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Mutations were introduced into the EAF plasmid by homologous recombination after first introducing the gene-specific changes into the respective fragments subcloned into pBlueScript (Stratagene). We used a limited polymerase chain reaction (PCR)-based mutational strategy to reduce the chance of introducing secondary mutations; minimal regions of the PCR-amplified products containing the desired changes were ligated into existing subclones. The limited PCR regions could easily be sequenced and shown to be free of extraneous mutations. To generate the desired alteration on the EAF plasmid in B171-8, we performed suicide vector-directed homologous double recombination as described in (7, 11). We generated B171-8ΔAcm by replacing the pilinencoding bfpA gene with a chloramphenicol acetyltransferase gene; transcription of the remaining bfp operon genes was unaffected (11, 16). B171-8T::Gm has sustained an insertional disruption of bfpT (13). Wild-type phenotypes were restored by providing the mutants with a normal copy of the respective gene in trans on a low-copy-number plasmid.
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37
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600 for each inoculation. Volunteers took nothing by mouth for 2 hours before challenge. They ingested 150 ml of a 1.3% sodium bicarbonate solution 1 minute before ingesting 30 ml of the dose of challenge bacteria in phosphate-buffered saline plus 1.3% sodium bicarbonate. All stool samples were collected for 48 hours. The nursing staff were unaware of the nature of the challenge bacteria and scored the stool specimens as formed/ semiformed or liquid. The end point of this study was development of diarrhea as indicated by the volume and number of liquid stools produced in the first 48 hours. Forty-eight hours after ingesting the bacteria the volunteers were treated with ciprofloxacin and were released from the GCRC after their stools were determined to be negative for E. coli.
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Amino acid residues are abbreviated as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, Xaa; Y, Tyr.
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ma) were generated by a modification of the Stratagene QuikChange mutagenesis strategy. A pair of primers carrying the intended nucleotide changes but otherwise complementary to the sequences encoding the Walker box A region of either protein were used for amplification. To minimize unintentional changes in the nucleotide sequence, we used the limited PCR-based strategy (22) to replace the homologous wild-type fragment that had been cloned into a modified version of suicide plasmid pGP704 (31). The appropriate loci of the EAF plasmid were replaced by the intended mutations by using the suicide vector-driven homologous recombination strategy employed above. Complementation of the D and F mutations was accomplished by providing a copy of the wild-type gene on a low-copy-number plasmid, which restored the normal LA and autoaggregation phenotypes.
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Scanning electron microscopy was performed at the Microscope and Graphic Imaging Center, California State University, Hayward (N. R. Smith, Director) and at the SETI Institute, NASA Ames Research Center. The studies of volunteers were performed in the General Clinical Research Center (GCRC) at Stanford University Medical Center. Supported by Health and Human Services grants 1R03-DK52038 and 1R01-Al39521 from the National Institutes of Health and by Health and Human Services grant M01-RR00070 from the General Clinical Research Program, National Institutes of Health. We thank D. Kaiser, B. Stocker, and B. W. Brown for helpful suggestions and critical reading of the manuscript; R. Valdivia and S. Falkow for providing the GFP plasmid; S. R. Kushner for providing pWKS plasmids; J. Giron for providing the BFP antiserum; J. Engel for the suggestion to use GFP to mark the individual bacteria; the nursing staff of the GCRC and the volunteers for their participation in the study; and N. Smith and K. Kato for their advice and generous use of the SEMs.
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