Cis-2-dodecenoic acid receptor RpfR links quorum-sensing signal perception with regulation of virulence through cyclic dimeric guanosine monophosphate turnover

Abstract

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

Fig. 1.

Influence of RpfF_Bc and RpfR on intracellular c-di-GMP level. (A) Detection of intracellular c-di-GMP level by HPLCy (HPLC) assay. Relative amount of c-di-GMP was calculated on the basis of their peak areas. For the convenience of comparison, c-di-GMP level of B. cenocepacia wild-type strain H111 was arbitrarily defined as 100% and used to normalize the c-di-GMP level ratios of the other strains. Data shown are means of two repeats and error bars indicate SDs. (B) Genomic organization of the region encoding RpfF_Bc and RpfR in B. cenocepacia H111 and domain structure analysis of RpfR.

Fig. 2.

Influence of RpfR on BDSF-regulated phenotypes. Similar to the BDSF synthase mutant, deletion of rpfR caused a reduction of swarming motility (A), biofilm formation (B), and protease production (C). Data shown are means of three replicates and error bars indicate SDs.

Fig. 3.

Effect of in trans protein expression on BDSF-regulated phenotypes. In trans expression of the encoding region of RpfR, the GGDEF, and EAL domains, and the EAL domain only in the BDSF synthase⁻ mutant ∆rpfF_Bc rescued its phenotype defects in swarming motility (A), biofilm formation (B), and protease production (C). Data shown are means of two replicates and error bars indicate SDs.

Fig. 4.

ITC analysis of the molecular interaction between BDSF and RpfR. (A) ITC titration of 20 μM RpfR with 200 μM BDSF in PBS buffer at 21 °C. (B) ITC titration of 20 μM RpfR with 200 μM of the saturated isomer of BDSF in PBS buffer at 21 °C.

Fig. 5.

Impact of BDSF on RpfR enzyme activity. Exogenous addition of BDSF to a RpfR protein solution increased its c-di-GMP phosphodiesterase activity. For the convenience of comparison, peak of c-di-GMP in the RpfR solution without BDSF at 0 min was defined as 100% and used to normalize the c-di-GMP level ratios at different time points. Data shown are means of two replicates and error bars indicate SDs.

Fig. 6.

Influence of RpfF_Bc and RpfR on the virulence of B. cenocepacia against C. elegans. (A) Mutants ∆rpfF_Bc(▲) and ∆rpfR(●) showed reduced virulence compared with their parental wild-type strain H111 (■) and ∆rpfR complemented strain (▼) in slow killing (A) and fast killing (B) assays. Data presented are the mean of triplicate experiments and error bars represent SDs.