Abstract
A sub-group of LuxR family of proteins that plays important roles in quorum sensing, a process of cell-cell communication, is widespread in proteobacteria. These proteins have a typical modular structure consisting of N-ter autoinducer binding and C-ter helix-turn-helix (HTH) DNA binding domains. The autoinducer binding domain recognizes signaling molecules which are most often N-acyl homoserine lactones (AHLs) but could also be other novel and yet unidentified molecules. In this study we carried out a series of specific domain swapping and promoter activation experiments as a first step to engineer synthetic signaling modules, taking advantage of the modularity and the versatile/diverse signal specificities of LuxR proteins. In our experiments the N-ter domains from different LuxR homologs were either interchanged or placed in tandem followed by a C-ter domain. The rational design of the hybrid proteins was supported by a structure-based homology modeling studies of three members of the LuxR family (i.e., LasR, RhlR, and OryR being chosen for their unique ligand binding specificities) and of selected chimeras. Our results reveal that these LuxR homologs were able to activate promoter elements that were not their usual targets; we also show that hybrid LuxR proteins retained the ability to recognize the signal specific for their N- ter autoinducer binding domain. However, the activity of hybrid LuxR proteins containing two AHL binding domains in tandem appears to depend on the organization and nature of the introduced domains. This study represents advances in the understanding of the modularity of LuxR proteins and provides additional possibilities to use hybrid proteins in both basic and applied synthetic biology based research.
Highlights
Quorum sensing (QS) is a system of bacterial communication which involves the synthesis and detection of chemical molecules called autoinducers to regulate community-specific traits in a cell density dependent manner (Engebrecht et al, 1983; Kaplan and Greenberg, 1987; Fuqua et al, 1994; Fuqua and Greenberg, 2002)
Regulate Foreign Promoters In order to assess whether PAB LuxR solos could bind and activate target promoters of a canonical LuxR protein, βgalactosidase assays were carried out with specific promoterlacZ reporter fusion constructs as described in the Materials and Methods section
We tested the ability of the PAB LuxR solo (OryR) of Xanthomonas oryzae pv. oryzae (Xoo) to activate promoter regions of lasI and rhlI in E. coli
Summary
Quorum sensing (QS) is a system of bacterial communication which involves the synthesis and detection of chemical molecules called autoinducers to regulate community-specific traits in a cell density dependent manner (Engebrecht et al, 1983; Kaplan and Greenberg, 1987; Fuqua et al, 1994; Fuqua and Greenberg, 2002). LuxR domain swapping molecules [generally N-acyl homoserine lactones (AHLs)] synthesized by the LuxI homolog to regulate gene expression (Fuqua et al, 1994; Zhu and Winans, 2001; Fuqua and Greenberg, 2002; Schaefer et al, 2013). Most of the LuxR homologs require binding to the signal molecule for correct folding/dimerization/stabilization and subsequent activation of target promoter regions (Zhu and Winans, 2001; Vannini et al, 2002; Zhang et al, 2002). Some LuxR proteins are able to fold/dimerize in the absence of the autoinducer to repress the promoter region of target genes but are de-repressed on binding the ligand due to conformational changes (Pearson et al, 1997; Minogue et al, 2002; Medina et al, 2003; Ventre et al, 2003; Steindler et al, 2009). The two domains of the LuxR protein, the presence of appropriate ligand and important features of the target promoter element are central for LuxR mediated gene regulation
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