The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors

Abstract

The widely conserved second messenger cyclic diguanosine monophosphate (c-di-GMP) plays a key role in quorum-sensing (QS)-dependent production of virulence factors in Xanthomonas campestris pv. campestris. The detection of QS diffusible signal factor (DSF) by the sensor RpfC leads to the activation of response regulator RpfG, which activates virulence gene expression by degrading c-di-GMP. Here, we show that a global regulator in the X. campestris pv. campestris QS regulatory pathway, Clp, is a c-di-GMP effector. c-di-GMP specifically binds to Clp with high affinity and induces allosteric conformational changes that abolish the interaction between Clp and its target gene promoter. Clp is similar to the cyclic AMP (cAMP) binding proteins Crp and Vfr and contains a conserved cyclic nucleotide monophosphate (cNMP) binding domain. Using site-directed mutagenesis, we found that the cNMP binding domain of Clp contains a glutamic acid residue (E99) that is essential for c-di-GMP binding. Substituting the residue with serine (E99S) resulted in decreased sensitivity to changes in the intracellular c-di-GMP level and attenuated bacterial virulence. These data establish the direct role of Clp in the response to fluctuating c-di-GMP levels and depict a novel mechanism by which QS links the second messenger with the X. campestris pv. campestris virulence regulon.

FIG. 1.

ITC analysis of c-di-GMP (A) and cAMP (B) binding to Clp. The top of each panel shows the ITC titration of 20 μM Clp with 5-μl aliquots of 500 μM c-di-GMP or 10 mM cAMP in Tris-HCl buffer at 20°C. The bottom of each panel shows the binding isotherm for the titration represented in the top of the panel; the solid line is the best fit of the data to a one-site binding model.

FIG. 2.

The effect of c-di-GMP on the conformational structure and promoter binding activity of Clp. (A) EMSA analysis of the impact of c-di-GMP on Clp binding to the engXCA promoter. DIG-labeled promoter fragments were incubated with purified Clp proteins in the presence or absence of nucleotide, as indicated. The final concentration of nucleotide in the reaction mixture was 10 μM or 100 μM. (B and C) Far-UV CD spectra of Clp and the control, glutathione-S-transferase (GST), in the presence or absence of c-di-GMP, as indicated. deg, degrees; com², cm².

FIG. 3.

Modulation of the intracellular level of c-di-GMP changes the expression pattern of engXCA. (A) The effect of rpfG and clp mutations on engXCA transcription in X. campestris pv. campestris (Xcc). (B) Effect of c-di-GMP synthase (PA5487) and degradation enzyme (PA3947) on engXCA expression. The genes for in trans expression were cloned under the control of the lac promoter in expression vector pDSK519 (pDSK); the empty vector was introduced into wild-type strain 8004 (WT) and its ΔrpfG deletion mutant as controls. (C) RT-PCR analysis of engXCA with the strains described above. The relative signal intensity for each strain was derived after normalization against the corresponding 16S rRNA loading control. In panels A and B, the data are the means of three repeats, and the error bars indicate standard deviation.

FIG. 4.

The role of conserved amino acid residues implicated in cAMP binding in the functionality of Clp. (A) The effect of substituting the conserved amino acid residues on the regulatory activity of Clp in modulating engXCA expression. (B) The effect of substituting the conserved amino acid residues on the regulatory activity of Clp in modulating EPS production. The data are the means of three repeats, and the error bars indicate standard deviation.

FIG. 5.

The impact of amino acid substitution on the sensitivity of Clp to its inhibitory ligand c-di-GMP. (A) EMSA analysis of Clp and its derivatives (25 nM) in the presence or absence of c-di-GMP. (B) Effect of in trans expression of c-di-GMP synthase (PA5487) on engXCA expression in X. campestris pv. campestris strains expressing wild-type Clp or substituted alleles. (C) The effect of in trans expression of c-di-GMP synthase (PA5487) on the virulence of X. campestris pv. campestris strains expressing wild-type Clp or substituted alleles against cabbage. The experiment was repeated three times, and the photograph, taken 4 days after inoculation, shows a representative set of results.

FIG. 6.

The E99 residue of Clp may be a signature residue of c-di-GMP effectors with a cNMP binding domain. (A) Clp homologues can be grouped into two categories based on the signature amino acid E99. Colored shading indicates the conserved residues corresponding to E99 and T149 of Clp. XCV0519, Xanthomonas campestris pv. vesicatoria (NCBI accession no. CAJ22150); XAC0483, Xanthomonas axonopodis pv. citri (NCBI accession no. AAM35374); XOO_3933, Xanthomonas oryzae pv. oryzae MAFF 311018 (NCBI accession no. BAE70688); Clp, Xanthomonas campestris pv. campestris strain 8004 (NCBI accession no. AAY47667); XfasM23_0792, Xylella fastidiosa M23 (NCBI accession no. ACB92231); Clp_Sm, Stenotrophomonas maltophilia K279a (NCBI accession no. YP_001973974); PaerPA_01000634, Pseudomonas aeruginosa (NCBI accession no. ZP_01363536.1); Vfr, Pseudomonas aeruginosa PAO1 (NCBI accession no. AAG04041); Avin_46100, Azotobacter vinelandii DJ (NCBI accession no. ACO80719); PSPTOT1_1546, Pseudomonas syringae pv. tomato T1 (NCBI accession no. ZP_03395776); Crp, Escherichia coli strain K-12 substrain MG1655 (NCBI accession no. AAC76382). (B) ITC analysis of c-di-GMP (500 μM) binding to Clp_Sm. The dissociation constant (K_d) was estimated as 2.16 ± 5.4⁻⁷ M. (C) ITC analysis of Clp_Sm binding cAMP (1 mM).

FIG. 7.

Schematic representation of the role of Clp and c-di-GMP in the DSF-mediated QS regulation of virulence at low (A) and high (B) population density. The Clp-dependent virulence regulon is represented by vir, which includes engXCA and other virulence genes, as depicted in our previous study (7).