Identification of genes regulated by the cepIR quorum-sensing system in Burkholderia cenocepacia by high-throughput screening of a random promoter library.
The Burkholderia cenocepacia cepIR quorum-sensing system regulates expression of extracellular proteases, chitinase, and genes involved in ornibactin biosynthesis, biofilm formation, and motility. In a genome-wide screen we identified cepIR-regulated genes by screening a random promoter library of B. cenocepacia K56-2 constructed in a luminescence reporter detection plasmid for differential expression in response to N-octanoyl-l-homoserine lactone (OHL). Eighty-nine clones were identified; in 58 of these clones expression was positively regulated by cepIR, and in 31 expression was negatively regulated by cepIR. The expression profiles of the 89 promoter clones were compared in the cepI mutant K56-dI2 in medium supplemented with 30 pM OHL and K56-2 to confirm that the presence of OHL restored expression to wild-type levels. To validate the promoter library observations and to determine the effect of a cepR mutation on expression of selected genes, the mRNA levels of nine genes whose promoters were predicted to be regulated by cepR were quantitated by quantitative reverse transcription-PCR in the wild type and cepI and cepR mutants. The expression levels of all nine genes were similar in the cepI and cepR mutants and consistent with the promoter-lux reporter activity. The expression of four selected cepIR-regulated gene promoters was examined in a cciIR mutant, and two of these promoters were also regulated by cciIR. This study extends our understanding of genes whose expression is influenced by cepIR and indicates the global regulatory effect of the cepIR system in B. cenocepacia.
Subsin
Benchamas
B
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, 3330 Hospital Dr. NW, Calgary, Alberta, Canada T2N 4N1.
Chambers
Catherine E
CE
Visser
Michelle B
MB
Sokol
Pamela A
PA
D001426:Bacterial Proteins / Q000235:genetics; D016956:Burkholderia cepacia / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D011401:Promoter Regions, Genetic / Q000235:genetics*; D053038:Quorum Sensing / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D001426:Bacterial Proteins