Quorum-sensing (QS) regulates the production of key virulence factors in Pseudomonas aeruginosa and other important pathogenic bacteria. In this report, extracts of leaves and bark of Combretum albiflorum (Tul.) Jongkind (Combretaceae) were found to quench the production of QS-dependent factors in P. aeruginosa PAO1. Chromatographic fractionation of the crude active extract generated several active fractions containing flavonoids, as shown by their typical spectral features. Purification and structural characterization of one of the active compounds led to the identification of the flavan-3-ol catechin [(2R,3S)-2-(3,4-dihydroxyphenyl)-3,4-dihydro-1(2H)-benzopyran-3,5,7-triol]. The identity of catechin as one of the active molecules was confirmed by comparing the high-pressure liquid chromatography profiles and the mass spectrometry spectra obtained for a catechin standard and for the active C. albiflorum fraction. Moreover, standard catechin had a significant negative effect on pyocyanin and elastase productions and biofilm formation, as well as on the expression of the QS-regulated genes lasB and rhlA and of the key QS regulatory genes lasI, lasR, rhlI, and rhlR. The use of RhlRand LasR-based biosensors indicated that catechin might interfere with the perception of the QS signal N-butanoyl-L-homoserine lactone by RhlR, thereby leading to a reduction of the production of QS factors. Hence, catechin, along with other flavonoids produced by higher plants, might constitute a first line of defense against pathogenic attacks by affecting QS mechanisms and thereby virulence factor production.Pseudomonas aeruginosa is a gram-negative bacterium infecting insects, plants, animals, and humans (65). As an opportunistic pathogen, P. aeruginosa is a major cause of nosocomial diseases and mortality in immunocompromised patients and particularly in patients with cystic fibrosis, diffused panbronchitis, and pulmonary deficiencies (21, 54). Successful infection of diverse hosts is due to the profusion and diversity of virulence factors secreted by P. aeruginosa such as proteases, exopolysaccharides and redox-active compounds, as well as to its capacity to form biofilms (9, 60, 62).Many pathogenic bacteria trigger the production of their virulence factors in a population density-dependent manner, a cell-to-cell communication mechanism known as quorum sensing (QS) (24). This mechanism enables bacteria to detect their population density through the production, release, and perception of small diffusible molecules called autoinducers and to coordinate gene expression accordingly (7,9,13,24,84). In P. aeruginosa, two QS systems (las and rhl) drive the production (by the synthetases LasI and RhlI) and the perception (by the transcription factors LasR and RhlR) of the acyl-homoserine lactones (AHL) N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), respectively (9, 62). Once LasR interacts with 3-oxo-C12-HSL, it induces the las system (by increasing lasI expression) and triggers the p...

Preliminary screening of the Malagasy plant Combretum albiflorum for compounds attenuating the production of quorum sensing (QS)-controlled virulence factors in bacteria led to the identification of active fractions containing flavonoids. In the present study, several flavonoids belonging to the flavone, flavanone, flavonol and chalcone structural groups were screened for their capacity to reduce the production of QS-controlled factors in the opportunistic pathogen Pseudomonas aeruginosa (strain PAO1). Flavanones (i.e. naringenin, eriodictyol and taxifolin) significantly reduced the production of pyocyanin and elastase in P. aeruginosa without affecting bacterial growth. Consistently, naringenin and taxifolin reduced the expression of several QS-controlled genes (i.e. lasI, lasR, rhlI, rhlR, lasA, lasB, phzA1 and rhlA) in P. aeruginosa PAO1. Naringenin also dramatically reduced the production of the acylhomoserine lactones N-(3-oxododecanoyl)-Lhomoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), which is driven by the lasI and rhlI gene products, respectively. In addition, using mutant strains deficient for autoinduction (DlasI and DrhlI) and LasR-and RhlR-based biosensors, it was shown that QS inhibition by naringenin not only is the consequence of a reduced production of autoinduction compounds but also results from a defect in the proper functioning of the RlhR-C4-HSL complex. Widely distributed in the plant kingdom, flavonoids are known for their numerous and determinant roles in plant physiology, plant development and in the success of plant-rhizobia interactions, but, as shown here, some of them also have a role as inhibitors of the virulence of pathogenic bacteria by interfering with QS mechanisms. INTRODUCTIONIn many pathogenic bacteria the production of virulence factors is triggered in a population density-dependent manner through quorum sensing (QS), a cell-to-cell communication mechanism that enables bacteria to coordinate virulence factor production by means of the synthesis, release and perception of small diffusible molecules called autoinducers (Antunes et al., 2010;Bjarnsholt et al., 2010;Case et al., 2008;Ng & Bassler, 2009). For instance, in the plant and mammal opportunistic pathogen Pseudomonas aeruginosa, two main QS systems (lasI/R and rhlI/R), responsible for the synthesis and perception of the acylhomoserine lactones (AHLs) N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), respectively , control the expression Abbreviations: AHL, acylhomoserine lactone; C4-HSL, N-butanoyl-Lhomoserine lactone; ESI-MS, electrospray ionization-MS; HHL, Nhexanoyl-L-homoserine lactone; 3-oxo-C12-HSL, N-(3-oxododecanoyl)-L-homoserine lactone; PI, propidium iodide; QS, quorum sensing.3These authors contributed equally to this work. of an arsenal of virulence factors. The transcription factors LasR and RhlR interact with and are activated by 3-oxo-C12-HSL and C4-HSL, respectively, triggering the production of biofilms, LasB elastase...

Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus.

The role and metabolism of indole-3-acetic acid in gram-negative bacteria is well documented, but little is known about indole-3-acetic acid biosynthesis and regulation in gram-positive bacteria. The phytopathogen Rhodococcus fascians, a gram-positive organism, incites diverse developmental alterations, such as leafy galls, on a wide range of plants. Phenotypic analysis of a leafy gall suggests that auxin may play an important role in the development of the symptoms. We show here for the first time that R. fascians produces and secretes the auxin indole-3-acetic acid. Interestingly, whereas noninfected-tobacco extracts have no effect, indole-3-acetic acid synthesis is highly induced in the presence of infected-tobacco extracts when tryptophan is not limiting. Indole-3-acetic acid production by a plasmid-free strain shows that the biosynthetic genes are located on the bacterial chromosome, although plasmid-encoded genes contribute to the kinetics and regulation of indole-3-acetic acid biosynthesis. The indole-3-acetic acid intermediates present in bacterial cells and secreted into the growth media show that the main biosynthetic route used by R. fascians is the indole-3-pyruvic acid pathway with a possible rate-limiting role for indole-3-ethanol. The relationship between indole-3-acetic acid production and the symptoms induced by R. fascians is discussed.The phytopathogenic actinomycete Rhodococcus fascians induces the development of a wide range of malformations on its numerous monocotyledonous and dicotyledonous host plants: leaf deformation, growth inhibition, witches' brooms, fasciations, and leafy galls (reference 23 and references therein). The leafy-gall structure, considered an extreme form of apical dominance (59), is the most severe outcome of the R. fasciansplant interaction. Consisting of small misshapen leaves and numerous buds that are inhibited for further outgrowth, the leafy gall is believed to result from the alteration of the endogenous hormone balance of the host plant (23). Leafy-gall features, such as leaf wrinkling, shoot multiplication, and delayed senescence, are typical cytokinin effects. In this respect, R. fascians pathogenicity has been linked to a linear plasmid (pFiD188) that carries different virulence genes. On pFiD188 an isopentenyl transferase (ipt) homologue is located that is essential for symptom development and has been found in all virulent strains examined thus far (14,15,53). Moreover, it is known that R. fascians can produce at least 11 different cytokinins in vitro (2,18,24,28,48,51). In R. fascians, cytokinins probably originate from two sources, either release from tRNA (36, 38) and/or condensation of isopentenyl pyrophosphate and 5Ј-AMP (14). Nevertheless, hitherto no clear correlation between known cytokinins and symptom development could be established (16,18), suggesting that new cytokinin-like molecules could be involved in the formation of the leafy gall (23).Formation of a leafy gall cannot be attributed solely to the action of cytokinin. Some leafy-gall charac...

The MIR396 family, composed of ath-miR396a and ath-miR396b in Arabidopsis, is conserved among plant species and is known to target the Growth-Regulating Factor (GRF) gene family. ath-miR396 overexpressors or grf mutants are characterised by small and narrow leaves and show embryogenic defects such as cotyledon fusion. Heterologous expression of ath-miR396a has been reported in tobacco and resulted in reduction of the expression of three NtGRF genes. In this study, the precursor of the Populus trichocarpa ptc-miR396c, with a mature sequence identical to ath-miR396b, was expressed under control of the CaMV35S promoter in tobacco. Typical phenotypes of GRF down-regulation were observed, including cotyledon fusion and lack of shoot apical meristem (SAM). At later stage of growth, transgenic plants had delayed development and altered specification of organ type during flower development. The third and fourth whorls of floral organs were modified into stigmatoid anthers and fasciated carpels, respectively. Several NtGRF genes containing a miR396 binding site were found to be down-regulated, and the cleavage of their corresponding mRNA at the miR396 binding site was confirmed for two of them using RACE-PCR analysis. The data obtained agree with the functional conservation of the miR396 family in plants and suggest a role for the miR396/GRF network in determination of floral organ specification.