Direct binding of the quorum sensing regulator CepR of Burkholderia cenocepacia to two target promoters in vitro.
Burkholderia cenocepacia is an opportunistic human pathogen that can aggressively colonize the cystic fibrosis lung. This organism has a LuxR/LuxI-type quorum sensing system that enables cell-cell communication via exchange of acyl homoserine lactones (AHLs). The CepR and CepI proteins constitute a global regulatory system, controlling expression of at least 40 genes, including those controlling swarming motility and biofilm formation. In this study, we isolated seven lacZ fusions in a clinical isolate of B. cenocepacia that are inducible by octanoyl-HSL. Induction of all of these genes requires CepR. The cepI promoter was tested for induction by a set of 33 synthetic autoinducers and analogues, and was most strongly induced by long-chain AHLs lacking 3-oxo substitutions. Expression of this promoter was inhibited by high concentrations of three different autoinducers, each having six-carbon acyl chains. When CepR protein was overproduced in Escherichia coli, it accumulated in a soluble form in the presence of octanoyl-HSL, but accumulated only as insoluble inclusion bodies in its absence. Purified CepR-OHL complexes bound to specific DNA sequences at the cepI and aidA promoters with high specificity. These binding sites included a 16-nucleotide imperfect dyad symmetry. Both CepR binding sites are centred approximately 44 nucleotides upstream of the respective transcription start sites.
Weingart
Christine L
CL
Department of Biology, Denison University, Granville, OH 43023, USA. weingartc@denison.edu
White
Catharine E
CE
Liu
Suping
S
Chai
Yunrong
Y
Cho
Hongbaek
H
Tsai
Ching-Sung
CS
Wei
Yuping
Y
Delay
Nicholas R
NR
Gronquist
Matthew R
MR
Eberhard
Anatol
A
Winans
Stephen C
SC
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism*; D001665:Binding Sites; D019117:Burkholderia / Q000235:genetics / Q000502:physiology*; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D015964:Gene Expression Regulation, Bacterial; D008025:Ligases / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D024363:Transcription Initiation Site
D001426:Bacterial Proteins; C117246:CepR protein, Burkholderia; C088386:homoserine lactone; D008025:Ligases; C449816:N-acylhomoserine lactone synthase; D015107:4-Butyrolactone