3-hydroxypalmitic Acid Methyl Ester Research Articles

Pseudomonas forestsoilum sp. nov. and P. tohonis biocontrol bacterial wilt by quenching 3-hydroxypalmitic acid methyl ester.

Bacterial wilt caused by Ralstonia solanacearum ranks the second top important bacterial plant disease worldwide. It is also the most important bacterial disease threatening the healthy development of Casuarina equisetifolia protection forest. 3-hydroxypalmitic acid methyl ester (3-OH PAME) functions as an important quorum sensing (QS) signal regulating the expression of virulence genes in R. solanacearum, and has been regarded as an ideal target for disease prevention and control. To screen native microorganisms capable of degrading 3-OH PAME, samples of C. equisetifolia branches and forest soil were collected and cultured in the medium containing 3-OH PAME as the sole carbon source. Bacteria with over 85% degradation rates of 3-OH PAME after 7-day incubation were further separated and purified. As a result, strain Q1-7 isolated from forest soil and strain Q4-3 isolated from C. equisetifolia branches were obtained and identified as Pseudomonas novel species Pseudomonas forestsoilum sp. nov. and P. tohonis, respectively, according to whole genome sequencing results. The degradation efficiencies of 3-OH PAME of strains Q1-7 and Q4-3 were 95.80% and 100.00% at 48 h, respectively. Both strains showed high esterase activities and inhibited R. solanacearum exopolysaccharide (EPS) and cellulase production. Application of strains Q1-7 and Q4-3 effectively protects C. equisetifolia, peanut and tomato plants from infection by R. solanacearum. Findings in this study provide potential resources for the prevention and control of bacterial wilt caused by R. solanacearum, as well as valuable materials for the identification of downstream quenching genes and the research and development of quenching enzymes for disease control.

Soil metagenome-derived 3-hydroxypalmitic acid methyl ester hydrolases suppress extracellular polysaccharide production in Ralstonia solanacearum

Autoinducers are indispensable for bacterial cell–cell communication. However, due to the reliance on culture-based techniques, few autoinducer-hydrolyzing enzymes are known. In this study, we characterized soil metagenome-derived unique enzymes capable of hydrolyzing 3-hydroxypalmitic acid methyl ester (3-OH PAME), an autoinducer of the plant pathogenic bacterium Ralstonia solanacearum. Among 146 candidate lipolytic clones from a soil metagenome library, 4 unique enzymes capable of hydrolyzing the autoinducer 3-OH PAME, termed ELP86, ELP96, ELP104, and EstDL33, were selected and characterized. Phylogenetic analysis revealed that metagenomic enzymes were novel esterase/lipase candidates as they clustered as novel subfamilies of family I, V, X, and family XI. The purified enzymes displayed various levels of hydrolytic activities towards 3-OH PAME with optimum activity at 40–50 °C and pH 7–10. Interestingly, ELP104 also displayed N-(3-oxohexanoyl)-L-homoserine lactone hydrolysis activity. Heterologous expression of the gene encoding 3-OH PAME hydrolase in R. solanacearum significantly decreased exopolysaccharide production without affecting bacterial growth. mRNA transcription analysis revealed that genes regulated by quorum-sensing, such as phcA and xpsR, were significantly down-regulated in the stationary growth phase of R. solanacearum. Therefore, metagenomic enzymes are capable of quorum-quenching by hydrolyzing the autoinducer 3-OH PAME, which could be used as a biocontrol strategy against bacterial wilt.

Characterization of bacteria degrading 3-hydroxy palmitic acid methyl ester (3OH-PAME), a quorum sensing molecule of Ralstonia solanacearum.

Bacterial wilt pathogen Ralstonia solanacearum causes severe crop loss of eggplant, which is of economic importance in India. 3-hydroxy palmitic acid methyl ester (3OH-PAME) is the main quorum sensing molecule governing the expression of virulence factors in R.solanacearum. Ability of 164 bacterial isolates from the xylem of eggplant (Solanum melongena L.), chilli pepper (Capsicum annuum L.) and wild eggplant (Solanum torvum Sw.) to degrade 3OH-PAME was tested by disc diffusion assay. Enzymatic degradation of 3OH-PAME by five bacteria was confirmed by High-Performance Liquid Chromatography-Mass Spectrometry analysis. 3OH-PAME degrading bacteria were identified as Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Rhodococcus corynebacterioides. 3OH-PAME degrading bacteria reduced the expression of virulence factors (exopolysaccharides and endoglucanase) of R.solanacearum in vitro and reduced wilt incidence in eggplant seedlings under greenhouse conditions. Isolates with quorum quenching activity successfully re-colonized eggplant seedlings. Quorum quenching bacteria produced antagonistic compounds, which may act synergistically with quorum quenching in reducing bacterial wilt in eggplant. This is the first report on endophytic bacteria of class Gammaproteobacteria and phylum Actinobacteria having 3OH-PAME degrading activity. This study demonstrates the potential use of endophytic bacteria as quorum quenching biocontrol agents for management of bacterial wilt in eggplant.

Purification and characterization of a novel esterase (?-hydroxypalmitate methyl ester hydrolase) and prevention of the expression of virulence by Ralstonia solanacearum

To screen novel micro-organisms and enzymes capable of degrading 3-hydroxypalmitic acid methyl ester (3-OH PAME), the quorum-sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. Ideonella sp. 0-0013, a betaproteobacterium isolated from soil using the selective-enrichment culture method, was grown on plates containing 3-OH PAME as its main carbon source. beta-Hydroxypalmitate methyl ester hydrolase (betaHPMEH) purified from the supernatant of the Ideonella sp. 0-0013 culture exhibited high hydrolysing activity towards the ester bond of 3-OH PAME and eliminated the 3-OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the betahpmeh gene expression vector degraded 3-OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). The ability of betaHPMEH to hydrolyse 3-OH PAME inhibited the production of EPS by the R. solanacearum wild-type strain, indicating that betaHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. This enzyme is the first protein that has been found to degrade a quormone other than N-acyl homoserine lactone.

Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system.

In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self-produced signal molecule [3-hydroxypalmitic acid methyl ester (3-OH PAME)], an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase). Analysis of a phcA-null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.

Illuminating quorum sensing

Quorum sensing is a cell-to-cell signaling mechanism by which bacteria coordinate gene expression in a density-dependent manner. Bacteria diffuse small signaling molecules that interact with transcription activators or sensor kinases to regulate gene transcription. By far the most extensively investigated family of intercellular signaling molecules are the N-acylhomoserine lactones (AHLs). These molecules, known as autoinducers, were first associated with the regulation of bioluminescence in Vibrio fischeri. In V. fischeri, above a threshold concentration AHLs interact with the transcription activator LuxR, activating the lux operon and thereby producing light. It has been shown that a wide range of bacteria produce autoinducers to regulate a variety of phenotypes, such as plasmid conjugal transfer and stationary-phase survival. However, not all bacteria communicate using AHLs alone – it has been shown that Ralstonia solanacearum uses a 3-hydroxypalmitic acid methyl ester in conjunction with AHLs to modulate pathogenicity, and in Gram-positive bacteria, many signaling molecules are in fact peptides.

Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum.

Ralstonia solanacearum, a phytopathogenic bacterium, uses an environmentally sensitive and complex regulatory network to control expression of multiple virulence genes. Part of this network is an unusual autoregulatory system that produces and senses 3-hydroxypalmitic acid methyl ester. In culture, this autoregulatory system ensures that expression of virulence genes, such as those of the eps operon encoding biosynthesis of the acidic extracellular polysaccharide, occurs only at high cell density (>10(7) cells/ml). To determine if regulation follows a similar pattern within tomato plants, we first developed a quantitative immunofluorescence (QIF) method that measures the relative amount of a target protein within individual bacterial cells. For R. solanacearum, QIF was used to determine the amount of beta-galactosidase protein within wild-type cells containing a stable eps-lacZ reporter allele. When cultured cells were examined to test the method, QIF accurately detected both low and high levels of eps gene expression. QIF analysis of R. solanacearum cells recovered from stems of infected tomato plants showed that expression of eps during pathogenesis was similar to that in culture. These results suggest that there are no special signals or conditions within plants that override or short-circuit the regulatory processes observed in R. solanacearum in culture. Because QIF is a robust, relatively simple procedure that uses generally accessible equipment, it should be useful in many situations where gene expression in single bacterial cells must be determined.

Autoregulator-dependent control of extracellular polysaccharide production in phytopathogenic bacteria

Extracellular polysaccharides (EPSs) likely provide phytopathogenic bacteria a selective advantage both inside and outside plants. Despite the relatively scant knowledge about EPS biosynthesis in phytopathogenic bacteria, it clearly is a well controlled, complex, energy-intensive process. Unexpectedly, three phytopathogenic bacteria have been found to autoregulate EPS production in response to extracellular signal compounds (pheromones) that they produce. Like many bacteria, Pantoea stewartii subsp. stewartii produces a N-acyl-homoserine lactone (AHL) autoinducer. However, unlike most AHL-dependent autoinduction systems, that in P. stewartii subsp. stewartii somehow represses EPS production in the absence of autoinducer. Instead of an AHL-dependent system (which it also has), Ralstonia solanacearum uses a novel autoregulator identified as 3-hydroxypalmitic acid methyl ester to regulate EPS biosynthesis. A lack of this autoregulator in R. solanacearum results in repression of EPS biosynthesis by a complex two-component sensor/response regulator signal cascade. Xanthomonas campestris pv. campestris has two partially overlapping autoregulatory systems. The autoregulators are incompletely characterized, but one diffusible signal factor (DSF) is thought to be a fatty acid derivative and the other diffusible factor (DF) may be a butyrolactone. The autoregulation pathways in X. campestris pv. campestris are essentially unknown, but EPS production is controlled by both the DSF and DF systems, whereas production of extracellular enzymes and pigment production are regulated independently. In a confined micro-environment, population density and intercellular concentrations of an autoregulator will increase in parallel, so autoregulation is one way that bacteria can coordinate gene expression to synthesize EPS only at high cell density. However, because there is often limited evidence that it is actually cell density that is being detected, researchers should not assume a priori that autoregulation must function for quorum sensing. Some possible reasons for why phytopathogenic bacteria would benefit from delaying EPS production are discussed.

An RpoS (sigmaS) homologue regulates acylhomoserine lactone-dependent autoinduction in Ralstonia solanacearum.

Many bacteria sense an appropriate growth condition or a critical population density for gene expression by producing acylhomoserine lactones (acyl-HSLs) that act as intercellular autoinduction signals. We recently showed that, in Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium, acyl-HSL production requires soll, which encodes a putative acyl-HSL synthase, and that its expression is positively regulated by the acyl-HSL-responsive SolR transcriptional regulator. This acyl-HSL-dependent autoinduction system is noteworthy because (i) it is regulated by a 'higher level' autoinducer system (responsive to 3-hydroxypalmitic acid methyl ester) via PhcA, a LysR-type transcriptional regulator and (ii) acyl-HSL production requires two additional unlinked loci. As reported here, cloning and sequencing of one of these other loci revealed that it encodes a homologue of RpoS, an alternative sigma factor (sigmaS) that in other bacteria activates gene expression during stationary phase or in response to stress conditions. R. solanacearum RpoS (RpoS(Rso)) was demonstrated to function as a sigma factor because when introduced in trans into an Escherichia coli rpoS mutant it largely restored expression of the RpoS-dependent bolAp1 gene. Mutation of rpoS(Rso) in R. solanacearum reduced survival during starvation and low pH conditions, but did not affect survival during exposure to hydrogen peroxide, high osmolarity or high temperature. This mutant was also altered in its production of several virulence factors and wilted tomato plants several days more slowly than the wild-type parent. Transcription of solR and soll were decreased in an rpoS(Rso) background (thereby reducing acyl-HSL production), but neither mutations in solR, soll or phcA nor addition of acyl-HSLs affected rpoS(Rso) expression. Therefore, in R. solanacearum the acyl-HSL-dependent autoinduction system is controlled both by a second autoinduction system and by the RpoS(Rso) sigma factor.

Hierarchical autoinduction in Ralstonia solanacearum: control of acyl-homoserine lactone production by a novel autoregulatory system responsive to 3-hydroxypalmitic acid methyl ester.

Bacteria employ autoinduction systems to sense the onset of appropriate cell density for expression of developmental genes. In many gram-negative bacteria, autoinduction involves the production of and response to diffusible acylated-homoserine lactones (acyl-HSLs) and is mediated by members of the LuxR and LuxI families. Ralstonia (Pseudomonas) solanacearum, a phytopathogenic bacterium that appears to autoregulate its virulence genes, produces compounds that promote expression of several heterologous acyl-HSL-responsive reporter gene constructs. High-pressure liquid chromatography of highly concentrated ethyl acetate extracts revealed that culture supernatants of strain AW1 contained two compounds with retention times similar to N-hexanoyl- and N-octanoyl-HSL. To investigate the role of these acyl-HSLs in R. solanacearum virulence gene expression, transposon mutants that were deficient for inducing an acyl-HSL-responsive reporter in Agrobacterium tumefaciens were generated. Three loci involved in normal acyl-HSL production were identified, one of which was shown to contain the divergently transcribed solR and solI genes, the luxR and luxI homologs, respectively. A 4.1-kb fragment containing solR and solI enabled all of the mutants (regardless of the locus inactivated) and a naturally acyl-HSL-defective strain of R. solanacearum to produce acyl-HSLs. Inactivation of solI abolished production of all detectable acyl-HSLs but affected neither the expression of virulence genes in culture nor the ability to wilt tomato plants. AW1 has a functional autoinduction system, because (i) expression of solI required SolR and acyl-HSL and (ii) expression of a gene linked to solR and solI, designated aidA, was acyl-HSL dependent. Because AidA has no homologs in the protein databases, its discovery provided no clues as to the role of acyl-HSLs in R. solanacearum gene regulation. However, expression of solR and solI required the global LysR-type virulence regulator PhcA, and both solR and solI exhibited a cell density-associated pattern of expression similar to other PhcA-regulated genes. The acyl-HSL-dependent autoinduction system in R. solanacearum is part of a more complex autoregulatory hierarchy, since the transcriptional activity of PhcA is itself controlled by a novel autoregulatory system that responds to 3-hydroxypalmitic acid methyl ester.

Identification of 3-hydroxypalmitic acid methyl ester as a novel autoregulator controlling virulence in Ralstonia solanacearum.

Expression of virulence genes in Ralstonia solanacearum, a phytopathogenic bacterium, is controlled by a complex regulatory network that integrates multiple signal inputs. Production of several virulence determinants is coordinately reduced by inactivation of phcB, but is restored by growth in the presence of a volatile extracellular factor (VEF) produced by wild-type strains of R. solanacearum. The VEF was purified from spent culture broth by distillation, solvent extraction, and liquid chromatography. Gas chromatography and mass spectroscopy identified 3-hydroxypalmitic acid methyl ester (3-OH PAME) as the major component in the single peak of VEF activity. Authentic 3-OH PAME and the purified VEF were active at < or =1 nM, and had nearly equivalent specific activities for stimulating the expression of eps (the biosynthetic locus for extracellular polysaccharide) in a phcB mutant. Authentic 3-OH PAME also increased the production of three virulence factors by a phcB mutant over 20-fold to wild-type levels, restored normal cell density-associated expression of eps and increased expression of eps when delivered via the vapour phase. Reanalysis of the PhcB amino acid sequence suggested that it is a small-molecule S-adenosylmethionine-dependent methyltransferase, which might catalyse synthesis of 3-OH PAME from a naturally occurring fatty acid. Biologically active concentrations of extracellular 3-OH PAME were detected before the onset of eps expression, suggesting that it is an intercellular signal that autoregulates virulence gene expression in wild-type R. solanacearum. Other than acyl-homoserine lactones, 3-OH PAME is the only endogenous fatty acid derivative shown to be an autoregulator and may be the first example of a new family of compounds that can mediate long-distance intercellular communication.

A two-component system in Ralstonia (Pseudomonas) solanacearum modulates production of PhcA-regulated virulence factors in response to 3-hydroxypalmitic acid methyl ester.

Expression of virulence factors in Ralstonia solanacearum is controlled by a complex regulatory network, at the center of which is PhcA, a LysR family transcriptional regulator. We report here that expression of phcA and production of PhcA-regulated virulence factors are affected by products of the putative operon phcBSR(Q). phcB is required for production of an extracellular factor (EF), tentatively identified as the fatty acid derivative 3-hydroxypalmitic acid methyl ester (3-OH PAME), but a biochemical function for PhcB could not be deduced from DNA sequence analysis. The other genes in the putative operon are predicted to encode proteins homologous to members of two-component signal transduction systems: PhcS has amino acid similarity to histidine kinase sensors, whereas PhcR and OrfQ are similar to response regulators. PhcR is quite unusual because its putative output domain strongly resembles the histidine kinase domain of a sensor protein. Production of the PhcA-regulated factors exopolysaccharide I, endoglucanase, and pectin methyl esterase was reduced 10- to 100-fold only in mutants with a nonpolar insertion in phcB [which express phcSR(Q) in the absence of the EF]; simultaneously, expression of phcA was reduced fivefold. Both a wild-type phenotype and phcA expression were restored by addition of 3-OH PAME to growing cultures. Mutants with polar insertions in phcB or lacking the entire phcBSR(Q) region produced wild-type levels of PhcA-regulated virulence factors. The genetic data suggest that PhcS and PhcR function together to regulate expression of phcA, but the biochemical mechanism for this is unclear. At low levels of the EF, it is likely that PhcS phosphorylates PhcR, and then PhcR interacts either with PhcA (which is required for full expression of phcA) or an unknown component of the signal cascade to inhibit expression of phcA. When the EF reaches a threshold concentration, we suggest that it reduces the ability of PhcS to phosphorylate PhcR, resulting in increased expression of phcA and production of PhcA-regulated factors.

Differential Expression of Virulence Genes and Motility in Ralstonia (Pseudomonas) solanacearum during Exponential Growth

A complex network regulates virulence in Ralstonia solanacearum (formerly Pseudomonas solanacearum); central to this system is PhcA, a LysR-type transcriptional regulator. We report here that two PhcA-regulated virulence factors, endoglucanase (Egl) and acidic exopolysaccharide I (EPS I), and motility are expressed differentially during exponential growth in batch cultures. Tests with strains carrying lacZ fusions in a wild-type genetic background revealed that expression (on a per-cell basis) of phcA was constant but expression of egl and epsB increased 20- to 50-fold during multiplication from 1 x 10(sup7) to 5 x 10(sup8) CFU/ml. Expression of xpsR, an intermediate regulator downstream of PhcA in the regulatory cascade for eps expression, was similar to that of epsB and egl. Motility track photography revealed that all strains were essentially nonmotile at 10(sup6) CFU/ml. As cell density increased, 30 to 50% of wild-type cells were motile between 10(sup7) and 10(sup8) CFU/ml, but this population was again nonmotile at 10(sup9) CFU/ml. In contrast, about 60% of the cells of phcB and phcA mutants remained motile at 10(sup9) CFU/ml. Expression of phcB, which is not positively regulated by PhcA, was the inverse of epsB, egl, and xpsR (i.e., it decreased 20-fold at high cell density). PhcB is essential for production of an extracellular factor, tentatively identified as 3-hydroxypalmitic acid methyl ester (3-OH PAME), that might act as an exponential-phase signal to activate motility or expression of virulence genes. However, growth of the lacZ fusion strains in medium containing excess 3-OH PAME did not result in motility or expression of virulence genes at dramatically lower cell densities, suggesting that 3-OH PAME is not the only factor controlling these traits.