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


CONCLUSIONS
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.
Chambers
Catherine E
CE
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Center, Calgary, Alberta, Canada. psokol@ucalgary.ca <psokol@ucalgary.ca>
Lutter
Erika I
EI
Visser
Michelle B
MB
Law
Peggy P Y
PP
Sokol
Pamela A
PA
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D016956:Burkholderia cepacia / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D019295:Computational Biology; D004251:DNA Transposable Elements; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D053038:Quorum Sensing; D015398:Signal Transduction; D014158:Transcription, Genetic
D001426:Bacterial Proteins; C117246:CepR protein, Burkholderia; D004251:DNA Transposable Elements; C088386:homoserine lactone; D015107:4-Butyrolactone