The Xanthomonas oryzae pv. oryzae PilZ Domain Proteins Function Differentially in Cyclic di-GMP Binding and Regulation of Virulence and Motility.
ABSTRACT: The PilZ domain proteins have been demonstrated to be one of the major types of receptors mediating cyclic di-GMP (c-di-GMP) signaling pathways in several pathogenic bacteria. However, little is known about the function of PilZ domain proteins in c-di-GMP regulation of virulence in the bacterial blight pathogen of rice Xanthomonas oryzae pv. oryzae. Here, the roles of PilZ domain proteins PXO_00049 and PXO_02374 in c-di-GMP binding, regulation of virulence and motility, and subcellular localization were characterized in comparison with PXO_02715, identified previously as an interactor with the c-di-GMP receptor Filp to regulate virulence. The c-di-GMP binding motifs in the PilZ domains were conserved in PXO_00049 and PXO_02374 but were less well conserved in PXO_02715. PXO_00049 and PXO_02374 but not PXO_02715 proteins bound to c-di-GMP with high affinity in vitro, and the R(141) and R(10) residues in the PilZ domains of PXO_00049 and PXO_02374, respectively, were crucial for c-di-GMP binding. Gene deletion of PXO_00049 and PXO_02374 resulted in significant increases in virulence and hrp gene transcription, indicating their negative regulation of virulence via type III secretion system expression. All mutants showed significant changes in sliding motility but not exopolysaccharide production and biofilm formation. In trans expression of the full-length open reading frame (ORF) of each gene in the relevant mutants led to restoration of the phenotype to wild-type levels. Moreover, PXO_00049 and PXO_02374 displayed mainly multisite subcellular localizations, whereas PXO_02715 showed nonpolar distributions in the X. oryzae pv. oryzae cells. Therefore, this study demonstrated the different functions of the PilZ domain proteins in mediation of c-di-GMP regulation of virulence and motility in X. oryzae pv. oryzae.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. Xoo produces a range of virulence-related factors to facilitate its pathogenesis in rice, however, the regulatory mechanisms of Xoo virulence expression have been not fully elucidated. Recent studies have revealed that virulence factor production is regulated via cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway that is well-conserved in Xoo and other Xanthomonas species. A set of GGDEF, EAL, HD-GYP, and PilZ domain proteins with diverse signal sensory domains for c-di-GMP synthesis, hydrolysis, and binding is encoded in the Xoo genome. Bioinformatic, genetic, and biochemical analysis has identified an array of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), as well as degenerate GGDEF/EAL, PilZ domain proteins along with a transcription regulator. These signaling components have been characterized to regulate various bacterial cellular processes, such as virulence, exopolysaccharide (EPS) production, biofilm formation, motility, and adaptation at the transcriptional, post-translational, and protein-protein interaction levels. This review summarized the recent progress in understanding the importance and complexity of c-di-GMP signaling in regulating bacterial virulence expression, highlighting the identified key signal elements and orthologs found in Xanthomonads, discussing the diverse functions of GGDEF/EAL/HD-GYP domains, existence of a complicated multifactorial network between DGCs, PDEs, and effectors, and further exploration of the new c-di-GMP receptor domains. These findings and knowledge lay the groundwork for future experimentation to further elucidate c-di-GMP regulatory circuits involved in regulation of bacterial pathogenesis.
Project description:In Xanthomonas oryzae pv. oryzae, the bacterial blight pathogen of rice, there are over 20 genes encoding GGDEF, EAL, and HD-GYP domains, which are potentially involved in the metabolism of second messenger c-di-GMP. In this study, we focused on the characterization of an EAL domain protein, EdpX1. Deletion of the edpX1 gene resulted in a 2-fold increase in the intracellular c-di-GMP levels, which were restored to the wild-type levels in the complemented ?edpX1(pB-edpX1) strain, demonstrating that EdpX1 is an active phosphodiesterase (PDE) in X. oryzae pv. oryzae. In addition, colorimetric assays further confirmed the PDE activity of EdpX1 by showing that the E153A mutation at the EAL motif strongly reduced its activity. Virulence assays on the leaves of susceptible rice showed that the ?edpX1 mutant was severely impaired in causing disease symptoms. In trans expression of wild-type edpX1, but not edpX1 E153A, was able to complement the weakened virulence phenotype. These results indicated that an active EAL domain is required for EdpX1 to regulate the virulence of X. oryzae pv. oryzae. We then demonstrated that the ?edpX1 mutant was defective in secreting exopolysaccharide (EPS) and forming biofilms. The expression of edpX1 in the ?edpX1 mutant, but not edpX1 E153A, restored the defective phenotypes to near-wild-type levels. In addition, we observed that EdpX1-green fluorescent protein (EdpX1-GFP) exhibited multiple subcellular localization foci, and this pattern was dependent on its transmembrane (TM) region, which did not seem to directly contribute to the regulatory function of EdpX1. Thus, we concluded that EdpX1 exhibits PDE activity to control c-di-GMP levels, and its EAL domain is necessary and sufficient for its regulation of virulence in X. oryzae pv. oryzae.IMPORTANCE Bacteria utilize c-di-GMP as a second messenger to regulate various biological functions. The synthesis and degradation of c-di-GMP are catalyzed by GGDEF domains and an EAL or HD-GYP domain, respectively. Multiple genes encoding these domains are often found in one bacterial strain. For example, in the genome of X. oryzae pv. oryzae PXO99A, 26 genes encoding proteins containing these domains were identified. Therefore, to fully appreciate the complexity and specificity of c-di-GMP signaling in X. oryzae pv. oryzae, the enzymatic activities and regulatory functions of each GGDEF, EAL, and HD-GYP domain protein need to be elucidated. In this study, we showed that the EAL domain protein EdpX1 is a major PDE to regulate diverse virulence phenotypes through the c-di-GMP signaling pathway.
Project description:Cyclic di-GMP [(bis-(3'-5')-cyclic di-guanosine monophosphate)] is an almost ubiquitous second messenger in bacteria that is implicated in the regulation of a range of functions that include developmental transitions, aggregative behaviour, adhesion, biofilm formation and virulence. Comparatively little is known about the mechanism(s) by which cyclic di-GMP exerts these various regulatory effects. PilZ has been identified as a cyclic di-GMP binding protein domain; proteins with this domain are involved in regulation of specific cellular processes, including the virulence of animal pathogens. Here we have examined the role of PilZ domain proteins in virulence and the regulation of virulence factor synthesis in Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot of crucifers. The Xcc genome encodes four proteins (XC0965, XC2249, XC2317 and XC3221) that have a PilZ domain. Mutation of XC0965, XC2249 and XC3221 led to a significant reduction of virulence in Chinese radish. Mutation of XC2249 and XC3221 led to a reduction in motility whereas mutation of XC2249 and XC0965 affected extracellular enzyme production. All mutant strains were unaffected in biofilm formation in vitro. The reduction of virulence following mutation of XC3221 could not be wholly attributed to an effect on motility as mutation of pilA, which abolishes motility, has a lesser effect on virulence.
Project description:The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ?XOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ?XOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ?XOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.
Project description:Cyclic diguanylate (c-di-GMP) is an allosteric activator and second messenger implicated in the regulation of a variety of biological processes in diverse bacteria. In Vibrio cholerae, c-di-GMP has been shown to inversely regulate biofilm-specific and virulence gene expression, suggesting that c-di-GMP signaling is important for the transition of V. cholerae from the environment to the host. However, the mechanism behind this regulation remains unknown. Recently, it was proposed that the PilZ protein domain represents a c-di-GMP-binding domain. Here we show that V. cholerae PilZ proteins bind c-di-GMP specifically and are involved in the regulation of biofilm formation, motility, and virulence. These findings confirm a role for PilZ proteins as c-di-GMP-sensing proteins within the c-di-GMP signaling network.
Project description:Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice blight disease as well as a serious phytopathogen worldwide. It is also one of the model organisms for studying bacteria-plant interactions. Current progress in bacterial signal transduction pathways has identified cyclic di-GMP as a major second messenger molecule in controlling Xanthomonas pathogenicity. However, it still remains largely unclear how c-di-GMP regulates the secretion of bacterial virulence factors in Xoo. In this study, we focused on the important roles played by DgcA (XOO3988), one of our previously identified diguanylate cyclases in Xoo, through further investigating the phenotypes of several dgcA-related mutants, namely, the dgcA-knockout mutant ?dgcA, the dgcA overexpression strain OdgcA, the dgcA complemented strain CdgcA and the wild-type strain. The results showed that dgcA negatively affected virulence, EPS production, bacterial autoaggregation and motility, but positively triggered biofilm formation via modulating the intracellular c-di-GMP levels. RNA-seq data further identified 349 differentially expressed genes controlled by DgcA, providing a foundation for a more solid understanding of the signal transduction pathways in Xoo. Collectively, the present study highlights DgcA as a major regulator of Xoo virulence, and can serve as a potential target for preventing rice blight diseases.
Project description:PilZ domain is one of the key receptors for the newly discovered secondary messenger molecule cyclic di-GMP (c-di-GMP). To date, several monomeric PilZ domain proteins have been identified. Some exhibit strong c-di-GMP binding activity, while others have barely detectable c-di-GMP binding activity and require an accessory protein such as FimX to indirectly respond to the c-di-GMP signal. We now report a novel tetrameric PilZ domain structure of XCC6012 from the plant pathogen Xanthomonas campestris pv. campestris (Xcc). It is one of the four PilZ domain proteins essential for Xcc pathogenicity. Although the monomer adopts a structure similar to those of the PilZ domains with very weak c-di-GMP binding activity, it is nevertheless interrupted in the middle by two extra long helices. Four XCC6012 proteins are thus self-assembled into a tetramer via the extra heptad repeat ?3 helices to form a parallel four-stranded coiled-coil, which is further enclosed by two sets of inclined ?2 and ?4 helices. We further generated a series of XCC6012 variants and measured the unfolding temperatures and oligomeric states in order to investigate the nature of this novel tetramer. Discovery of this new PilZ domain architecture increases the complexity of c-di-GMP-mediated regulation.
Project description:HD-GYP domain cyclic dimeric GMP (c-di-GMP) phosphodiesterases are implicated in motility and virulence in bacteria. Borrelia burgdorferi possesses a single set of c-di-GMP-metabolizing enzymes, including a putative HD-GYP domain protein, BB0374. Recently, we characterized the EAL domain phosphodiesterase PdeA. A mutation in pdeA resulted in cells that were defective in motility and virulence. Here we demonstrate that BB0374/PdeB specifically hydrolyzed c-di-GMP with a K(m) of 2.9 nM, confirming that it is a functional phosphodiesterase. Furthermore, by measuring phosphodiesterase enzyme activity in extracts from cells containing the pdeA pdeB double mutant, we demonstrate that no additional phosphodiesterases are present in B. burgdorferi. pdeB single mutant cells exhibit significantly increased flexing, indicating a role for c-di-GMP in motility. Constructing and analyzing a pilZ pdeB double mutant suggests that PilZ likely interacts with chemotaxis signaling. While virulence in needle-inoculated C3H/HeN mice did not appear to be altered significantly in pdeB mutant cells, these cells exhibited a reduced ability to survive in Ixodes scapularis ticks. Consequently, those ticks were unable to transmit the infection to naïve mice. All of these phenotypes were restored when the mutant was complemented. Identification of this role of pdeB increases our understanding of the c-di-GMP signaling network in motility regulation and the life cycle of B. burgdorferi.
Project description:Diguanylate cyclase and phosphodiesterase enzymatic activities control c-di-GMP levels modulating planktonic versus sessile lifestyle behavior in bacteria. The PilZ domain is described as a sensor of c-di-GMP intracellular levels and the proteins containing a PilZ domain represent the best studied class of c-di-GMP receptors forming part of the c-di-GMP signaling cascade. In P. fluorescens F113 we have found two diguanylate cyclases (WspR, SadC) and one phosphodiesterase (BifA) implicated in regulation of swimming motility and biofilm formation. Here we identify a flgZ gene located in a flagellar operon encoding a protein that contains a PilZ domain. Moreover, we show that FlgZ subcellular localization depends on the c-di-GMP intracellular levels. The overexpression analysis of flgZ in P. fluorescens F113 and P. putida KT2440 backgrounds reveal a participation of FlgZ in Pseudomonas swimming motility regulation. Besides, the epistasis of flgZ over wspR and bifA clearly shows that c-di-GMP intracellular levels produced by the enzymatic activity of the diguanylate cyclase WspR and the phosphodiesterase BifA regulates biofilm formation through FlgZ.
Project description:Bacterial blight of rice is an important serious bacterial diseases of rice in many rice-growing regions, caused by Xanthomonas oryzae pv. oryzae (Xoo). The thiG gene from Xoo strain ZJ173, which is involved with thiazole moiety production in the thiamine biosynthesis pathway, is highly conserved among the members of Xanthomonas. The thiG deletion mutant displayed impaired virulence and growth in thiamine-free medium but maintained its normal growth rate in the rice tissues, indicating that the thiG gene is involved in Xoo virulence. Compared to the wild type strain, the formation of cell-cell aggregates was affected in thiG deletion mutants. Although biofilm formation was promoted, motility and migration in rice leaves were repressed in the thiG mutants, and therefore limited the expansion of pathogen infection in rice. Quorum sensing and extracellular substance are two key factors that contribute to the formation of cell-cell aggregates. Our study found that in the thiG mutant the expression of two genes, rpfC and rpfG, which form a two-component regulatory signal system involved in the regulation of biofilm formation by a second messenger cyclic di-GMP is down-regulated. In addition, our study showed that xanthan production was not affected but the expression of some genes associated with xanthan biosynthesis, like gumD, gumE, gumH and gumM, were up-regulated in thiG mutants. Taken together, these findings are the first to demonstrate the role of the thiazole biosynthsis gene, thiG, in virulence and the formation of aggregates in Xanthomonas oryzae pv. oryzae.