酶产生缺陷的独立幽门螺杆菌突变体，突出了这种诱变策略的潜力。
