Analysis of Vibrio cholerae ToxR function by construction of novel fusion proteins.
The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), beta-lactamase (monomeric, ToxR-Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR transmembrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function.
Ottemann
K M
KM
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Mekalanos
J J
JJ
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D015169:Colony Count, Microbial; D003470:Culture Media / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting; D005809:Genes, Regulator; D007421:Intestine, Small / Q000382:microbiology; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010405:Penicillinase / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D027981:Symporters; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014734:Vibrio cholerae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity
D001426:Bacterial Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D027981:Symporters; D014157:Transcription Factors; C499324:toxR protein, Vibrio cholerae; C066719:toxR protein, bacteria; C021499:lactose permease; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D010405:Penicillinase