Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection.
A complete understanding of host-parasite interactions must necessarily include the identification and characterization of gene products expressed by both parties during the infectious process. We have developed a new screen to identify bacterial genes that are transcriptionally induced during infection of a host animal. The method is based on pre-selection of strains carrying tnpR operon fusions (encoding resolvase, a site-specific DNA recombinase) which are not expressed in vitro, followed by screening for a subset of these strains that subsequently express resolvase within the host environment. The latter subset was recognized as recombinants that had deleted a resolvase-specific reporter construct. Thirteen transcription units of Vibrio cholerae were identified that were induced during infection in an infant mouse model of cholera. Five of these were predicted to encode polypeptides with diverse functions in metabolism, biosynthesis and motility; one encoded a secreted lipase; two appear to be antisense to genes involved in motility; and five are predicted to encode polypeptides of unknown function. Three of the transcripts were shown to be required for full virulence in infant mice, as assessed by competition experiments.
Camilli
A
A
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA. acamilli@opal.tufts.edu
Mekalanos
J J
JJ
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D002465:Cell Movement / Q000235:genetics; D002633:Chemotaxis / Q000235:genetics; D002771:Cholera / Q000235:genetics*; D003001:Cloning, Molecular; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D016373:DNA, Antisense / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005798:Genes, Bacterial; D019426:Integrases; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014158:Transcription, Genetic; D019895:Transposases; D014734:Vibrio cholerae / Q000235:genetics* / Q000502:physiology; D014774:Virulence / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D016373:DNA, Antisense; D010455:Peptides; D011993:Recombinant Fusion Proteins; D045522:Recombinases; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D019895:Transposases; C115006:integron integrase IntI1; D008049:Lipase; D001616:beta-Galactosidase