SWIM analysis allows rapid identification of residues involved in invasin-mediated bacterial uptake.
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.
Krukonis
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Tufts University School of Medicine, Howard Hughes Medical Institute, Department of Molecular Biology, Microbiology, 136 Harrison Avenue M&V 409, Boston, MA 02111, USA.
Isberg
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D018829:Adhesins, Bacterial; D000409:Alanine / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D018407:Bacterial Physiological Phenomena / Q000187:drug effects; D001426:Bacterial Proteins / Q000737:chemistry*; D004926:Escherichia coli / Q000502:physiology*; D008322:Mammals; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010587:Phagocytosis / Q000502:physiology*; D017422:Sequence Analysis, DNA; D014407:Tumor Cells, Cultured; D015011:Yersinia pseudotuberculosis / Q000737:chemistry*
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; C053476:invasin, Yersinia; D001224:Aspartic Acid; D000409:Alanine