FIG. 3.

STM screen for KLA-attenuated mutants. (A) Schematic diagram showing the STM screen conducted in the oral infection model. A total of 2,880 mutants that produced the wild-type capsule were selected and arrayed in 60 MPs. Three 8-week-old BALB/c mice were orally administered 10⁷ CFU of bacterial culture of a particular MP pool which contains 48 uniquely tagged mutants. Livers and spleens were harvested from these mice, adequate dilutions of tissue homogenates were plated onto kanamycin-LB agar, and at least 3,000 colonies for each output were pooled for the preparation of bacterial genomic DNA. The tag mixtures carried by the mutants in each of the inocula and the output pools were PCR amplified and spotted on 48 replica blots. Each unique fluorescein-labeled tag was subsequently used as a probe for hybridization to those blots, and the mutants whose associated tags were detected in the inocula but not in the pools recovered from either the liver or the spleen were identified as KLA attenuated, indicated by circles in panel B.