Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence Chunxi Yanga,b,c,d,1, Chaoyu Cuia,b,c,1, Qiumian Yea,b, Jinhong Kane, Shuna Fua,b, Shihao Songa,b, Yutong Huanga,b, Fei Hec, Lian-Hui Zhanga,c, Yantao Jiaf, Yong-Gui Gaod, Caroline S. Harwoodb,g,2, and Yinyue Denga,b,c,2 aState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, South China Agricultural University, Guangzhou 510642, China; bGuangdong Innovative Research Team of Sociomicrobiology, College of Agriculture, South China Agricultural University, Guangzhou 510642, China; cIntegrative Microbiology Research Centre, South China Agricultural University, Guangzhou 510642, China; dSchool of Biological Sciences, Nanyang Technological University, Singapore 637551; eCenter for Crop Germplasm Resources, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; fState Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; and gDepartment of Microbiology, University of Washington, Seattle, WA 98195 Contributed by Caroline S. Harwood, October 30, 2017 (sent for review June 1, 2017; reviewed by Maxwell J. Dow and Tim Tolker-Nielsen) Quorum sensing (QS) signals are used by bacteria to regulate N-octanoyl homoserine lactone (C8-HSL). The two QS systems biological functions in response to cell population densities. Cyclic have both distinct and overlapping effects on gene expression diguanosine monophosphate (c-di-GMP) regulates cell functions in (16, 17). response to diverse environmental chemical and physical signals One of the ways in which QS systems can act is by controlling that bacteria perceive. In Burkholderia cenocepacia, the QS signal levels of intracellular cyclic diguanosine monophosphate (c-di-GMP) receptor RpfR degrades intracellular c-di-GMP when it senses the in bacteria (18–21). c-di-GMP regulates various biological func- QS signal cis-2-dodecenoic acid, also called Burkholderia diffusible tions such as motility, biofilm formation, and virulence by a variety signal factor (BDSF), as a proxy for high cell density. However, it of mechanisms including binding to effector proteins that are parts was unclear how this resulted in control of BDSF-regulated phe- of flagella or exopolysaccharide secretion systems, by binding to notypes. Here, we found that RpfR forms a complex with a regu- transcription factors, and by binding to riboswitches (19, 21–25). lator named GtrR (BCAL1536) to enhance its binding to target gene c-di-GMP is synthesized by diguanylate cyclases with GGDEF promoters under circumstances where the BDSF signal binds to domains and degraded by phosphodiesterases with EAL or HD- RpfR to stimulate its c-di-GMP phosphodiesterase activity. In the GYP domains (18, 25, 26). In DSF-type QS, perception of DSF absence of BDSF, c-di-GMP binds to the RpfR-GtrR complex and by the transmembrane receptor RpfC stimulates its autophos- inhibits its ability to control gene expression. Mutations in rpfR phorylation and phosphotransfer to RpfG, a HD-GYP domain- and gtrR had overlapping effects on both the B. cenocepacia tran- containing protein, to activate its c-di-GMP phosphodiesterase scriptome and BDSF-regulated phenotypes, including motility, bio- activity (18, 19, 27–30). Low intracellular levels of c-di-GMP in film formation, and virulence. These results show that RpfR is a QS turn allow the c-di-GMP–binding protein Clp to become an ac- signal receptor that also functions as a c-di-GMP sensor. This pro- tive transcription factor that turns on gene expression (19). The tein thus allows B. cenocepacia to integrate information about its BDSF-type QS system found in B. cenocepacia also con- physical and chemical surroundings as well as its population den- trols intracellular levels of c-di-GMP, but by a totally different sity to control diverse biological functions including virulence. This type of QS system appears to be widely distributed in beta and Significance gamma proteobacteria. Many bacteria sense their population density by a process quorum sensing | c-di-GMP | bacterial virulence | BDSF signal called quorum sensing (QS). In addition, individual bacterial cells sense their chemical and physical environment by adjust- uorum sensing (QS) is a cell-to-cell communication system ing levels of the intracellular compound cyclic diguanosine Qthat is widely employed by bacterial cells to coordinate monophosphate (c-di-GMP). Here, we show that RpfR, a QS behaviors that are advantageous to populations of cells, in- signal receptor protein from the pathogenic bacterium Bur- cluding exoenzyme production, biofilm formation, and antibiotic kholderia cenocepacia, forms a complex with c-di-GMP and a production (1–3). QS involves the production and detection of regulator named GtrR. This complex is not proficient to activate diffusible signal molecules and the initiation of appropriate re- expression of virulence genes. However, upon binding its QS sponses in a cell density-dependent manner (1, 4). The original signal, RpfR degrades c-di-CMP, leading to activation of gene concept of QS was developed based on N-acylhomoserine lac- expression by a RpfR–GtrR complex. This work describes a tone (AHL) signal molecules, which are constitutively produced system where a pathogen uses a single protein to integrate at basal levels until they reach a critical threshold concentration information about its physical and chemical surroundings and and then bind to and activate a cognate receptor to control target its population density to control virulence. gene expression (1, 2, 5). Recently, diffusible signal factor Author contributions: C.S.H. and Y.D. designed research; C.Y., C.C., Q.Y., J.K., S.F., S.S., (DSF)-type QS signals have been recognized as another impor- Y.H., and Y.D. performed research; C.Y., C.C., Q.Y., J.K., S.F., F.H., L.-H.Z., Y.J., Y.-G.G., tant and common type of QS system (3, 6–8). DSF was originally C.S.H., and Y.D. analyzed data; and C.Y., C.C., Q.Y., C.S.H., and Y.D. wrote the paper. identified in the gamma proteobacterium Xanthomonas cam- Reviewers: M.J.D., University College Cork; and T.T.-N., Copenhagen University. pestris pv. campestris (Xcc) and is involved in regulating biofilm The authors declare no conflict of interest. – dispersal, motility, and virulence (9 11). More recently, a related Published under the PNAS license. signal, cis-2-dodecenoic acid, also called Burkholderia diffusible 1C.Y. and C.C. contributed equally to this work. signal factor (BDSF), was identified in the human pathogen and 2To whom correspondence may be addressed. Email: [email protected] or ydeng@ beta proteobacterium Burkholderia cenocepacia, where it con- scau.edu.cn. – trols similar phenotypes (3, 12 15). B. cenocepacia also has This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. an AHL QS system composed of CepI and CepR proteins and 1073/pnas.1709048114/-/DCSupplemental. 13006–13011 | PNAS | December 5, 2017 | vol. 114 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1709048114 Downloaded by guest on September 27, 2021 mechanism. In this system, a soluble receptor protein named RpfR that includes PAS, GGDEF, and EAL domains, degrades c-di- GMP when it binds BDSF (16, 24); however, the downstream events were unclear. Here we identify a global regulator, which we name GtrR, that binds to RpfR and controls expression of genes in B. cenocepacia when BDSF levels are relatively high. In this circumstance, in- tracellular c-di-GMP levels are low due to the phosphodiesterase (PDE) activity of RpfR-BDSF. In the absence of BDSF, RpfR has low PDE activity, and a GtrR-RpfR-c-di-GMP complex forms that is not proficient to regulate gene expression. Thus, RpfR serves as a sensor of both BDSF and c-di-GMP. RpfR and GtrR homologs are present in diverse Gram-negative bacteria, sug- Fig. 2. Complementation of the rpfR mutant with rpfR and gtrR. In trans gesting that the BDSF-type QS system is widespread. expression of RpfR and GtrR complemented swarming motility (A)and biofilm formation in microtiter trays (B) in the RpfR-deficient mutant. The Results data shown are the mean of three replicates, and error bars indicate the SD. GtrR Is a Global Regulator That Controls BDSF-Regulated Phenotypes in B. cenocepacia. To identify regulatory components of the BDSF the BDSF receptor rpfR mutant and the gtrR mutant and found QS system, we screened a Tn5 mutant library of B. cenocepacia strain H111 carrying a lectin-encoding bclACB operon-lacZ substantial overlap in the genes that were affected in expression promoter fusion plasmid for colonies that were light blue on LB (SI Appendix,TableS1). Quantitative RT-PCR analysis of the al- plates supplemented with X-gal. The bclACB operon is con- teredexpressionofselectgenesconfirmedtheRNA-Seqresults trolled by both BDSF and C8-HSL (17, 31). We screened (SI Appendix,TableS2). From this we concluded that GtrR is likely ∼40,000 colonies and identified mutants with insertions in the a key downstream component of the BDSF-signaling system in known regulatory genes rpfR and cepR. In addition, we identified B. cenocepacia. a gene annotated as a Fis family transcriptional regulator (BCAL1536; I35_RS07130). We named this regulator global GtrR Regulates Target Gene Expression by Directly Binding to Promoters. transcriptional regulator downstream RpfR (GtrR). GtrR has an To study regulation by GtrR, we constructed PbclACB-lacZ and AAA+ATPase σ54-interaction domain and a C-terminal helix- PcepI-lacZ reporter systems in a gtrR mutant (SI Appendix,TableS3). turn-helix DNA-binding motif. It has previously been reported Both of these target operons are positively controlled by BDSF to be regulated by the BDSF system (17) and is important for survival of B. cenocepacia K56-2 in a rat lung infection model (32).