SWIM ANALYSIS ALLOWS RAPID IDENTIFICATION OF RESIDUES INVOLVED IN INVASIN-MEDIATED BACTERIAL UPTAKE

The Yersinia pseudotuberculosis invasin protein promotes bacterial upt ake into normally non-phagocytic cells. Combinations of six alanine su bstitutions in a region of invasin previously shown to be important fo r bacterial internalization were analyzed using binomial and codon mut agenesis 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 ma intained activity. Results of the single sequencing run performed on t he pool indicated that strains harboring the D911A substitution were a bsent after enrichment, confirming the importance of an aspartate resi due at this site. When single clones were subsequently isolated from t he pool, those containing multiple alanine substitutions in invasin sh owed uptake defects that were additive, with the exception of S904A/M9 12A and S910A/M912A double mutants. Binomial mutagenesis combined with a pooled enrichment and sequencing strategy, called 'SWIM' mutagenesi s (selection without isolation of mutants), could be applied to any sy stem for which there exists an enrichment scheme, using a single oligo nucleotide pool to analyze multiple residues. (C) 1998 Elsevier Scienc e B.V. All rights reserved.