Ried J L, Collmer A
Department of Botany, University of Maryland, College Park 20742.
Gene. 1987;57(2-3):239-46. doi: 10.1016/0378-1119(87)90127-2.
A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.
在菊欧文氏菌中展示了一种用于标记交换-消除诱变的技术，该技术能够在革兰氏阴性细菌中构建定向的无标记突变。该技术采用一个携带在3.8 kb BamHI片段上的nptI-sacB-sacR盒，在菊欧文氏菌中赋予卡那霉素（Km）抗性和蔗糖敏感性（由于sacB产生果聚糖蔗糖酶）。将该盒插入克隆的菊欧文氏菌pelC基因（编码果胶酸裂解酶同工酶PLc）的一个Sau3A位点，然后通过基因交换重组导入欧文氏菌基因组。得到的突变体对Km呈抗性、对蔗糖敏感且缺乏PLc。然后通过PstI切割从质粒携带的pelC基因中切除该盒，留下一个28 bp的移码插入。通过选择蔗糖耐受性，将含有28 bp插入的pelC等位基因与含有nptI-sacB-sacR盒的染色体等位基因进行交换。得到的菊欧文氏菌突变体对Km敏感且缺乏PLc。该技术允许构建具有许多定向突变的复杂菌株，而无需引入相应数量的抗生素抗性标记，并且应该证明是有用的，例如，在探索菊欧文氏菌中多个pel基因的作用方面。
