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Volumn 280, Issue 5372, 1998, Pages 2114-2118

Type IV pili, transient bacterial aggregates, and virulence of enteropathogenic escherichia coli

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL VIRULENCE; BACTERIUM ADHERENCE; BACTERIUM PILUS; DIARRHEA; ESCHERICHIA COLI; NONHUMAN; PRIORITY JOURNAL; REVIEW;

EID: 0032568984     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5372.2114     Document Type: Review
Times cited : (391)

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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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    • 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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