Research Article: Cyclic-di-GMP binds to histidine kinase RavS to control RavS-RavR phosphotransfer and regulates the bacterial lifestyle transition between virulence and swimming

Date Published: August 13, 2019

Publisher: Public Library of Science

Author(s): Shou-Ting Cheng, Fang-Fang Wang, Wei Qian, Nian Wang.


The two-component signalling system (TCS) comprising a histidine kinase (HK) and a response regulator (RR) is the predominant bacterial sense-and-response machinery. Because bacterial cells usually encode a number of TCSs to adapt to various ecological niches, the specificity of a TCS is in the centre of regulation. Specificity of TCS is defined by the capability and velocity of phosphoryl transfer between a cognate HK and a RR. Here, we provide genetic, enzymology and structural data demonstrating that the second messenger cyclic-di-GMP physically and specifically binds to RavS, a HK of the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. The [c-di-GMP]-RavS interaction substantially promotes specificity between RavS and RavR, a GGDEF–EAL domain-containing RR, by reinforcing the kinetic preference of RavS to phosphorylate RavR. [c-di-GMP]-RavS binding effectively decreases the phosphorylation level of RavS and negatively regulates bacterial swimming. Intriguingly, the EAL domain of RavR counteracts the above regulation by degrading c-di-GMP and then increasing the level of phosphorylated RavS. Therefore, RavR acts as a bifunctional phosphate sink that finely controls the level of phosphorylated RavS. These biochemical processes interactively modulate the phosphoryl flux between RavS-RavR and bacterial lifestyle transition. Our results revealed that c-di-GMP acts as an allosteric effector to dynamically modulate specificity between HK and RR.

Partial Text

The two-component signalling system (TCS) is one of the predominant molecular machineries used by almost all bacteria to monitor and adaptively respond to environmental cues [1, 2]. The prototypical TCS is composed of a membrane-bound histidine kinase (HK) and a cytosolic response regulator (RR). Upon detecting a stimulus, HK autophosphorylates an invariant histidine residue within its dimerization and histidine phosphotransfer (DHp) domain and then catalyses the transfer of the phosphoryl group onto a conserved aspartic acid within the receiver (REC) domain of the cognate RR [3]. The activated RR then modulates bacterial adaptation by controlling gene transcription or cellular behaviour [4, 5]. There is a high level of specificity between a HK and its cognate RR, which is quantified by the kinetic preference during phosphotransfer [6, 7]. The complexity of TCS regulation was revealed after three decades of extensive investigations. Bacterial cells dynamically and elegantly regulate time, rhythm, space and flux of the phosphotransfer between the HK and RR to adapt to diverse ecological niches [8]. For example, hybrid-type HK-mediated phosphorelay, phosphatases, auxiliary proteins and small RNAs are involved in TCS regulation [9–13].

Kinetic preferences between HKs and RRs define the specificity of TCS regulation [7, 44], which is quantified by the velocity of a HK to phosphorylate a RR [6]. In the present study, genetic, biophysical and biochemical data revealed that c-di-GMP substantially enhanced the specificity between RavS and RavR of X. campestris pv. campestris. RavS physically interacts with c-di-GMP via its CA region. This interaction remarkably accelerated phosphoryl transfer from RavS~P towards RavR, resulting in efficient dephosphorylation of RavS~P. Intriguingly, the EAL domain-containing RavR acts as a phosphate sink of RavS~P. Stimulated by c-di-GMP, the REC domain of RavR receives a phosphoryl group from RavS to decrease the RavS~P level, which negatively modulates bacterial swimming. However, the EAL domain of RavR degrades c-di-GMP. Degradation of c-di-GMP revokes the accelerating effect of c-di-GMP on RavS-RavR phosphotransfer, leading to a high level of RavS~P, which positively regulates bacterial swimming (Fig 8). Therefore, these results suggest that RavS is a pivotal node for integrating the regulatory pathways of c-di-GMP and TCS, and RavR is a bifunctional regulator that finely controls the phosphorylation state of RavS (Fig 8). The crosstalk between c-di-GMP and RavS–RavR phosphoryl transfer regulates a bacterial lifestyle transition from virulence to freely swimming.




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