Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome

BMC Microbiology Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome Catherine E Chambers 0 Erika I Lutter 0 Michelle B Visser 0 Peggy PY Law 0 Pamela A Sokol 0 0 Address: Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center , Calgary, Alberta , Canada Background: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. Results: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The firstgeneration consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. Conclusion: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system. - Background Burkholderia cenocepacia, belongs to a group of nine related species with common phenotypes, but distinct genotypes collectively named the "Burkholderia cepacia complex" (Bcc) [1,2]. The Bcc are opportunistic pathogens in immunocompromised and cystic fibrosis (CF) patients but have also been isolated from plant rhizopheres as well as urban and suburban soils [1-3]. The ability of bacteria to adapt to diverse environments is dependent on the coordinate regulation of factors required to survive and proliferate in each niche. The CepIR quorum sensing system is one regulatory network that contributes to the response of B. cenocepacia to environmental signals (reviewed in [4,5]). Quorum sensing allows bacterial populations to coordinate gene expression in response to population density. CepIR belongs to a group of more than 50 quorum sensing systems that are homologous to the LuxIR system of Vibrio fishceri [6,7]. LuxI homologs are N-acyl homoserine lactone (AHL) synthases that generate AHL signal molecules that are released into the environment. LuxR homologs are transcriptional regulators that complex with AHL and typically bind to a lux-box overlapping the -35 sequence of a promoter to regulate transcription. The lux-box consensus sequence recognized by LuxR homologs typically consists of an inverted repeat with significant consensus among quorum sensing systems [6,8-10]. The CepIR system was originally identified in B. cenocepacia (formerly B. cepacia) K56-2 [11] and has subsequently been shown to be widely distributed throughout the Bcc [12,13]. CepI directs the synthesis of N-octanoyl homoserine lactone (OHL) and N-hexanoyl homoserine lactone (HHL) and cepR encodes for the transcriptional regulator [11-14]. CepR has been shown to negatively regulate its own expression, but positively regulate cepI expression at the transcriptional level [14]. The cepIR genes are involved in the regulation of the pvdA gene required for ornibactin biosynthesis [14], the zmpA and zmpB extracellular metalloproteases [15,16], the aidA gene involved in virulence in Caenorhabditis elegans [17-20], swarming motility and in at least some systems a functional CepIR quorum sensing system is necessary for biofilm formation [21-23]. The CepIR system has been shown to contribute to virulence in both plant and animal models. In B. cepacia ATCC 25416 mutations in cepI and cepR attenuated maceration in the onion-rot model [24]. The contribution of CepIR to the severity of B. cenocepacia infections has been demonstrated in two different animal models, a chronic respiratory infection model in rats and an acute intranasal infection model in Cftr(-/-) mice [16]. CepIR have also been shown to be important for virulence in C. elegans [25]. Proteomics and promoter based approaches have been used to identify genes regulated by the CepIR quorum sensing system. Proteome analysis was used to compare the protein profiles of B. cenocepacia strain H111 and an H111 cepI mutant [19]. Differences in expression were observed for 55 out of 585 proteins and partial N-terminal amino acid sequences were determined for peptide fragments of 11 proteins including AidA, FimA, and SodB. A promoter trap approach was used to identify positively regulated OHL-CepR dependent promoters in B. cepacia ATCC 25416 [17]. A library of ATCC 25416 fragments cloned upstream of a promoterless lacZ gene in a vector that also contained cepR was screened in E. coli in the presence and absence of OHL. Twenty-eight clones with genes upregulated in the presence of OHL were identified. The genes belonged to several functional classes; however, the only overlap in genes identified between the two studies was aidA [17,19]. Mutagenesis with a transposon containing a promoterless lacZ reporter was used to identify seven genes positively regulated by the cepIR quorum sensing system in B. cenocepacia strain K56-2, including cepI and aidA [20]. Identification of genes directly and indirectly regulated by CepR is a key step to understanding this regulatory system and the regulatory hierarchies that mediate the adaptation B. cenocepacia to the diverse environments it encounters. The above approaches search for genes regulated under defined in vitro conditions and therefore may not identify genes induced only in specific environmental niches including the plant or animal host. Only the study by Aguilar et al. [17] attempted to identify genes that are regulated by the direct interaction of CepR at the promoter. LuxR homologs have been shown to bind to specific sequences referred to as lux boxes or the boxes for the gene des (...truncated)