Selection of motile nonchemotactic mutants of Escherichia coli by field-flow fractionation.

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RESUMO

We have developed a chromatographic method for isolating bacterial cells that are motile but nonchemotactic. Separation of strains of different phenotype occurs along a thin horizontal channel between two stirred chambers, the lower one containing a chemical attractant. The channel is bounded above and below by rigid filters, permeable to the attractant but not to the bacteria. The lower part of the channel is occupied by a porous plate comprising a vertical array of capillary tubes. An aliquot of cells is injected at one end of the channel and eluted by continuous flow of cell-free medium. Fluid leaving the other end of the channel is collected in a fraction collector. Cells that respond to the gradient swim to the bottom of the channel where they are retarded by the capillary array. Nonmotile cells sink to the bottom and are trapped in a similar manner. Motile cells that fail to respond to the gradient diffuse across the full height of the channel and, thus, travel through the apparatus at the average velocity of the eluent. When mixed with wild-type cells at a ratio of 1:1000 and subjected to an aspartate gradient, aspartate-blind cells were recovered quantitatively. The enrichment was approximately 200 to 1. The wild-type cells that survived the selection had a poorly motile phenotype.

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