Krukonis E S, Isberg R R
Tufts University School of Medicine, Howard Hughes Medical Institute, Department of Molecular Biology, Microbiology, 136 Harrison Avenue M&V 409, Boston, MA 02111, USA.
Gene. 1998 Apr 28;211(1):109-16. doi: 10.1016/s0378-1119(98)00087-0.
The Yersinia pseudotuberculosis invasin protein promotes bacterial uptake into normally non-phagocytic cells. Combinations of six alanine substitutions in a region of invasin previously shown to be important for bacterial internalization were analyzed using binomial and codon mutagenesis strategies. A single pool of mutants, potentially containing 64 derivatives with various combinations of alanine substitutions, was enriched by one passage through HEp2 cells. DNA was isolated from the resulting pool of internalization-competent bacteria and sequenced in a single set of reactions to determine which alanine substitutions maintained activity. Results of the single sequencing run performed on the pool indicated that strains harboring the D911A substitution were absent after enrichment, confirming the importance of an aspartate residue at this site. When single clones were subsequently isolated from the pool, those containing multiple alanine substitutions in invasin showed uptake defects that were additive, with the exception of S904A/M912A and S910A/M912A double mutants. Binomial mutagenesis combined with a pooled enrichment and sequencing strategy, called 'SWIM' mutagenesis (selection without isolation of mutants), could be applied to any system for which there exists an enrichment scheme, using a single oligonucleotide pool to analyze multiple residues.
耶尔森氏假结核菌侵袭蛋白可促进细菌被正常非吞噬细胞摄取。利用二项式和密码子诱变策略，分析了侵袭蛋白中一个先前已证明对细菌内化很重要的区域内六个丙氨酸取代的组合。通过在HEp2细胞中传代一次，富集了一个可能包含64种具有不同丙氨酸取代组合衍生物的单一突变体库。从产生的具有内化能力的细菌库中分离DNA，并在一组单一反应中进行测序，以确定哪些丙氨酸取代保持活性。对该库进行的单次测序结果表明，富集后含有D911A取代的菌株不存在，证实了该位点天冬氨酸残基的重要性。随后从该库中分离出单个克隆时，侵袭蛋白中含有多个丙氨酸取代的那些克隆表现出累加的摄取缺陷，但S904A/M912A和S910A/M912A双突变体除外。二项式诱变与一种称为“SWIM”诱变（不分离突变体进行选择）的汇集富集和测序策略相结合，可应用于任何存在富集方案的系统，使用单一寡核苷酸库来分析多个残基。
