Cell-cell signaling in Xanthomonas campestris involves an HD-GYP domain protein that functions in cyclic di-GMP turnover

Abstract

HD-GYP is a protein domain of unknown biochemical function implicated in bacterial signaling and regulation. In the plant pathogen Xanthomonas campestris pv. campestris, the synthesis of virulence factors and dispersal of biofilms are positively controlled by a two-component signal transduction system comprising the HD-GYP domain regulatory protein RpfG and cognate sensor RpfC and by cell-cell signaling mediated by the diffusible signal molecule DSF (diffusible signal factor). The RpfG/RpfC two-component system has been implicated in DSF perception and signal transduction. Here we show that the role of RpfG is to degrade the unusual nucleotide cyclic di-GMP, an activity associated with the HD-GYP domain. Mutation of the conserved H and D residues of the isolated HD-GYP domain resulted in loss of both the enzymatic activity against cyclic di-GMP and the regulatory activity in virulence factor synthesis. Two other protein domains, GGDEF and EAL, are already implicated in the synthesis and degradation respectively of cyclic di-GMP. As with GGDEF and EAL domains, the HD-GYP domain is widely distributed in free-living bacteria and occurs in plant and animal pathogens, as well as beneficial symbionts and organisms associated with a range of environmental niches. Identification of the role of the HD-GYP domain thus increases our understanding of a signaling network whose importance to the lifestyle of diverse bacteria is now emerging.

Fig. 1.

Expression of the cyclic di-GMP phosphodiesterase PA2567 restores extracellular enzyme and EPS virulence factor production to Xcc rpf mutants. PA2567 cloned in pLAFR6 was introduced into rpfG and rpfGHC mutants. (A) The level of protease production assessed by zones of clearing produced after growth of bacteria on skimmed milk agar plates. (B) The production of xanthan by bacteria grown on NYG agar supplemented with 2% (wt/vol) glucose. Xanthan production is evident by the mucoid appearance of the colonies, which is reduced in the rpf mutants. The relative level of xanthan production in different strains was confirmed by gravimetric analysis of polysaccharide production by bacteria grown in NYGB medium with 2% (wt/vol) glucose. (C and D) The relative levels of mannanase (C) and endoglucanase (D) in culture supernatants of bacterial strains grown in NYGB medium to an OD at 600 nm of 2.0. Values given are the means and standard errors of triplicate measurements.

Fig. 2.

Effect of expression of RpfG on swarming in P. aeruginosa. (A) Swarming phenotypes of P. aeruginosa rsmA mutant and wild-type PA01. (B) Expression of RpfG partially restores swarming to the rsmA mutant of P. aeruginosa (upper right) and enhances swarming motility in wild type P. aeruginosa (lower right). These effects are not observed when rpfG is cloned in the incorrect orientation to be driven by the vector promoter (left).

Fig. 3.

The isolated HD-GYP domain of RpfG is active in regulation and possesses cyclic di-GMP phosphodiesterase activity. (A and B) The HD-GYP domain with a C-terminal His₆ tag can partially restore mannanase (A) and endoglucanase (B) synthesis to both rpfG and rpfGHC mutants. Bacterial strains were grown in NYGB medium to an OD at 600 nm of 2.0. Values given are the means and standard errors of triplicate measurements. (C) SDS/PAGE of the HD-GYPHis₆ protein purified by nickel affinity chromatography shows a single band of the expected size of 27 kDa. (D) The purified protein has enzymatic activity against cyclic di-GMP. Reverse-phase HPLC analysis of aliquots of reaction mixtures boiled immediately after addition of the enzyme and after 300 min of incubation shows the degradation of cyclic di-GMP to a compound with the same mobility as the GMP standard. The identity of the product was confirmed by mass spectrometry.

Fig. 4.

Mutation of the signature H and D residues in the HD-GYP domain abolishes its regulatory function. (A and B) Variant proteins with H231A and D232A alteration (labeled AD-GYP and HA-GYP) are unable to restore production of mannanase (A) and endoglucanase (B) to rpfG and rpfGHC mutants of Xcc. Values given are the means and standard errors of triplicate measurements. (C) Western blot analysis with an anti-His₆ antiserum shows that all variant proteins are expressed in Xcc. Lanes: 1, 8004 (HD-GYPHis₆); 2, 8004 (pLAFR3); 3, rpfG (pLAFR3); 4, rpfG (HD-GYPHis₆); 5, rpfG (AD-GYPHis₆); 6, rpfG (HA-GYPHis₆); 7, rpfG (AA-GYPHis₆). (D) Variant proteins H231A and D232A are unable to restore production of protease to rpfG of Xcc. The level of protease production was assessed by zones of clearing produced after growth of bacteria on skimmed milk agar plates. The H231AD232A variant is also inactive, and similar results are seen in the rpfGHC mutant background (data not shown).

Fig. 5.

Reconstruction of the Rpf/DSF signaling pathway in P. aeruginosa. Expression of RpfG and RpfC together (from the rpfGHC operon) partially restores swarming motility to the rsmA mutant of P. aeruginosa (upper left). Addition of DSF to the medium in increasing amounts allows a full restoration to wild-type motility.