Burkholderia Cenocepacia Integrates Cis-2-Dodecenoic Acid and Cyclic Dimeric Guanosine Monophosphate Signals to Control Virulence

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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, intracellular 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). An in-frame deletion mutant of gtrR that we constructed had reduced motility and biofilm formation, two phenotypes controlled by BDSF (Fig. 1). We then compared the transcriptome profiles of the wild-type strain and the gtrR mutant by RNA-Seq and saw changes in the expression levels of several hundred genes (SI Appendix, Table S1). These genes are asso- ciated with a range of biological functions, including motility and cell attachment, stress tolerance, virulence, regulation, transcriptional regulators, membrane components, transport, multi- drug resistance, detoxification, and signal transduction (SI Appendix,TableS1).

GtrR Is a Downstream Component of the BDSF QS System. The expression of gtrR in trans in the rpfR mutant fully restored the BDSF- regulated phenotypes of motility (Fig. 2A) and biofilm formation (Fig. 2B). Based on this, we compared the transcriptome profiles of

Fig. 3. Effects of GtrR at BDSF-controlled promoters. The effects of MICROBIOLOGY GtrR on bclACB (A)andcepI (B) gene expression were measured by assessing β-galactosidase activities of the bclACB-lacZ and cepI-lacZ transcriptional fusions in the H111 wild-type, ΔrpfR, and ΔgtrR strains. (C)EMSA analysis of in vitro binding of GtrR to the promoter of bclACB, in which a 32P-labeled 506-bp bclACB promoter DNA probe was used for protein- Fig. 1. Effects of GtrR on BDSF-regulated phenotypes. The wild-type strain, binding assay. A protein–DNA complex, represented by a band shift, was a gtrR mutant, and a complemented gtrR mutant were tested for the BDSF- formed when different concentrations of GtrR protein were incubated with regulated phenotypes of swarming motility (A) and biofilm formation in the probe at room temperature for 20 min. EMSA analysis of the in vitro microtiter trays (B). The data shown are the mean of three replicates, and binding of GtrR to the cepI promoter (D). Identical to C,a32P-labeled 210-bp error bars indicate the SD. cepI promoter DNA probe was incubated with GtrR.

Yang et al. PNAS | December 5, 2017 | vol. 114 | no. 49 | 13007 Downloaded by guest on September 27, 2021 estimated dissociation constant (KD) of 3.98 ± 0.33 μM, and the association of the two proteins with each other was not inhibited by addition of either BDSF or c-di-GMP (SI Appendix, Fig. S3). We then tested if RpfR affects GtrR binding to target promoter DNA by performing EMSA. As shown in Fig. 4 B and C, the binding of the GtrR to the bclACB and cepI promoter probes was enhanced when RpfR was present in the reaction mixtures. The amounts of the probes that bound to GtrR increased with increasing amounts of RpfR. To confirm our observed interaction between RpfR and GtrR, we used a bacterial two-hybrid system to detect protein–protein interactions (33). A positive interaction between gtrR fused to the alpha subunit RNA polymerase gene and rpfR fused to the lambda repressor protein was revealed by growth on His-deficient medium containing 5 mM 3-amino-1,2,4-triazole (3-AT) and 12.5 μg/mL streptomycin (Fig. 4D).

RpfR Binds c-di-GMP. Since RpfR has c-di-GMP PDE activity, Fig. 4. Interactions between RpfR and GtrR. MST analysis indicated that it can be surmised that it binds c-di-GMP. To quantify this, weperformedMSTandwealsotestedifGtrRcouldbindc-di-GMP. GtrR binds to RpfR with a KD of 3.98 ± 0.33 μM(A). Effect of RpfR on binding of GtrR to the bclACB promoter (B) and cepI promoter (C). Bacterial two- As shown in Fig. 5, there was no detectable binding between hybrid analysis of interaction of RpfR and GtrR in vivo (D). “Fnorm (1/1000)” c-di-GMP and GtrR, but c-di-GMP bound to RpfR with an indicates that the fluorescence time traces changes in the MST response. estimated dissociation constant (KD) of 2.92 ± 0.26 μM. In addition, we purified polypeptides encompassing only the PAS-GGDEF or EAL domains of RpfR and tested their (SI Appendix, Tables S1 and S2) (16, 17). In agreement with the binding to c-di-GMP. We found that only the EAL domain RNA-Seq and qPCR results, deletion of gtrR resulted in re- was required for c-di-GMP binding (SI Appendix,Fig.S4). duced expression levels of both bclACB and cepI (Fig. 3 A and This finding agrees with our previous results in which the B). To test if transcriptional regulation of these target operons EAL domain of RpfR was required for c-di-GMP PDE ac- is achieved by direct binding of GtrR to their promoters, tivity (24). electrophoretic mobility shift analyses (EMSA) were performed. A PCR-amplified 506-bp DNA fragment from the c-di-GMP Inhibits Binding of the RpfR–GtrR Complex to Target bclACB promoter and a 210-bp DNA fragment from the cepI Promoter DNA. To determine how the binding of c-di-GMP to promoter were used as probes (SI Appendix,TableS4). GtrR, RpfR might affect the activity of GtrR, we examined the effects which has 463 amino acids and a calculated molecular weight of of c-di-GMP on binding of the RpfR–GtrR complex to the 51 kDa, was purified using affinity chromatography (SI Appen- bclACB promoter by EMSA. The addition of c-di-GMP at a dix,Fig.S1A). As shown in Fig. 3 C and D,thebclACB and cepI relatively high concentration (150 μM) resulted in a decrease promoter DNA fragments formed stable DNA-protein com- in the ability of RpfR–GtrR to bind target promoter DNA (Fig. plexes with GtrR and migrated at slower rates than unbound 6A). RpfR did not retain its c-di-GMP PDE activity in the probes. The amount of the probe that bound to GtrR increased EMSA. In contrast, the addition of BDSF did not affect binding with increasing amounts of GtrR (Fig. 3 C and D). The amount of the RpfR–GtrR complex to target promoter DNA (SI Ap- of labeled probe bound to GtrR decreased in the presence of pendix,Fig.S5). We obtained the same result when we used the 100 times unlabeled probe (SI Appendix,Fig.S2). cepI promoter probe in gel shift assays (Fig. 6B). c-di-GMP did not affect GtrR binding to target promoters in the absence of RpfR Enhances GtrR Binding to Target Promoter DNA. Although RpfR (SI Appendix,Fig.S6). These results are consistent with GtrR appeared to be a downstream component of the BDSF the idea that binding of c-di-GMP by RpfR lowers the affinity system, the relationship between BDSF sensing by RpfR and of the RpfR–GtrR complex for target promoter DNA, ulti- activation of gene expression by GtrR was unclear. To explore mately decreasing target gene expression. Neither BDSF nor the possibility that these two proteins might interact, we purified c-di-GMP affected the binding of RpfR to GtrR in vitro (SI each of them (SI Appendix, Fig. S1) and used microscale ther- Appendix,Fig.S3). To further assess if c-di-GMP could dis- mophoresis (MST) to see if they had an affinity for each rupt the RpfR–GtrR interaction, we carried out the bacterial other. As shown in Fig. 4A, RpfR interacted with GtrR with an two-hybrid assay described above and in Fig. 4D,butwiththe additional expression of the diguanylate cyclase gene, wspR. As expected, in trans expression of wspR caused a significant increase in intracellular c-di-GMP levels in the Escherichia

Fig. 5. MST analysis of the binding of c-di-GMP to RpfR (A) and GtrR (B). The

Kd for the binding between c-di-GMP and RpfR was calculated to be 2.92 ± 0.264 μM. No binding of c-di-GMP to GtrR was detected. “Fnorm (1/1000)” Fig. 6. The effects of c-di-GMP on RpfR–GtrR complex binding to the pro- indicates the fluorescence time traces changes in the MST response. moter DNA of bclACB (A) and cepI (B) were assessed by performing EMSA.

13008 | www.pnas.org/cgi/doi/10.1073/pnas.1709048114 Yang et al. Downloaded by guest on September 27, 2021 receptor-negative mutants of B. cenocepacia (13, 24, 31). To investigate whether GtrR was required for virulence, we tested both rpfR and gtrR mutants in cell line and mouse models. As expected, deletion of either rpfR or gtrR led to a reduction in bacterial virulence in both models (Fig. 7). This result is consistent with previous findings (32). Cytotoxicity was measured by quantifying the release of lactate dehydrogenase (LDH) into the supernatants of cultured cells. When cells were incubated with rpfR and gtrR mutant strains, cytotoxicity levels were 46 and 51%, respectively, of the levels achieved when cells were incubated with the wild-type B. cenocepacia H111 strain at 8 h post- inoculation (Fig. 7A). Mouse model assays demonstrated similar Fig. 7. The effects of gtrR and rpfR on the pathogenicity of B. cenocepacia results. The mortality of mice infected with the wild-type strain were determined using cell line (A) and mouse infection (B) models. Cell or the rpfR or the gtrR mutant strains was 80, 20, and 30%, re- cytotoxicity was detected and measured as LDH release. LDH release by B. spectively, at 5 d postinfection (Fig. 7B). Complementation of cenocepacia wild-type strain H111 was arbitrarily defined as 100% and used the gtrR mutant with a cloned gtrR gene restored the pathoge- to normalize the LDH release ratios of the gtrR and rpfR mutants and the nicity of the mutant to wild-type levels in both the cell line and complemented strains. The data shown are the mean of three replicates, and mouse models. Further investigation was conducted to analyze error bars indicate the SDs. For the mouse model, mortality was calculated after BALB/c mice were infected using 1 × 109 cfu of B. cenocepacia strains the symptoms of mouse lungs infected by the mutant strains over a 5-d period. because the lung is the most important niche for B. cenocepacia. At 2 d postinfection, the macroscopic pathological findings indicated severe pulmonary inflammation when the mice were coli reporter strain (SI Appendix,Fig.S7). However, this did not infected with the wild-type or complemented mutant strains, but cause a significant disruption of the RpfR–GtrR interaction, as there was no or only mild inflammation with decreased in- indicated by the ability of cells to grow on M9 medium sup- flammatory cell infiltration and serosanguinous exudation when plemented with streptomycin and 3-AT. Although c-di-GMP the mice were infected with the rpfR and gtrR mutants (SI Ap- does not affect RpfR–GtrR complex formation in this assay, we pendix, Fig. S10). cannot exclude that binding of c-di-GMP to RpfR promotes conformational changes in the complex. Discussion To characterize the relationship between c-di-GMP PDE ac- From the results of this study, we can put together a model for how tivity and c-di-GMP sensing, we created point mutations in the BDSF-mediated signaling controls gene expression in B. cen- EAL domain of RpfR to see if we could disrupt c-di-GMP PDE ocepacia. At low population densities when the amount of BDSF is activity but not the binding of c-di-GMP to RpfR. Among nine low and c-di-GMP is present at basal levels in cells, c-di-GMP binds RpfR variant proteins that we tested, six completely lost their to RpfR–GtrR complexes, and this prevents GtrR from acti- ability to degrade c-di-GMP. The three remaining variant proteins vating transcription. At high population densities, when the exhibited c-di-GMP PDE activity (SI Appendix, Table S5). We concentration of BDSF that cells are exposed to is relatively identified one variant, RpfRE466A, that had a similar binding af- high, BDSF binds to RpfR to stimulate its c-di-GMP phospho- finity for c-di-GMP as the wild-type protein, but had no c-di-GMP diesterase activity. As a result, the concentration of intracellular PDE activity (SI Appendix, Fig. S8 and Table S5). Inclusion of c-di-GMP drops to a level sufficiently below its binding constant c-di-GMP in EMSA assays carried out with the RpfRE466A variant for RpfR as to allow RpfR and GtrR to form a transcriptionally at similar concentrations, as used in EMSA assays with wild-type active complex (Fig. 8). Several QS systems modulate c-di-GMP RpfR, inhibited binding of the RpfRE466A–GtrR complex to target levels in response to exposure to a QS signal, including the promoter DNA (SI Appendix,Fig.S9). RpfC–RpfG system in Xcc (19) and the LuxPQ and CpqS systems in Vibrio cholerae (34). However, the B. cenocepacia BDSF GtrR Contributes to B. cenocepacia Pathogenicity. Previous studies QS system is distinctive in that it regulates c-di-GMP levels in the characterized the attenuated virulence of BDSF synthase- and cells by degrading this molecule in response to BDSF and its MICROBIOLOGY

Fig. 8. Model for BDSF-mediated QS regulation of virulence at low (A) and high (B) cell population densities. RpfFBc is involved in synthesis of BDSF signals. At low cell density, a pool of intracellular c-di-GMP is determined by the activities of various diguanylate cyclases and phosphodiesterases in response to physical and chemical cues from the environment. When c-di-GMP is sufficiently high, it binds to RpfR–GtrR and prevents this complex from controlling gene expression. At high cell density, perception of BDSF by RpfR substantially enhances its c-di-GMP phosphodiesterases activity, causing a reduction of the intracellular c-di-GMP to a level sufficient to allow a RpfR–GtrR complex to bind to the target promoter DNA and enhance target gene expression and virulence in B. cenocepacia.

Yang et al. PNAS | December 5, 2017 | vol. 114 | no. 49 | 13009 Downloaded by guest on September 27, 2021 activity is negatively controlled by c-di-GMP. Moreover, a single Serratia, Enterobacter, Pantoea, and Cronobacter (SI Appendix, protein, RpfR, integrates sensory input from c-di-GMP and Table S6). BDSF. One can imagine that this circuitry endows the B. cen- In conclusion, our working model of the RpfR–GtrR complex ocepacia BDSF QS system with the ability to fine-tune gene responds to both BDSF signal and c-di-GMP to control gene expression depending on the relative concentrations of BSDF expression. Our findings will inform further studies seeking to and c-di-GMP that RpfR encounters in cells. B. cenocepacia develop new strategies to target the multifunctional protein H111 encodes six c-di-GMP phosphodiesterases, 12 diguanylate RpfR, which senses both QS signals and an intracellular second cyclases, and, in addition to RpfR, four hybrid diguanylate cyclase/ messenger to control diseases caused by bacterial pathogens. phosphodiesterase proteins and two HD-GYP domain proteins (potential c-di-GMP phosphodiesterases). As has been demon- Materials and Methods strated in many other bacteria, a subset of these proteins likely Bacterial Growth Conditions and Virulence Assays. The bacterial strains used in senses the physical and chemical characteristics of the extracel- this work are listed in SI Appendix, Table S3. B. cenocepacia H111 strains lular environment of B. cenocepacia to control the total in- were cultured at 37 °C in medium comprised of 5 g peptone (Difco), 3 g yeast extract (Difco), and 20 g glycerol per liter (35) or LB Lennox. The following tracellular concentration of c-di-GMP. Thus, one would expect − antibiotics were added when necessary: tetracycline, 100 μgmL1; tri- that there would be environmental circumstances where low in- −1 −1 – methoprim, 25 μgmL ; and gentamicin, 10 μgmL . Cell line and mouse tracellular c-di-GMP levels would allow for RpfR-GtrR directed infection models were used for virulence assays following the methods in- gene expression to occur in the absence of BDSF. In fact, it has dicated in SI Appendix. been shown that QS phenotypes of the BDSF synthase mutant rpfFBc can be rescued by in trans expression of a c-di-GMP Mutagenesis and Phenotype Analysis. B. cenocepacia H111 was used as the phosphodiesterase (24). parental strain to generate an in-frame deletion mutant of gtrR, following In contrast to the regulatory model that we have just presented the methods described previously (24). Swarming motility and biofilm for- for BDSF QS, the DSF-type QS system in Xcc and related mation assays were performed using previously described methods (36). bacteria operates with a different set of signal perception and Detailed descriptions of the above methods are provided in SI Appendix. regulatory proteins to regulate intracellular c-di-GMP levels, and its activity is not modulated by c-di-GMP as the BDSF-type QS Protein Purification and Analysis. Detailed descriptions are provided in system is (16, 19, 24, 27–29). The remarkable differences be- SI Appendix. Briefly, rpfR and gtrR were amplified with the primers listed in tween the QS perception and c-di-GMP sensing mechanisms SI Appendix, Table S4, and cloned into the expression vector pET-28a. The fusion gene constructs were transformed into E. coli strain BL21. Affinity suggest that the DSF system in Xcc and the BDSF system in purification of HIS-GtrR and HIS-RpfR fusion proteins and binding assays B. cenocepacia have evolved independently. The rpfF/rpfC/rpfG were performed following previously described methods (24). gene cluster of the DSF-type QS system is conserved in bacterial species belonging to the genera Xanthomonas, Xylella, Steno- ACKNOWLEDGMENTS. This work was financially supported by grants from trophomonas, Methylobacillus, Thiobacillus, and Leptospirillum the Guangdong Natural Science Funds for Distinguished Young Scholars (2014A030306015), the China National Key Project for Basic Research (973 (6). A BLAST search with the B. cenocepacia rpfFBc/rpfR gene Project 2015CB150600), the China National Natural Science Foundation cluster and gtrR revealed that the BDSF-type QS system likely (31571969), the Pearl River Nova Program of Guangzhou (201506010067), operates in a different set of bacteria, including members of the and the Introduction of Innovative R&D Team Program of Guangdong genera Burkholderia, Paraburkholderia, Achromobacter, Yersinia, Province (2013S034).

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