The genus Burkholderia encompasses a fascinating collection of diverse β-proteobacteria (Coenye and Vandamme, 2003). This genus includes over 50 species, some of which are potentially useful in bioremediation, while other members are capable of forming nitrogen-fixing root nodules with legumes (Bontemps et al., 2010; Chen et al., 2003). Some members protect host plants against fungal pathogens, while others are themselves pathogenic against plants, animals, and humans (Coenye and Vandamme, 2003; Jones and Webb, 2003). Seventeen pathogenic species are members of the Burkholderia cepacia complex, or BCC (Vandamme et al., 1997; Vanlaere et al., 2008; Vanlaere et al., 2009), two of which are described by the Center for Disease Control as category B select agents (Godoy et al., 2003). B. cenocepacia, previously known as B. cepacia genomovar III (Vandamme et al., 2003), is recognized as an opportunistic pathogen of humans and is a particular threat to cystic fibrosis (CF) patients (Mahenthiralingam et al., 2005; Vandamme et al., 1997). Colonization of the CF lung by B. cenocepacia (Vandamme et al., 2003) tends to occur in patients already infected with Pseudomonas aeruginosa, another opportunistic pathogen of the CF lung (Jones and Webb, 2003; Vandamme et al., 1997). An infection caused by both organisms can result in serious clinical complications. B. cenocepacia strains are resistant to most antibiotics, making them virtually impossible to eradicate (Nzula et al., 2002). Infections with B. cenocepacia may have variable clinical outcomes ranging from asymptomatic carriage to a sudden fatal deterioration in lung function (Mahenthiralingam et al., 2005). Four strains of B. cenocepacia have been sequenced in their entirety, one of which is described in a publication (Holden et al., 2009). The Joint Genome Institute is currently sequencing nine additional strains (JGI, 2010). All four sequenced isolates have three circular chromosomes that vary in size between 3.9 and 0.88 MB in length. Strains J2315 and HI2424 also have one plasmid, 93 KB and 165 KB in length, respectively. Many or possibly all Burkholderia spp. encode at least one regulatory system that resembles the LuxR and LuxI proteins of Vibrio fischeri, where LuxI synthesizes an acylhomoserine lactone (AHL)-type pheromone, also called an autoinducer, and LuxR is an AHL-dependent transcriptional regulator (Choi and Greenberg, 1992; Eberhard et al., 1981; Engebrecht and Silverman, 1984). Regulatory systems of this family are found in countless proteobacteria, where they are thought to allow individual bacteria to coordinate their physiology with their siblings. Collectively, these systems regulate diverse processes, including pathogenesis, biofilm formation, bacterial conjugation, and the production of antibiotics and other secondary metabolites (Whitehead et al., 2001). In general, target genes are transcribed preferentially at population densities high enough to favor AHL accumulation (Eberhard et al., 1991), a phenomenon referred to as quorum sensing (Fuqua et al., 1994). B. thailandiensis has three such systems, one of which is implicated in cell aggregation, while another is required for antibiotic production (Chandler et al., 2009; Duerkop et al., 2009). A plant growth promoting isolate of B. ambifaria uses quorum sensing to regulate the production of the anti-fungal compound pyrrolnitrin (Schmidt et al., 2009). LuxR-type proteins have two domains, an N-terminal pheromone binding domain and a C-terminal DNA binding domain (Pappas et al., 2004). Purified LuxR, TraR of Agrobacterium tumefaciens, and LasR of Pseudomonas aeruginosa, when complexed with their respective AHLs, bind with high specificity to recognition sequences (referred to as lux, tra, or las boxes, respectively) that are found at target promoters (Schuster et al., 2004; Urbanowski et al., 2004; Zhu and Winans, 1999). LasR is also able to bind to sequences that have no obvious resemblance to canonical las boxes. A few members of this family bind DNA only in the absence of AHLs (Castang et al., 2006; Cui et al., 2005; Fineran et al., 2005; Minogue et al., 2005; Sjoblom et al., 2006). B. cenocepacia J2315 encodes three LuxR homologs and two LuxI homologs (Lewenza et al., 1999; Malott et al., 2005; Malott et al., 2009). Among these, CepR and CepI appear to be well conserved within the BCC (Venturi et al., 2004). CepI synthesizes primarily octanoylhomoserine lactone (OHL), and lower levels of hexanoylhomoserine lactone (HHL) (Aguilar et al., 2003b; Gotschlich et al., 2001; Huber et al., 2001; Lewenza et al., 1999). Null mutations in cepI or cepR increased the production of the siderophore ornibactin, and decreased the production of secreted lipases and metalloproteases ZmpA and ZmpB (Kooi et al., 2006; Lewenza et al., 1999; Lewenza and Sokol, 2001; Sokol et al., 2003). CepI and CepR are also required for swarming motility and biofilm formation (Huber et al., 2001) and for pathogenicity in several animal models (Kothe et al., 2003; Sokol et al., 2003). B. cenocepacia also expresses the CciI and CciR proteins, which are encoded on a genomic island called cci (cenocepacia island), that is associated with epidemic strains (Malott et al., 2005). The CepIR and CciIR systems extensively interact, in that CciR negatively regulates cepI, while CepR is required for expression of the cciIR operon (Malott et al., 2005). Transcriptional profiling studies indicate that CepR and CciR regulate many of the same genes, but do so in opposite ways (O'Grady et al., 2009). B. cenocepacia also encodes an orphan LuxR homolog called CepR2, which represses a cluster of genes that may direct production of an antibiotic or other secondary metabolite (Malott et al., 2009). In addition to transcriptional profiling several other approaches have been used to identify genes whose expression is influenced by CepR and/or OHL. In one study, the proteome of a wild type B. cenocepacia was compared to that of a cepR mutant. Fifty-five proteins were found to be differentially expressed in the two strains, approximately 10% of all detected proteins (Riedel et al., 2003). In a second study, fragments of a B. cepacia strain were cloned into a promoter trap plasmid and introduced into an E. coli strain that expressed CepR (Aguilar et al., 2003a). Twenty-eight promoter fragments were identified as being induced by OHL, and in all cases, induction required CepR. In a third study, a library of B. cenocepacia DNA fragments were introduced into a plasmid containing a promoterless luxCDABE operon (Subsin et al., 2007). That study identified 58 OHL-inducible promoters and 31 OHL-repressible promoters. Regulation of nine of these genes required CepR, while the others were not tested. Seven OHL-inducible genes were identified by screening a library of lacZ fusions (Weingart et al., 2005). Induction of all of these genes required CepR. Purified CepR-OHL complexes bound with high affinity and specificity to specific DNA sequences at two target promoters (Weingart et al., 2005). These binding sites contained a 16-nucleotide imperfect dyad symmetry and were centered approximately 44 nucleotides upstream of the transcription start sites. These two sites are to date the only experimentally confirmed CepR binding sites. Most of the studies described above do not distinguish whether a target promoter is controlled by CepR directly or indirectly. CepR could regulate a promoter indirectly, for example, by directly regulating an unknown regulatory gene whose product directly regulates that promoter. Alternatively, a CepR mutation might perturb cellular physiology in such a way that various promoters are affected by secondary effects. To date, the most comprehensive study attempting to define the optimal CepR binding site was done by Chambers, Sokol, and colleagues (Chambers et al., 2006), who approached this question with an impressive combination of genetics and bioinformatics. Mutagenesis of the known CepR binding site within the cepI promoter completely abolished induction (Chambers et al., 2006). The promoters of six genes known to be induced by OHL were used to formulate a consensus CepR binding motif (Chambers et al., 2006). This information was used to test eight additional candidate promoters, six of which were CepR-regulated. Ultimately, ten inducible promoters were used to refine the consensus sequence, and 57 possible CepR binding sites were identified upstream of various genes. The consensus motif identified in the Chambers study included the sequence CTG-N10-CAG, which has dyad symmetry. However, several other bases in the consensus did not preserve this symmetry, and some of those non-symmetric bases were said to be highly conserved (Chambers et al., 2006). The partial dyad symmetry suggests that CepR binds DNA as dimer and that the two DNA binding domains have rotational symmetry. Although we have no proof of this, structural studies of a related protein support this idea (Vannini et al., 2002; Zhang et al., 2002). Several other LuxR-type proteins are thought to decode dyad symmetrical sequences (Antunes et al., 2008; White and Winans, 2007; Whitehead et al., 2001). In the present study, we tested the ten putative CepR binding sites described above for the ability to bind purified CepR-OHL complexes. We also systematically resected and mutated a known CepR binding site, and use the resulting information to identify four new promoters that are regulated directly by CepR. All four promoters are regulated by CepR in vivo, require their binding sites for regulation, and bind with high affinity to CepR-OHL in vitro.