Amino-Terminal Protein Fusions to the TraR Quorum-Sensing Transcription Factor Enhance Protein Stability and Autoinducer-Independent Activity

Abstract

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 aminoterminal 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.Acyl-homoserine lactone (AHL)-based quorum-sensing systems are widespread cell-cell communication systems found throughout the proteobacteria. These systems control diverse sets of genes in a population density-dependent manner and regulate diverse biological functions, including bioluminescence, virulence, the formation of biofilms, exopolysaccharide production, and plasmid conjugation (10,21,35). These systems involve two major components. First, they have an AHL synthase, which usually resembles the LuxI protein of Vibrio fischeri and which synthesizes chemical signals that diffuse across the cell membrane. The other component is an AHL signal receptor and transcription factor that resembles the V. fischeri LuxR protein. These transcription factors consist of two domains, an N-terminal AHL binding domain and a Cterminal DNA binding domain (8,14). These transcription factors are thought to bind to cis-acting operator sequences located in the promoter region of target genes (36).Although a large number of putative LuxR family members have been identified, only a few of them have been extensively studied genetically, biochemically, and structurally. When overexpressed in Escherichia coli, several LuxR-type proteins accumulate only in an insoluble form in the absence of their cognate autoinducers but are highly soluble in their presence. These proteins inc...

Signal Discrimination by Differential Regulation of Protein Stability in Quorum Sensing

“…This observation led to the speculation that the N-terminal AHL binding domain of LuxR-type proteins is responsible for “masking” the DNA-binding activity of the C-terminal domain 32. However, a search for a constitutively active form of TraR via random mutagenesis resulted in N-terminal fusion of TraR to an aminoglycoside N-acetyltransferase that enhanced the overall protein stability and allowed TraR to activate downstream gene expression with its cognate signal 33. This indicates a potential clarification to the previously mentioned theory on the function of the N-terminus of LuxR analogs: the ability of the N-terminus to “mask” the DNA-binding activity of the C-terminus is potentially due to the destabilization or otherwise enhanced affinity for proteolysis it confers upon the protein rather than the capacity to physically cover the DNA-binding domain alone.…”

Characterization of a New Plasmid-Like Prophage in a Pandemic Vibrio parahaemolyticus O3:K6 Strain

“…NP_490571), Escherichia coli (45% identity) (GenBank accession no. YP_538713) and other species, and it regulates genes that are required for conjugation (10) and may also associate with the transfer of the plasmidlike genome of VP882.…”

Use of Bacterial Quorum-Sensing Components to Regulate Gene Expression in Plants

“…Crystal structure studies show that TraR contains a helix-turn-helix motif (Zhu and Winans, 1999;Zhang et al, 2002). The C-terminal region of the TraR protein is for DNA binding and transcriptional activation, whereas the N-terminal region is involved in dimerization and proteolysis signaling (Chai and Winans, 2005). A recent study also indicates that a stable homodimer forms only when TraR binds to a single molecule of OOHL in a 1:1 ratio (Zhang et al, 2002).…”