Site-directed mutagenesis of a LuxR-type quorum-sensing transcription factor: alteration of autoinducer specificity.
TraR is a quorum-sensing transcription factor from Agrobacterium tumefaciens that regulates replication and conjugation genes of the tumour-inducing (Ti) plasmid. TraR activity requires the autoinducer pheromone N-3-oxooctanoyl-l-homoserine lactone (OOHL). Structural studies of TraR-OOHL-DNA complexes showed that one molecule of OOHL is completely engulfed within the N-terminal domain of each TraR subunit. TraR is thought to bind OOHL via four hydrogen bonds, three of them direct and one water mediated, and by numerous hydrophobic interactions. Here, we show that all residues predicted to hydrogen bond with OOHL are essential for wild-type protein function. Mutants that failed to detect OOHL in vivo invariably failed to sequester exogenous OOHL. We showed previously that TraR is protected from cellular proteases by OOHL, and now show that mutants that failed to detect OOHL were also not protected from proteolysis by OOHL. We also describe several mutants with altered autoinducer specificity. Three mutants (T129V, T129A and T115I) detected 3-oxo-AHLs and 3-unsubstituted AHLs with equal sensitivity, indicating that these mutations perturb the water-mediated hydrogen bond to the 3-oxo moiety of OOHL. Three other mutants (A49I, A49M and Q58L) preferentially detected AHLs containing six or seven carbon atoms rather than eight. The bulkier residues in these mutations appear to have occupied a portion of the OOHL binding site, interfering with binding of the acyl chain of AHLs.
Chai
Yunrong
Y
Department of Microbiology, 360 A Wing Hall, Cornell University, Ithaca, New York 14853, USA.
Winans
Stephen C
SC
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010675:Pheromones / Q000737:chemistry / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D010675:Pheromones; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C064778:LuxR autoinducer binding proteins; D015107:4-Butyrolactone