Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.
ABSTRACT: 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 ?rpfF 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 ?rpfC 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.
Project description:Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ?rpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ?rpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.
Project description: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.
Project description:In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)-11-methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl-CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl-CoA ligase, Escherichia coli?FadD, in the E. coli ß-oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ?rpfB ?rpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3-hydroxyacyl-acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl-ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl-CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl-ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.
Project description:Xanthomonas hortorum pv. pelargonii (Xhp), the causal agent of bacterial blight in pelargonium, is the most threatening bacterial disease of this ornamental worldwide. To gain an insight into the regulation of virulence in Xhp, we have disrupted the quorum sensing (QS) genes, which mediate the biosynthesis and sensing of the diffusible signal factor (DSF). Mutations in rpfF (encoding the DSF synthase) and rpfC (encoding the histidine sensor kinase of the two-component system RfpC/RpfG) and overexpression of rpfF showed a significant reduction in incidence and severity of the disease on pelargonium. Confocal laser scanning microscopy images of inoculated plants with a green fluorescent protein (GFP)-labelled wild-type strain showed that the pathogen is homogeneously dispersed in the lumen of xylem vessels, reaching the apex and invading the intercellular spaces of the leaf mesophyll tissue within 21?days. In contrast, the rpfF and rpfC knockout mutants, as well as the rpfF-overexpressing strain, remained confined to the vicinity of the inoculation site. The rpfF and rpfC mutants formed large incoherent aggregates in the xylem vessels that might interfere with upward movement of the bacterium within the plant. Both mutants also formed extended aggregates under in?vitro conditions, whereas the wild-type strain formed microcolonies. Expression levels of putative virulence genes in?planta were substantially reduced within 48?h after inoculation with the QS mutants when compared with the wild-type. The results presented indicate that an optimal DSF concentration is crucial for successful colonization and virulence of Xhp in pelargonium.
Project description:Lysobacter species are emerging as novel sources of antibiotics, but the regulation of these antibiotics has not been thoroughly elucidated to date. In this work, we identified a small diffusible signaling factor (DSF) molecule (LbDSF) that regulates the biosynthesis of a novel Xanthomonas-specific antibiotic compound (XSAC) in Lysobacter brunescens OH23. LbDSF was isolated from the culture broth of L. brunescens OH23, and the chemical structure of the molecule was determined by NMR and MS. The LbDSF compound induced GUS expression in a reporter strain of Xanthomonas campestris pv. campestris FE58, which contained the gus gene under the control of a DSF-inducible engXCA promoter. LbDSF production was found to be linked to the enoyl-CoA hydratase RpfF and dependent on the two-component regulatory system RpfC (hybrid sensor histidine kinase)/RpfG (response regulator), and LbDSF production was increased 6.72 times in the ?rpfC compared to wild-type OH23. LbDSF-regulated XSAC production was dramatically decreased in ?rpfF, ?rpfC, and ?rpfG. Additionally, a significant reduction in surface motility and a number of changes in colony morphology was observed in the ?rpfF, ?rpfC, and ?rpfG compared to the wild-type OH23. The exogenous LbDSF significantly increased XSAC production in wild-type OH23 and recovered the XSAC biosynthetic ability in ?rpfF. Taken together, these results showed that LbDSF is a fatty-acid-derived DSF that positively regulates XSAC biosynthesis, cell morphology, and surface motility. Moreover, the RpfC/RpfG quorum-sensing signal transduction pathway mediates XSAC biosynthesis. These findings may facilitate antibiotic production through genetic engineering in Lysobacter spp.
Project description: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.
Project description:The rpf gene cluster of Xanthomonas campestris pathovar campestris (Xcc) is required for the pathogenesis of this bacterium to plants. Several rpf genes are involved in the coordinate positive regulation of the production of virulence factors mediated by the small diffusible molecule DSF (for diffusible signal factor). RpfF directs the synthesis of DSF, and a two-component sensory transduction system comprising RpfC and RpfG has been implicated in the perception of the DSF signal and signal transduction. In L medium, rpfF, rpfG, rpfC, and rpfGHC mutants grew as matrix-enclosed aggregates, whereas the wild type grew in a dispersed planktonic fashion. Synthesis of the extracellular polysaccharide xanthan was required for aggregate formation. Addition of DSF triggered dispersion of the aggregates formed by the rpfF strain, but not those of rpf strains defective in DSF signal transduction. An extracellular enzyme from Xcc whose synthesis was positively controlled by the DSF/rpf system could disperse the aggregates produced by all rpf strains. The enzyme was identified as the single endo-beta-1,4-mannanase encoded by the Xcc genome. This enzyme had no detectable activity against soluble xanthan. The endo-beta-1,4-mannanase was required for the full virulence of Xcc to plants. On the basis of this model system, we propose that one role of the beta-mannanase during disease is to promote transitions from an aggregated or biofilm lifestyle to a planktonic lifestyle in response to the DSF signal.
Project description:Cell density-dependent regulation of gene expression in Xylella fastidiosa that is crucial to its switching between plant hosts and insect vectors is dependent on RpfF and its production of 2-enoic acids known as diffusible signal factor (DSF). We show that X. fastidiosa produces a particularly large variety of similar, relatively long-chain-length 2-enoic acids that are active in modulating gene expression. Both X. fastidiosa itself and a Pantoea agglomerans surrogate host harboring X. fastidiosa RpfF (XfRpfF) is capable of producing a variety of both saturated and unsaturated free fatty acids. However, only 2-cis unsaturated acids were found to be biologically active in X. fastidiosa X. fastidiosa produces, and is particularly responsive to, a novel DSF species, 2-cis-hexadecanoic acid that we term XfDSF2. It is also responsive to other, even longer 2-enoic acids to which other taxa such as Xanthomonas campestris are unresponsive. The 2-enoic acids that are produced by X. fastidiosa are strongly affected by the cellular growth environment, with XfDSF2 not detected in culture media in which 2-tetradecenoic acid (XfDSF1) had previously been found. X. fastidiosa is responsive to much lower concentrations of XfDSF2 than XfDSF1. Apparently competitive interactions can occur between various saturated and unsaturated fatty acids that block the function of those agonistic 2-enoic fatty acids. By altering the particular 2-enoic acids produced and the relative balance of free enoic and saturated fatty acids, X. fastidiosa might modulate the extent of DSF-mediated quorum sensing.X. fastidiosa, having a complicated lifestyle in which it moves and multiplies within plants but also must be vectored by insects, utilizes DSF-based quorum sensing to partition the expression of traits needed for these two processes within different cells in this population based on local cellular density. The finding that it can produce a variety of DSF species in a strongly environmentally context-dependent manner provides insight into how it coordinates the many genes under the control of DSF signaling to successfully associate with its two hosts. Since the new DSF variant XfDSF2 described here is much more active than the previously recognized DSF species, it should contribute to plant disease control, given that the susceptibility of plants can be greatly reduced by artificially elevating the levels of DSF in plants, creating "pathogen confusion," resulting in lower virulence.
Project description:UNLABELLED: Cell-cell signaling in Xylella fastidiosa has been implicated in the coordination of traits enabling colonization in plant hosts as well as insect vectors. This cell density-dependent signaling has been attributed to a diffusible signaling factor (DSF) produced by the DSF synthase RpfF. DSF produced by related bacterial species are unsaturated fatty acids, but that of X. fastidiosa was thought to be different from those of other taxa. We describe here the isolation and characterization of an X. fastidiosa DSF (XfDSF) as 2(Z)-tetradecenoic acid. This compound was isolated both from recombinant Erwinia herbicola expressing X. fastidiosa rpfF and from an X. fastidiosa rpfC deletion mutant that overproduces DSF. Since an rpfF mutant is impaired in biofilm formation and underexpresses the hemagglutinin-like protein-encoding genes hxfA and hxfB, we demonstrate that these traits can be restored by ca. 0.5 µM XfDSF but not by myristic acid, the fully saturated tetradecenoic acid. A phoA-based X. fastidiosa biosensor that assesses DSF-dependent expression of hxfA or hxfB revealed a high level of molecular specificity of DSF signaling. IMPORTANCE: X. fastidiosa causes diseases in many important plants, including grape, where it incites Pierce's disease. Virulence of X. fastidiosa for grape is coordinated by cell-cell signaling molecules, designated DSF (Diffusible Signaling Factor). Mutants blocked in DSF production are hypervirulent for grape, suggesting that virulence is suppressed upon DSF accumulation and that disease could be controlled by artificial elevation of the DSF level in plants. In this work, we describe the isolation of the DSF produced by X. fastidiosa and the verification of its biological activity as an antivirulence factor. We also have developed X. fastidiosa DSF biosensors to evaluate the specificity of cell-cell signaling to be investigated.
Project description:Cell-cell signaling in Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, mediated by a fatty acid Diffusible Signaling Factor (DSF), is required to colonize insect vectors and to suppress virulence to grape. Here, we show that a hybrid two-component regulatory protein RpfC is involved in negative regulation of DSF synthesis by RpfF in X. fastidiosa. X. fastidiosa rpfC mutants hyperexpress rpfF and overproduce DSF and are deficient in virulence and movement in the xylem vessels of grape. The expression of the genes encoding the adhesins FimA, HxfA, and HxfB is much higher in rpfC mutants, which also exhibit a hyperattachment phenotype in culture that is associated with their inability to migrate in xylem vessels and cause disease. rpfF mutants deficient in DSF production have the opposite phenotypes for all of these traits. RpfC is also involved in the regulation of other signaling components including rpfG, rpfB, a GGDEF domain protein that may be involved in intracellular signaling by modulating the levels of cyclic-di-GMP, and the virulence factors tolC and pglA required for disease. rpfC mutants are able to colonize the mouthparts of insect vectors and wild-type strains but are not transmitted as efficiently to new host plants, apparently because of their high levels of adhesiveness. Because of the conflicting contributions of adhesiveness and other traits to movement within plants and vectoring to new host plants, X. fastidiosa apparently coordinates these traits in a population-size-dependent fashion involving accumulation of DSF.