Controlled synthesis of the DSF cell-cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell-cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence.     

A two-component system involving an HD-GYP domain protein links cell-cell signalling to pathogenicity gene expression in Xanthomonas campestris

The synthesis of extracellular enzymes and extracellular polysaccharide (EPS) in Xanthomonas campestris pv. campestris (Xcc) is regulated by a cluster of genes called rpf (for regulation of pathogenicity factors). Two of the genes, rpfF and rpfB, have previously been implicated in the synthesis of a diffusible regulatory molecule, DSF. Here, we describe a screen of transposon insertion mutants of Xcc that identified two DSF-overproducing strains. In each mutant, the gene disrupted is rpfC, which encodes a hybrid two-component regulatory protein in which the sensor and regulator domains are fused and which contains an additional C-terminal phosphorelay (HPt) domain. We show that rpfC is in an operon with rpfH and rpfG. The predicted protein RpfG has a regulatory input domain attached to a specialized version of an HD domain, previously suggested to function in signal transduction. The predicted protein RpfH is structurally related to the sensory input domain of RpfC. We show that RpfC and RpfG act positively to regulate the synthesis of extracellular enzymes and EPS, but that RpfC acts negatively to regulate the synthesis of DSF. We propose that RpfGHC is a signal transduction system that couples the synthesis of pathogenicity factors to sensing of environmental signals that may include DSF itself.     

A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal molecule

Mutations in the seven clustered rpf genes cause downregulated synthesis of extracellular enzymes and reduced virulence of Xanthomonas campestris pathovar campestris ( Xcc ). The phenotype of mutants in one of the genes, rpfF , can be restored by a diffusible extracellular factor (DSF) produced by all Xcc strains tested, apart from rpfF and rpfB mutants. DSF accumulates in early stationary phase (when synthesis of enzymes is maximal), but levels decline subsequently. Addition of DSF to exponentially-growing wild-type bacteria does not cause precocious enzyme synthesis. rpfB and rpfF are expressed throughout growth, but the rate increases in early stationary phase. RpfB is predicted to be a long-chain fatty acyl CoA ligase, and RpfF shows some relatedness to enoyl CoA hydratases. The properties of DSF suggest that it may be a fatty-acid derivative, and certain lipid preparations possess DSF activity at higher concentrations. These include lipid extracts and acid-hydrolysed lipopolysaccharide and lipid A from Xcc , and purified dodecanoic and hydroxydodecanoic acid. DSF production is confined to certain xanthomonads. We propose a model for the DSF system, which represents a novel mechanism for regulating virulence factor synthesis in response to physiological or environmental changes.     

Expression of the gum operon directing xanthan biosynthesis in Xanthomonas campestris and its regulation in planta

The gum gene cluster of Xanthomonas campestris pv. campestris comprises 12 genes whose products are involved in the biosynthesis of the extracellular polysaccharide xanthan. These genes are expressed primarily as an operon from a promoter upstream of the first gene, gumB. Although the regulation of xanthan synthesis in vitro has been well studied, nothing is known of its regulation in planta. A reporter plasmid was constructed in which the promoter region of the gum operon was fused to gusA. In liquid cultures, the expression of the gumgusA reporter was correlated closely with the production of xanthan, although a low basal level of beta-glucuronidase activity was seen in the absence of added carbon sources when xanthan production was very low. The expression of the gumgusA fusion also was subject to positive regulation by rpfF, which is responsible for the synthesis of the diffusible signal factor (DSF). The expression of the gumgusA fusion in bacteria recovered from inoculated turnip leaves was maximal at the later phases of growth and was subject to regulation by rpfF. These results provide indirect support for the operation of the DSF regulatory system in bacteria in planta.     

Contribution of rpfB to cell-to-cell signal synthesis, virulence, and vector transmission of Xylella fastidiosa

In Xylella fastidiosa the fatty acid signal molecule diffusible signaling factor (DSF) is produced and sensed by components of the regulation of pathogenicity factors (rpf) cluster; lack of DSF production in RpfF mutants results in a non-vector-transmissible phenotype yet cells are hypervirulent to grape. rpfB has not been characterized in Xylella fastidiosa, although its homolog has been suggested to be required for DSF synthesis in Xanthomonas campestris pv. campestris. We show that RpfB is involved in DSF processing in both Xylella fastidiosa and Xanthomonas campestris, affecting the profile of DSF-like fatty acids observed in thin-layer chromatography. Although three fatty acids whose production is dependent on RpfF were detected in Xylella fastidiosa and Xanthomonas campestris wild-type strains, their respective rpfB mutants accumulated primarily one chemical species. Although no quantifiable effect of rpfB on plant colonization by Xylella fastidiosa was found, insect colonization and transmission was reduced. Thus, RpfB apparently is involved in DSF processing, and like Xanthomonas campestris, Xylella fastidiosa also produces multiple DSF molecules. It is possible that Xylella fastidiosa coordinates host vector and plant colonization by varying the proportions of different forms of DSF signals via RpfB.     

rpfF mutants of Xanthomonas oryzae pv. oryzae are deficient for virulence and growth under low iron conditions

Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. In the related bacterium Xanthomonas campestris pv. campestris, the rpfF gene is involved in production of a diffusible extracellular factor (DSF) that positively regulates synthesis of virulence-associated functions like extracellular polysaccharide (EPS) and extracellular enzymes. Transposon insertions in the rpfF homolog of X. oryzae pv. oryzae are deficient for virulence and production of a DSF but are proficient for EPS and extracellular enzyme production. The rpfF X. oryzae pv. oryzae mutants exhibit an unusual tetracycline susceptibility phenotype in which exogenous iron supplementation is required for phenotypic expression of a tetracycline resistance determinant that is encoded on an introduced plasmid. The rpfF X. oryzae pv. oryzae mutants also overproduce one or more siderophores and exhibit a growth deficiency under low iron conditions as well as in the presence of reducing agents that are expected to promote the conversion of Fe+3 to Fe+2. Exogenous iron supplementation promotes migration of rpfF X. oryzae pv. oryzae mutants in rice leaves. The results suggest that rpfF may be involved in controlling an iron-uptake system of X. oryzae pv. oryzae and that an inability to cope with the conditions of low iron availability in the host may be the reason for the virulence deficiency of the rpfF X. oryzae pv. oryzae mutants.     

Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain-protein interaction

The hybrid sensor kinase RpfC positively regulates the expression of a range of virulent genes and negatively modulates the synthesis of the quorum sensing signal diffusible signal factor (DSF) in Xanthomonas campestris. Three conserved amino acid residues of RpfC implicated in phosphorelay (His(198) in the histidine kinase domain, Asp(512) in the receiver domain, and His(657) in the histidine phosphotransfer domain) were essential for activation of the production of extracellular enzymes and extracellular polysaccharide (EPS) virulence factors but were not essential for repression of DSF biosynthesis. Domain deletion and subsequent in trans expression analysis revealed that the receiver domain of RpfC alone was sufficient to repress DSF overproduction in an rpfC deletion mutant. Further deletion and alanine scanning mutagenesis analyses identified a peptide of 107 amino acids and three amino acid residues (Gln(496), Glu(504), and Ile(552)) involved in modulating DSF production. Co-immunoprecipitation and far Western blot analyses suggested an interaction between the receiver domain and RpfF, the enzyme involved in DSF biosynthesis. These data support a model in which RpfC modulates two different functions (virulence factor synthesis and DSF synthesis) by utilization of a conserved phosphorelay system and a novel domain-specific protein-protein interaction mechanism, respectively. This latter mechanism represents an added dimension to conventional two-component signaling paradigms.     

Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions

The bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) recruits a diffusible signal factor (DSF), which has recently been structurally characterized as cis-11-methyl-2-dodecenoic acid, as a cell-cell communication signal to synchronize virulence gene expression and biofilm dispersal. In this study, we showed that despite the existance of phenotype variations in different Xcc isolates, the DSF-mediated functions were in general conserved. To investigate the genomic profiles of DSF regulation, we designed and conducted oligomicroarray analysis by comparison of the gene expression patterns of wild-type strain XC1 and its DSF-deficient mutant XC1dF, as well as those of XC1dF in the presence or absence of DSF signals. The analyses led to identification of 165 genes, whose expression was significantly influenced by DSF signals. These genes encode proteins and enzymes belonging to at least 12 functional groups. In addition to those previously known DSF-dependent activities such as production of extracellular enzymes and extracellular polysaccharides, microarray analyses also revealed new functions mediated by DSF, such as flagellum synthesis, resistance to toxins and oxidative stress, and aerobic respiration. Phenotype analyses confirmed that DSF signalling contributed to resistance to toxin acriflavin and hydrogen peroxide, and to the survival of bacterial cells at different temperatures. We conclude that DSF cell-cell signalling is not only essential for co-ordinating the expression of virulence genes but also plays a vital role in keeping up the general competence of the pathogen in ecosystems.     

The Host Plant Metabolite Glucose Is the Precursor of Diffusible Signal Factor (DSF) Family Signals in Xanthomonas campestris

Plant pathogen Xanthomonas campestris pv. campestris produces cis-11-methyl-2-dodecenoic acid (diffusible signal factor [DSF]) as a cell-cell communication signal to regulate biofilm dispersal and virulence factor production. Previous studies have demonstrated that DSF biosynthesis is dependent on the presence of RpfF, an enoyl-coenzyme A (CoA) hydratase, but the DSF synthetic mechanism and the influence of the host plant on DSF biosynthesis are still not clear. We show here that exogenous addition of host plant juice or ethanol extract to the growth medium of X. campestris pv. campestris could significantly boost DSF family signal production. It was subsequently revealed that X. campestris pv. campestris produces not only DSF but also BDSF (cis-2-dodecenoic acid) and another novel DSF family signal, which was designated DSF-II. BDSF was originally identified in Burkholderia cenocepacia to be involved in regulation of motility, biofilm formation, and virulence in B. cenocepacia. Functional analysis suggested that DSF-II plays a role equal to that of DSF in regulation of biofilm dispersion and virulence factor production in X. campestris pv. campestris. Furthermore, chromatographic separation led to identification of glucose as a specific molecule stimulating DSF family signal biosynthesis in X. campestris pv. campestris. ¹³C-labeling experiments demonstrated that glucose acts as a substrate to provide a carbon element for DSF biosynthesis. The results of this study indicate that X. campestris pv. campestris could utilize a common metabolite of the host plant to enhance DSF family signal synthesis and therefore promote virulence.     

The HD-GYP domain of RpfG mediates a direct linkage between the Rpf quorum-sensing pathway and a subset of diguanylate cyclase proteins in the phytopathogen Xanthomonas axonopodis pv citri

Bacteria use extracellular levels of small diffusible autoinducers to estimate local cell-density (quorum-sensing) and to regulate complex physiological processes. The quorum-sensing signal transduction pathway of Xanthomonas spp. phytopathogens has special features that distinguish it from that of other pathogens. This pathway consists of RpfF, necessary for the production of the unique autoinducer 'diffusible signalling factor' (DSF), and RpfC and RpfG, a two-component system necessary for the DSF-dependent production of extracellular pathogenicity factors and cellular dispersion. Yeast two-hybrid and direct in vitro assays were used to identify interactions involving the Rpf group of proteins. We show that RpfC, a protein consisting of N-terminal transmembrane, histidine kinase, response-regulator and C-terminal histidine phosphotransfer domains interacts with both RpfG, a protein consisting of an N-terminal response regulator domain and a C-terminal HD-GYP domain, and with RpfF. We also show that RpfC interacts with the only known homologue of 'conditioned medium factor', which is involved in quorum-sensing in Dictyostelium discoideum under conditions of nutritional stress. Furthermore, RpfCG is shown to interact with a second two-component system made up of NtrB and NtrC homologues. Finally we show that the recently characterized HD-GYP phosphodiesterase domain of RpfG interacts directly with diguanylate cyclase GGDEF domain-containing proteins coded by the Xanthomonas axonopodis pv. citri genome, which in other bacteria produce cyclic diGMP, an important second messenger involved in the regulation of complex bacterial processes including biofilm production, virulence and motility. These results demonstrate a direct physical linkage between quorum-sensing and cyclic diGMP signalling pathways in bacteria.     

Cell-to-cell signaling in Xylella fastidiosa suppresses movement and xylem vessel colonization in grape

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein-marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.     

植物病原黄单胞菌退出群体感应生理状态的分子机制和生物学意义

黄单胞菌是一类引起多种作物病害的病原细菌总称。它们利用自身产生的DSF(Diffusible signaling factor)-家族群体感应(quorum sensing,QS)信号分子感应群体密度,调控致病相关基因的表达。当黄单胞菌培养达到对数生长后期时,培养体系中DSF信号分子浓度迅速降低,呈现一种典型的群体感应信号反转(turnover)现象。编码脂酰辅酶A连接酶的基因rpfB在黄单胞菌生长后期表达量显著提高,敲除rpfB导致DSF生物合成显著提高,因此,RpfB参与DSF-家族QS信号分子的降解,在群体感应后期引导黄单胞菌退出群体感应状态。DSF信号通过RpfC/RpfG双组分系统、环二鸟苷酸和全局性转录因子Clp负调控rpfB的表达。DSF群体感应信号关键降解基因rpfB存在于多种细菌中,表明该信号反转机制相对保守,但其调控的生物学功能因菌而异。     

Quorum sensing and virulence regulation in Xanthomonas campestris

It is now clear that cell–cell communication, often referred to as quorum sensing (QS), is the norm in the prokaryotic kingdom and this community-wide genetic regulatory mechanism has been adopted for regulation of many important biological functions. Since the 1980s, several types of QS signals have been identified, which are associated commonly with different types of QS mechanisms. Among them, the diffusible signal factor (DSF)-dependent QS system, originally discovered from bacterial pathogen Xanthomonas campestris pv. campestris , is a relatively new regulatory mechanism. The rapid research progress over the last few years has identified the chemical structure of the QS signal DSF, established the DSF regulon, and unveiled the general signaling pathways and mechanisms. Particular noteworthy are that DSF biosynthesis is modulated by a novel posttranslational autoinduction mechanism involving protein–protein interaction between the DSF synthase RpfF and the sensor RpfC, and that QS signal sensing is coupled to intracellular regulatory networks through a second messenger cyclic-di-GMP and a global regulator Clp. Genomic and genetic analyses show that the DSF QS-signaling pathway regulates diverse biological functions including virulence, biofilm dispersal, and ecological competence. Moreover, evidence is emerging that the DSF QS system is conserved in a range of plant and human bacterial pathogens.     

Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia

The genome of Stenotrophomonas maltophilia encodes a cell-cell signaling system that is highly related to the diffusible signal factor (DSF)-dependent system of the phytopathogen Xanthomonas campestris. Here we show that in S. maltophilia, DSF signaling controls factors contributing to the virulence and antibiotic resistance of this important nosocomial pathogen.