Amino-terminal protein fusions to the TraR quorum-sensing transcription factor enhance protein stability and autoinducer-independent activity.
TraR of Agrobacterium tumefaciens is a member of the LuxR family of quorum-sensing transcription factors and regulates genes required for conjugation and vegetative replication of the tumor-inducing (Ti) plasmid in the presence of the autoinducer 3-oxooctanoyl-homoserine lactone (OOHL). In the absence of OOHL, TraR is rapidly destroyed by proteolysis, suggesting that this ligand is required for TraR folding. To date, no TraR variant has been found that is active in the absence of OOHL. In this study, we conducted whole-cell and plasmid mutagenesis experiments to search for constitutive mutations of traR and identified two constitutive alleles. Surprisingly, neither contained a point mutation within the traR gene, but rather, both encoded fusion proteins between TraR and the N-terminal domain of an aminoglycoside N-acetyltransferase, encoded by a plasmid-borne antibiotic resistance gene present in the original strain. Data from Western immunoblot assays, pulse-chase assays, and immunoprecipitation assays show that these fusion proteins are far more stable to proteolysis than native apo-TraR. We also constructed a library of traR alleles encoding random amino-terminal fusions and selected for constitutive TraR activity. Five independent fusion proteins were identified by this approach. These fusion proteins accumulated to far higher levels than wild-type TraR in the absence of OOHL. One of these fusions was overexpressed in Escherichia coli and showed detectable tra box binding in the absence of OOHL. These data suggest that the native amino terminus of TraR may signal proteolysis and that fusing it to other proteins might sequester it from intracellular proteases.
Chai
Yunrong
Y
Department of Microbiology, Cornell University, Ithaca, NY 14853, USA.
Winans
Stephen C
SC
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006714:Homoserine / Q000031:analogs & derivatives / Q000378:metabolism; D009154:Mutation / Q000235:genetics / Q000502:physiology; D010957:Plasmids / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C080512:TraR protein, Agrobacterium tumefaciens; D006714:Homoserine; D000123:Acetyltransferases; C019681:aminoglycoside acetyltransferase