Diffusible Signal Factor Production Research Articles

Quorum sensing improves the plant growth-promoting ability of Stenotrophomonas rhizophila under saline-alkaline stress by enhancing its environmental adaptability.

Quorum sensing (QS) system has an essential function in plant growth-promoting rhizobacteria (PGPR) response to environmental stress and PGPR induction of plant tolerance to saline-alkaline stress. Nevertheless, there is a lack of understanding about how QS influences the growth-promoting effects of PGPR on plants. Stenotrophomonas rhizophila DSM14405T is a PGPR with a QS system, which can secrete diffusible signal factor (DSF), one of the QS signal molecules. In this study, we used the S. rhizophila wild type (WT) and an incompetent DSF production rpfF-knockout mutant strain to explore whether DSF-QS could affect the growth-promoting ability of PGPR in Brassica napus L. By measuring the seed germination rate, plant fresh weight, biomass, the total antioxidant capacity (T-AOC) level, and the content of chlorophyll in leaves, we found that DSF was unable to enhance the growth-promoting capacity of ΔrpfF and did not directly assist the plants in tolerating saline-alkaline stress. However, DSF aided S. rhizophila ΔrpfF in resisting stress during its effective period, and QS represents a continuous and precise regulatory mechanism. Altogether, our results show that DSF is helpful to improve the environmental adaptability and survival rate of S. rhizophila, thus indirectly improving the germination rate of seeds and helping plants grow under saline-alkaline stress. In this study, the mechanism of QS enhancing the environmental adaptability of PGPR was studied, which provided a theoretical basis for improving the application of PGPR to help plants better cope with saline-alkaline stress.

Diffusible signal factor enhances the saline-alkaline resistance and rhizosphere colonization of Stenotrophomonas rhizophila by coordinating optimal metabolism

Quorum sensing (QS) regulates various physiological processes in a cell density–dependent mode via cell–cell communication. Stenotrophomonas rhizophila DSM14405T having the diffusible signal factor (DSF)-QS system, is a plant growth-promoting rhizobacteria (PGPR) that enables host plants to tolerate saline-alkaline stress. However, the regulatory mechanism of DSF-QS in S. rhizophila is not fully understood. In this study, we used S. rhizophila DSM14405T wild-type (WT) and an incompetent DSF production rpfF-knockout mutant to explore the regulatory role of QS in S. rhizophila growth, stress responses, biofilm formation, and colonization under saline-alkaline stress. We found that a lack of DSF-QS reduces the tolerance of S. rhizosphere ΔrpfF to saline-alkaline stress, with a nearly 25-fold reduction in the ΔrpfF population compared with WT at 24 h under stress. Transcriptome analysis revealed that QS helps S. rhizophila WT respond to saline-alkaline stress by enhancing metabolism associated with the cell wall and membrane, oxidative stress response, cell adhesion, secretion systems, efflux pumps, and TonB systems. These metabolic systems enhance penetration defense, Na+ efflux, iron uptake, and reactive oxygen species scavenging. Additionally, the absence of DSF-QS causes overexpression of biofilm-associated genes under the regulation of sigma 54 and other transcriptional regulators. However, greater biofilm formation capacity confers no advantage on S. rhizosphere ΔrpfF in rhizosphere colonization. Altogether, our results show the importance of QS in PGPR growth and colonization; QS gives PGPR a collective adaptive advantage in harsh natural environments.

AmpR is a dual regulator in Stenotrophomonas maltophilia with a positive role in β-lactam resistance and a negative role in virulence, biofilm and DSF production

Stenotrophomonas maltophilia intrinsic resistance to β-lactams is mediated by two chromosomal β-lactamases, L1 and L2, whose induction depends on AmpR. Its quorum sensing (QS) signal, the diffusible signal factor (DSF), has a positive role in biofilm production, virulence and induction of β-lactamases. We hypothesized that AmpR has a role in virulence, biofilm production and QS system. Studies were done on S. maltophilia K279a, K279a ampRFS (ampR deficient mutant) and K279aM11 (constitutively active AmpR mutant). K279a ampRFS showed the highest biofilm biomass, thickness and 3D organization. Conversely, K279aM11 was the least efficient biofilm former strain. qRT-PCR showed that spgM, related to biofilm formation and virulence, was upregulated in K279a ampRFS and downregulated in K279aM11. A constitutively active AmpR led to a reduction of DSF production, while K279a ampRFS was the highest producer. Consequently, qRT-PCR showed that AmpR negatively regulated rpfF expression. K279a ampRFS presented the highest oxidative stress resistance, overexpressed sodA gene and showed the highest virulence in the Galleria mellonella killing assay. This is the first evidence of the function of AmpR as a dual regulator in S. maltophilia with a positive role in β-lactam resistance and a negative role in DSF production, biofilm formation, oxidative stress resistance and virulence.

Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing leptospirilli

Bioleaching of metal sulfide ores involves acidophilic microbes that catalyze the chemical dissolution of the metal sulfide bond that is enhanced by attached and planktonic cell mediated oxidation of iron(II)-ions and inorganic sulfur compounds. Leptospirillum spp. often predominate in sulfide mineral-containing environments, including bioheaps for copper recovery from chalcopyrite, as they are effective primary mineral colonizers and oxidize iron(II)-ions efficiently. In this study, we demonstrated a functional diffusible signal factor interspecies quorum sensing signaling mechanism in Leptospirillum ferriphilum and Leptospirillum ferrooxidans that produces (Z)-11-methyl-2-dodecenoic acid when grown with pyrite as energy source. In addition, pure diffusible signal factor and extracts from supernatants of pyrite grown Leptospirillum spp. inhibited biological iron oxidation in various species, and that pyrite grown Leptospirillum cells were less affected than iron grown cells to self inhibition. Finally, transcriptional analyses for the inhibition of iron-grown L. ferriphilum cells due to diffusible signal factor was compared with the response to exposure of cells to N- acyl-homoserine-lactone type quorum sensing signal compounds. The data suggested that Leptospirillum spp. diffusible signal factor production is a strategy for niche protection and defense against other microbes and it is proposed that this may be exploited to inhibit unwanted acidophile species.

BDSF Is the Predominant In-Planta Quorum-Sensing Signal Used During Xanthomonas campestris Infection and Pathogenesis in Chinese Cabbage.

Xanthomonas campestris pv. campestris uses the diffusible signal factor (DSF) family of quorum-sensing (QS) signals to coordinate virulence and adaptation. DSF family signals have been well-characterized using laboratory-based cell cultures. The in-planta QS signal used during X. campestris pv. campestris infection remains unclear. To achieve this goal, we first mimic in-planta X. campestris pv. campestris growth conditions by supplementing the previously developed XYS medium with cabbage hydrolysate and found that the dominant signal produced in these conditions was BDSF. Secondly, by using XYS medium supplemented with diverse plant-derived compounds, we examined the effects of diverse plant-derived compounds on the biosynthesis of DSF family signals. Several compounds were found to promote biosynthesis of BDSF. Finally, using an X. campestris pv. campestris ΔrpfB-Chinese cabbage infection model and an ultra-performance liquid chromatographic-time of flight-mass spectrometry-based assay, BDSF was found to comprise >70% of the DSF family signals present in infected cabbage tissue. BDSF at a concentration of 2.0 μM induced both protease activity and engXCA expression. This is the first report to directly show that BDSF is the predominant in-planta QS signal used during X. campestris pv. campestris infection. It provides a better understanding of the molecular interactions between X. campestris pv. campestris and its cruciferous hosts and also provides the logical target for designing strategies to counteract BDSF signaling and, thus, infection. Further studies are needed to get an exact idea about the DSF production dynamics of the wild-type strain inside the plant.

Ectopic Expression of Xylella fastidiosa rpfF Conferring Production of Diffusible Signal Factor in Transgenic Tobacco and Citrus Alters Pathogen Behavior and Reduces Disease Severity.

The pathogenicity of Xylella fastidiosa is associated with its ability to colonize the xylem of host plants. Expression of genes contributing to xylem colonization are suppressed, while those necessary for insect vector acquisition are increased with increasing concentrations of diffusible signal factor (DSF), whose production is dependent on RpfF. We previously demonstrated that transgenic citrus plants ectopically expressing rpfF from a citrus strain of X. fastidiosa subsp. pauca exhibited less susceptibility to Xanthomonas citri subsp. citri, another pathogen whose virulence is modulated by DSF accumulation. Here, we demonstrate that ectopic expression of rpfF in both transgenic tobacco and sweet orange also confers a reduction in disease severity incited by X. fastidiosa and reduces its colonization of those plants. Decreased disease severity in the transgenic plants was generally associated with increased expression of genes conferring adhesiveness to the pathogen and decreased expression of genes necessary for active motility, accounting for the reduced population sizes achieved in the plants, apparently by limiting pathogen dispersal through the plant. Plant-derived DSF signal molecules in a host plant can, therefore, be exploited to interfere with more than one pathogen whose virulence is controlled by DSF signaling.

Iron is a signal for Stenotrophomonas maltophilia biofilm formation, oxidative stress response, OMPs expression, and virulence.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen. In many bacteria iron availability regulates, through the Fur system, not only iron homeostasis but also virulence. The aim of this work was to assess the role of iron on S. maltophilia biofilm formation, EPS production, oxidative stress response, OMPs regulation, quorum sensing (QS), and virulence. Studies were done on K279a and its isogenic fur mutant F60 cultured in the presence or absence of dipyridyl. This is the first report of spontaneous fur mutants obtained in S. maltophilia. F60 produced higher amounts of biofilms than K279a and CLSM analysis demonstrated improved adherence and biofilm organization. Under iron restricted conditions, K279a produced biofilms with more biomass and enhanced thickness. In addition, F60 produced higher amounts of EPS than K279a but with a similar composition, as revealed by ATR-FTIR spectroscopy. With respect to the oxidative stress response, MnSOD was the only SOD isoenzyme detected in K279a. F60 presented higher SOD activity than the wt strain in planktonic and biofilm cultures, and iron deprivation increased K279a SOD activity. Under iron starvation, SDS-PAGE profile from K279a presented two iron-repressed proteins. Mass spectrometry analysis revealed homology with FepA and another putative TonB-dependent siderophore receptor of K279a. In silico analysis allowed the detection of potential Fur boxes in the respective coding genes. K279a encodes the QS diffusible signal factor (DSF). Under iron restriction K279a produced higher amounts of DSF than under iron rich condition. Finally, F60 was more virulent than K279a in the Galleria mellonella killing assay. These results put in evidence that iron levels regulate, likely through the Fur system, S. maltophilia biofilm formation, oxidative stress response, OMPs expression, DSF production and virulence.

Decoding the genetic and functional diversity of the DSF quorum-sensing system in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia uses the Diffusible Signal Factor (DSF) quorum sensing (QS) system to mediate intra- and inter-specific signaling and regulate virulence-related processes. The components of this system are encoded by the rpf cluster, with genes rpfF and rpfC encoding for the DSF synthase RpfF and sensor RpfC, respectively. Recently, we have shown that there exist two variants of the rpf cluster (rpf-1 and rpf-2), distinguishing two groups of S. maltophilia strains. Surprisingly, only rpf-1 strains produce detectable DSF, correlating with their ability to control biofilm formation, swarming motility and virulence. The evolutive advantage of acquiring two different rpf clusters, the phylogenetic time point and mechanism of this acquisition and the conditions that activate DSF production in rpf-2 strains, are however not known. Examination of this cluster in various species suggests that its variability originated most probably by genetic exchange between rhizosphere bacteria. We propose that rpf-2 variant strains make use of a strategy recently termed as “social cheating.” Analysis of cellular and extracellular fatty acids (FAs) of strains E77 (rpf-1) and M30 (rpf-2) suggests that their RpfFs have also a thioesterase activity that facilitates the release of unspecific FAs to the medium in addition to DSF. Production of DSF in rpf-1 strains appears in fact to be modulated by some of these extracellular FAs in addition to other factors such as temperature and nutrients, while in rpf-2 strains DSF biosynthesis is derepressed only upon detection of DSF itself, suggesting that they require cohabitation with DSF-producer bacteria to activate their DSF regulatory machinery. Finally, we show that the mixed rpf-1/rpf-2 population presents synergism in DSF production and virulence capacity in an in vivo infection model. Recovery and quantification of DSF from co-infected animals correlates with the observed mortality rate.

Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.

The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ΔrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ΔrpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes.

Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

In silico model of DSF synthase RpfF protein from Xanthomonas oryzae pv. Oryzae: a novel target for bacterial blight of rice disease.

Background: Rice plant diseases play a major role as biological constraints on production. One of such rice disease is bacterial leaf blight, caused by Xanthomonas oryzae pv. Oryzae (Xoo). The diffusible signal factor (DSF) synthesized by Xoo has a major role in virulence to rice plant. The DSF synthase RpfF protein, which is related to crotonase superfamily is responsible for the maintaining concentration of DSF. DSF-dependent quorum sensing (QS) system adopts protein- protein interaction mechanism to auto regulates the production of DSF. The antibacterial activity of pesticides against Xoo has not yet been completely understood. Three dimensional structure of RpfF protein was predicted using homology modeling method by MODELLER 9V9 software, SWISS MODEL and GENO3D online tools and structures were validated by Ramachandran plot, TM-Score and RMSD. 3D structure of RpfF (accession number AAL06345) was predicted using DSF synthase of Xanthomonas campestris pv. campestris (Xcc) (PDB ID: 3M6M) as a template. The stereo chemical check reveals the structure developed from the modeller was the best one and the potential ligand binding sites were identified by CASTp Server. The predicted RpfF model provides insight into its structure, active sites and aid in the development of novel inhibitors to control bacterial leaf blight in rice plant. DSF synthase RpfF protein could be used as a novel target to control infection

The rsmA‐like gene rsmAXoo of Xanthomonas oryzae pv. oryzae regulates bacterial virulence and production of diffusible signal factor

The plant-pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, one of the most destructive diseases of rice. A nonpolar mutant of the rsmA-like gene rsmA(Xoo) of the Xoo Chinese strain 13751 was constructed by homologous integration with a suicide plasmid. Virulence tests on a host plant, namely the hybrid rice cultivar Teyou 63, showed that the mutant had lost its virulence almost completely, whereas tests on a nonhost, namely castor-oil plant (Ricinus communis), showed that the mutant had also lost the ability to induce a hypersensitive response in the nonhost. In addition, the rsmA(Xoo) mutant produced significantly smaller amounts of the diffusible signal factor, extracellular endoglucanase, amylase and extracellular polysaccharide, but showed significantly higher glycogen accumulation, bacterial aggregation and cell adhesion. The expression of most hrp genes, genes encoding AvrBs3/PthA family members, rpfB, xrvA, glgA, eglXoB and XOO0175 (encoding an α-amylase) was down-regulated in the rsmA(Xoo) mutant. All phenotypes and expression levels of the tested genes in the rsmA(Xoo) mutant were restored to their levels in the wild-type by the presence of rsmA(Xoo) in trans. These results indicate that rsmA(Xoo) is essential for the virulence of Xoo.

Structural Basis of the Sensor-Synthase Interaction in Autoinduction of the Quorum Sensing Signal DSF Biosynthesis

The diffusible signal factor (DSF)-dependent quorum sensing (QS) system adopts a novel protein-protein interaction mechanism to autoregulate the production of signal DSF. Here, we present the crystal structures of DSF synthase RpfF and its complex with the REC domain of sensor protein RpfC. RpfF is structurally similarity to the members of the crotonase superfamily and contains an N-terminal α/β spiral core domain and a C-terminal α-helical region. Further structural and mutational analysis identified two catalytic glutamate residues, which is the conserved feature of the enoyl-CoA hydratases/dehydratases. A putative substrate-binding pocket was unveiled and the key roles of the residues implicated in substrate binding were verified by mutational analysis. The binding of the REC domain may lock RpfF in an inactive conformation by blocking the entrance of substrate binding pocket, thereby negatively regulating DSF production. These findings provide a structural model for the RpfC-RpfF interaction-mediated QS autoinduction mechanism.

The xrvA gene of Xanthomonas oryzae pv. oryzae, encoding an H-NS-like protein, regulates virulence in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight disease in rice, one of the most serious rice diseases. The xrvA gene from Xoo strain 1,3751 encodes a protein containing a histone-like nucleoid-structuring protein (H-NS) domain. The expression of xrvA in strain 1,3751 was enhanced in XOM2 minimal medium. Mutation of the xrvA gene of strain 1,3751 led to a significant reduction in virulence in the host plant rice, a delayed hypersensitive response in the nonhost castor-oil plant, a decrease in extracellular polysaccharide and diffusible signal factor production, and an increase in intracellular glycogen accumulation. Northern hybridization analyses revealed that the virulence-associated genes hrpG, hrpX, rpfC, rpfF, rpfG and gumB were downregulated in the xrvA mutant compared to the wild-type and complemented strains. Interestingly, increase of copy number of xrvA in the wild-type strain 1,3751 resulted in a strain showing similar phenotypes as the xrvA mutant and a reduction of the expression of gumB, hrpX, rpfC, rpfF and rpfG. These findings indicate that the xrvA gene, which is highly conserved in the sequenced strains of Xanthomonas, encodes an important regulatory factor for the virulence of Xoo.

Abstracts Submitted for Presentation at the 2008 APS Centennial Meeting

Xanthomonas albilineans produces albicidin, a unique and specific toxin that causes foliar symptoms of sugarcane leaf scald disease. In the related species Xanthomonas campestris pv. campestris, a cluster of genes called rpf (for regulation of pathogenicity factors) is involved in control of various cellular processes. Marker exchange mutants of the rpfF gene in strain XaFL07-1 of X. albilineans from Florida were generated, and the mutants were verified by PCR analysis and by use of an X. campestris diffusible signal factor (DSF) reporter strain. Both albicidin and protease production by these mutants appeared significantly reduced compared to XaFL07-1, suggesting that DSF is involved in regulation of albicidin biosynthesis by X. albilineans. However, sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, exhibited pencil line symptoms indicative of albicidin production on emerging leaves after inoculation of stalks by the decapitation method. Preliminary experiments indicated that at least some rpfF mutants colonized sugarcane stalks less efficiently, both spatially and in intensity, than wild type X. albilineans. Additional inoculation experiments are in progress to assess disease severity caused by rpfF mutants, and to study the role of DSF production in sugarcane stalk colonization by X. albilineans. (Resume d'auteur)

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.