Salicylate Biosynthesis Research Articles

Sequential activation of strigolactone and salicylate biosynthesis promotes leaf senescence.

Leaf senescence is a complex process strictly regulated by various external and endogenous factors. However, the key signaling pathway mediating leaf senescence remains unknown. Here, we show that Arabidopsis SPX1/2 negatively regulate leaf senescence genetically downstream of the strigolactone (SL) pathway. We demonstrate that the SL receptor AtD14 and MAX2 mediate the age-dependent degradation of SPX1/2. Intriguingly, we uncover an age-dependent accumulation of SLs in leaves via transcriptional activation of SL biosynthetic genes by the transcription factors (TFs) SPL9/15. Furthermore, we reveal that SPX1/2 interact with the WRKY75 subclade TFs to inhibit their DNA-binding ability and thus repress transcriptional activation of salicylic acid (SA) biosynthetic gene SA Induction-Deficient 2, gating the age-dependent SA accumulation in leaves at the leaf senescence onset stage. Collectively, our new findings reveal a signaling pathway mediating sequential activation of SL and salicylate biosynthesis for the onset of leaf senescence in Arabidopsis.

Salicylic Acid Biosynthesis and Metabolism: A Divergent Pathway for Plants and Bacteria.

Salicylic acid (SA) is an active secondary metabolite that occurs in bacteria, fungi, and plants. SA and its derivatives (collectively called salicylates) are synthesized from chorismate (derived from shikimate pathway). SA is considered an important phytohormone that regulates various aspects of plant growth, environmental stress, and defense responses against pathogens. Besides plants, a large number of bacterial species, such as Pseudomonas, Bacillus, Azospirillum, Salmonella, Achromobacter, Vibrio, Yersinia, and Mycobacteria, have been reported to synthesize salicylates through the NRPS/PKS biosynthetic gene clusters. This bacterial salicylate production is often linked to the biosynthesis of small ferric-ion-chelating molecules, salicyl-derived siderophores (known as catecholate) under iron-limited conditions. Although bacteria possess entirely different biosynthetic pathways from plants, they share one common biosynthetic enzyme, isochorismate synthase, which converts chorismate to isochorismate, a common precursor for synthesizing SA. Additionally, SA in plants and bacteria can undergo several modifications to carry out their specific functions. In this review, we will systematically focus on the plant and bacterial salicylate biosynthesis and its metabolism.

Identification and analysis of key genes involved in methyl salicylate biosynthesis in different birch species.

Species of the perennial woody plant genus Betula dominate subalpine forests and play a significant role in preserving biological diversity. In addition to their conventional benefits, birches synthesize a wide range of secondary metabolites having pharmacological significance. Methyl salicylate (MeSA) is one of these naturally occurring compounds constitutively produced by different birch species. MeSA is therapeutically important in human medicine for muscle injuries and joint pain. However, MeSA is now mainly produced synthetically due to a lack of information relating to MeSA biosynthesis and regulation. In this study, we performed a comprehensive bioinformatics analysis of two candidate genes mediating MeSA biosynthesis, SALICYLIC ACID METHYLTRANSFERASE (SAMT) and SALICYLIC ACID-BINDING PROTEIN 2 (SABP2), of high (B. lenta, B. alleghaniensis, B. medwediewii, and B. grossa) and low (B. pendula, B. utilis, B. alnoides, and B. nana) MeSA-producing birch species. Phylogenetic analyses of SAMT and SABP2 genes and homologous genes from other plant species confirmed their evolutionary relationships. Multiple sequence alignments of the amino acid revealed the occurrence of important residues for substrate specificity in SAMT and SABP2. The analysis of cis elements in different birches indicated a functional multiplicity of SAMT and SABP2 and provided insights into the regulation of both genes. We successfully developed six prominent single nucleotide substitution markers that were validated with 38 additional birch individuals to differentiate high and low MeSA-producing birch species. Relative tissue-specific expression analysis of SAMT in leaf and bark tissue of two high and two low MeSA-synthesizing birches revealed a high expression in the bark of both high MeSA-synthesizing birches. In contrast, SABP2 expression in tissues revealed indifferent levels of expression between species belonging to the two groups. The comparative expression and bioinformatics analyses provided vital information that could be used to apply plant genetic engineering technology in the mass production of organic MeSA.

OXI1 and DAD Regulate Light-Induced Cell Death Antagonistically through Jasmonate and Salicylate Levels.

Singlet oxygen produced from triplet excited chlorophylls in photosynthesis is a signal molecule that can induce programmed cell death (PCD) through the action of the OXIDATIVE STRESS INDUCIBLE 1 (OXI1) kinase. Here, we identify two negative regulators of light-induced PCD that modulate OXI1 expression: DAD1 and DAD2, homologs of the human antiapoptotic protein DEFENDER AGAINST CELL DEATH. Overexpressing OXI1 in Arabidopsis (Arabidopsis thaliana) increased plant sensitivity to high light and induced early senescence of mature leaves. Both phenomena rely on a marked accumulation of jasmonate and salicylate. DAD1 or DAD2 overexpression decreased OXI1 expression, jasmonate levels, and sensitivity to photooxidative stress. Knock-out mutants of DAD1 or DAD2 exhibited the opposite responses. Exogenous applications of jasmonate upregulated salicylate biosynthesis genes and caused leaf damage in wild-type plants but not in the salicylate biosynthesis mutant Salicylic acid induction-deficient2, indicating that salicylate plays a crucial role in PCD downstream of jasmonate. Treating plants with salicylate upregulated the DAD genes and downregulated OXI1 We conclude that OXI1 and DAD are antagonistic regulators of cell death through modulating jasmonate and salicylate levels. High light-induced PCD thus results from a tight control of the relative activities of these regulating proteins, with DAD exerting a negative feedback control on OXI1 expression.

Constructing an efficient salicylate biosynthesis platform by Escherichia coli chromosome integration

Salicylate (SA) is an important platform chemical widely used in cosmetic and pharmaceutical industries. In this study, an efficient SA producing strain was constructed by step-by-step chromosome integration. First, the SA biosynthetic module controlled by promoters PT7 or Ptac was integrated into the chromosome of E. coli, generating the basal strain SA producing strains. PT7 performed better than Ptac and led to higher SA titers, the best of which was 233.6 ± 6.6 mg/L. Disrupting pheA/tyrA eliminated accumulation of phenylalanine and tyrosine, improving SA titer to 492.4 ± 9.3 mg/L. Strengthening the upstream pathway by enhancing expression of aroG further improved SA titer to 679.9 ± 27.1 mg/L. Furthermore, pykA/pykF were disrupted to conserve phosphoenolpyruvate, resulting in a final strain that can produce 769.8 ± 12.6 mg/L of SA using glucose and glycerol as the mixed carbon source. When using sole glycerol instead, the titer was significantly improved to 1560.6 ± 50.2 mg/L. The platform strain was further used to produce muconic acid and salicyl alcohol by introducing the downstream SA-conversion modules. In this study, we constructed an effcient SA producing E. coli strain by chromosome integration, which can be used as a platform for the production of SA derivatives.

Biosensor-guided improvements in salicylate production by recombinant Escherichia coli

BackgroundSalicylate can be biosynthesized from the common metabolic intermediate shikimate and has found applications in pharmaceuticals and in the bioplastics industry. While much metabolic engineering work focused on the shikimate pathway has led to the biosynthesis of a variety of aromatic compounds, little is known about how the relative expression levels of pathway components influence salicylate biosynthesis. Furthermore, some host strain gene deletions that improve salicylate production may be impossible to predict. Here, a salicylate-responsive transcription factor was used to optimize the expression levels of shikimate/salicylate pathway genes in recombinant E. coli, and to screen a chromosomal transposon insertion library for improved salicylate production.ResultsA high-throughput colony screen was first developed based on a previously designed salicylate-responsive variant of the E. coli AraC regulatory protein (“AraC-SA”). Next, a combinatorial library was constructed comprising a series of ribosome binding site sequences corresponding to a range of predicted protein translation initiation rates, for each of six pathway genes (> 38,000 strain candidates). Screening for improved salicylate production allowed for the rapid identification of optimal gene expression patterns, conferring up to 123% improved production of salicylate in shake-flask culture. Finally, transposon mutagenesis and screening revealed that deletion of rnd (encoding RNase D) from the host chromosome further improved salicylate production by 27%.ConclusionsThese results demonstrate the effectiveness of the salicylate sensor-based screening platform to rapidly identify beneficial gene expression patterns and gene knockout targets for improving production. Such customized high-throughput tools complement other cell factory engineering strategies. This approach can be generalized for the production of other shikimate-derived compounds.

Effects of single or combined water deficit and aphid attack on tomato volatile organic compound (VOC) emission and plant-plant communication

Plants release a broad spectrum of volatile organic compounds (VOCs). The composition of the released VOC blend is dependent on the physiological status and, consequently, is affected by biotic and abiotic stresses. Stress-related VOCs can be perceived by different organisms, including natural enemies of herbivores and neighboring plants. Here, the responses of tomato plants (emitters) to single or combined abiotic (water stress) and biotic (aphid attack) stresses, and the effect of VOC released by emitters on neighboring unstressed plants (receivers), have been investigated. Emissions of α-pinene and methyl salicylate from plants exposed to single or combined stress, and of camphene from plants exposed to water or combined stress were significantly higher than in unstressed plants. In receivers, only the release of methyl salicylate increased when companion emitters were stressed. The expression of genes related to VOC biosynthesis and plant defense responses was unaffected or declined in water-stressed emitters, and was generally higher in receivers than in emitters. The gene coding for methyl salicylate biosynthesis was particularly active in aphid-attacked emitters and in receivers that were conditioned by the infested emitters. In addition, VOCs emitted by stressed plants induce VOC emission in unstressed receivers, and this increases attraction of parasitic wasps, which may improve protection against aphid attacks under conditions of reduced water availability.

Effect of modeled microgravity on UV-C-induced interplant communication of Arabidopsis thaliana

Controlled ecological life support systems (CELSS) will be an important feature of long-duration space missions of which higher plants are one of the indispensable components. Because of its pivotal role in enabling plants to cope with environmental stress, interplant communication might have important implications for the ecological stability of such CELSS. However, the manifestations of interplant communication in microgravity conditions have yet to be fully elucidated. To address this, a well-established Arabidopsis thaliana co-culture experimental system, in which UV-C-induced airborne interplant communication is evaluated by the alleviation of transcriptional gene silencing (TGS) in bystander plants, was placed in microgravity modeled by a two-dimensional rotating clinostat. Compared with plants under normal gravity, TGS alleviation in bystander plants was inhibited in microgravity. Moreover, TGS alleviation was also prevented when plants of the pgm-1 line, which are impaired in gravity sensing, were used in either the UV-C-irradiated or bystander group. In addition to the specific TGS-loci, interplant communication-shaped genome-wide DNA methylation in bystander plants was altered under microgravity conditions. These results indicate that interplant communications might be modified in microgravity. Time course analysis showed that microgravity interfered with both the production of communicative signals in UV-C-irradiated plants and the induction of epigenetic responses in bystander plants. This was further confirmed by the experimental finding that microgravity also prevented the response of bystander plants to exogenous methyl jasmonate (JA) and methyl salicylate (SA), two well-known airborne signaling molecules, and down-regulated JA and SA biosynthesis in UV-C-irradiated plants.

Jasmonate-mediated defence responses, unlike salicylate-mediated responses, are involved in the recovery of grapevine from bois noir disease

BackgroundBois noir is an important disease of grapevine (Vitis vinifera L.), caused by phytoplasmas. An interesting, yet elusive aspect of the bois noir disease is “recovery”, i.e., the spontaneous and unpredictable remission of symptoms and damage. Because conventional pest management is ineffective against bois noir, deciphering the molecular bases of recovery is beneficial. The present study aimed to understand whether salicylate- and jasmonate-defence pathways might have a role in the recovery from the bois noir disease of grapevine.ResultsLeaves from healthy, bois noir-diseased and bois noir-recovered plants were compared, both in the presence (late summer) and absence (late spring) of bois noir symptoms on the diseased plants. Analyses of salicylate and jasmonate contents, as well as the expression of genes involved in their biosynthesis, signalling and action, were evaluated. In symptomatic diseased plants (late summer), unlike symptomless plants (late spring), salicylate biosynthesis was increased and salicylate-responsive genes were activated. In contrast, jasmonate biosynthesis and signalling genes were up-regulated both in recovered and diseased plants at all sampling dates. The activation of salicylate signalling in symptomatic plants might have antagonised the jasmonate-mediated defence response by suppressing the expression of jasmonate-responsive genes.ConclusionsOur results suggest that grapevine reacts to phytoplasma infection through salicylate-mediated signalling, although the resultant full activation of a salicylate-mediated response is apparently ineffective in conferring resistance against bois noir disease. Activation of the salicylate signalling pathway that is associated with the presence of bois noir phytoplasma seems to antagonise the jasmonate defence response, by failing to activate or suppressing both the expression of some jasmonate responsive genes that act downstream of the jasmonate biosynthetic pathway, as well as the first events of the jasmonate signalling pathway. On the other hand, activation of the entire jasmonate signalling pathway in recovered plants suggests the potential importance of jasmonate-regulated defences in preventing bois noir phytoplasma infections and the subsequent development of bois noir disease. Thus, on one hand, recovery could be achieved and maintained over time by preventing the activation of defence genes associated with salicylate signalling, and on the other hand, by activating jasmonate signalling and other defence responses.

Methyl Salicylate Level Increase in Flax after Fusarium oxysporum Infection Is Associated with Phenylpropanoid Pathway Activation.

Flax (Linum usitatissimum) is a crop plant valued for its oil and fiber. Unfortunately, large losses in cultivation of this plant are caused by fungal infections, with Fusarium oxysporum being one of its most dangerous pathogens. Among the plant's defense strategies, changes in the expression of genes of the shikimate/phenylpropanoid/benzoate pathway and thus in phenolic contents occur. Among the benzoates, salicylic acid, and its methylated form methyl salicylate play an important role in regulating plants' response to stress conditions. Upon treatment of flax plants with the fungus we found that methyl salicylate content increased (4.8-fold of the control) and the expression profiles of the analyzed genes suggest that it is produced most likely from cinnamic acid, through the β-oxidative route. At the same time activation of some genes involved in lignin and flavonoid biosynthesis was observed. We suggest that increased methyl salicylate biosynthesis during flax response to F. oxysporum infection may be associated with phenylpropanoid pathway activation.

Unconventionally secreted effectors of two filamentous pathogens target plant salicylate biosynthesis.

Plant diseases caused by fungi and oomycetes pose an increasing threat to food security and ecosystem health worldwide. These filamentous pathogens, while taxonomically distinct, modulate host defense responses by secreting effectors, which are typically identified based on the presence of signal peptides. Here we show that Phytophthora sojae and Verticillium dahliae secrete isochorismatases (PsIsc1 and VdIsc1, respectively) that are required for full pathogenesis. PsIsc1 and VdIsc1 can suppress salicylate-mediated innate immunity in planta and hydrolyse isochorismate in vitro. A conserved triad of catalytic residues is essential for both functions. Thus, the two proteins are isochorismatase effectors that disrupt the plant salicylate metabolism pathway by suppressing its precursor. Furthermore, these proteins lack signal peptides, but exhibit characteristics that lead to unconventional secretion. Therefore, this secretion pathway is a novel mechanism for delivering effectors and might play an important role in host–pathogen interactions.

The Arabidopsis PEPR pathway couples local and systemic plant immunity

Recognition of microbial challenges leads to enhanced immunity at both the local and systemic levels. In Arabidopsis, EFR and PEPR1/PEPR2 act as the receptor for the bacterial elongation factor EF-Tu (elf18 epitope) and for the endogenous PROPEP-derived Pep epitopes, respectively. The PEPR pathway has been described to mediate defence signalling following microbial recognition. Here we show that PROPEP2/PROPEP3 induction upon pathogen challenges is robust against jasmonate, salicylate, or ethylene dysfunction. Comparative transcriptome profiling between Pep2- and elf18-treated plants points to co-activation of otherwise antagonistic jasmonate- and salicylate-mediated immune branches as a key output of PEPR signalling. Accordingly, as well as basal defences against hemibiotrophic pathogens, systemic immunity is reduced in pepr1 pepr2 plants. Remarkably, PROPEP2/PROPEP3 induction is essentially restricted to the pathogen challenge sites during pathogen-induced systemic immunity. Localized Pep application activates genetically separable jasmonate and salicylate branches in systemic leaves without significant PROPEP2/PROPEP3 induction. Our results suggest that local PEPR activation provides a critical step in connecting local to systemic immunity by reinforcing separate defence signalling pathways.

Regulation of ABA dependent wound induced spreading cell death by MYB108

Wounding results in the controlled cell death of a few rows of cells adjacent to disrupted cells resulting in physical wound closure, which combined with phenolic compound deposition, prevents water loss and pathogen entry. The control of these processes remains uncharacterized. Cell death in a mutant of Arabidopsis thaliana lacking BOTRYTIS SENSITIVE1/MYB108 (BOS1/MYB108) function was characterized utilizing physiological, cell biological and genetic methods. The bos1 mutant has a wound induced runaway cell death that includes enhanced reactive oxygen species (ROS) production that followed the extent of enhanced cell death. Exogenous abscisic acid (ABA) enhanced wound induced cell death in Col-0 plants and was sufficient to trigger cell death in bos1. Uncontrolled cell death was dependent of the production and perception of ABA. Furthermore, bos1 had altered sensitivity to and accumulation of ABA. Arabidopsis possesses a genetic program controlling the extent of wound inducible cell death. BOS1 acts as a negative regulator of ABA induced cell death, which functions in the control of this wound sealing program. This program is distinct from other known cell death programs in that it is ABA dependent, but independent of salicylate biosynthesis, ethylene, jasmonate, metacaspases and ROS derived from RBOHD and RBOHF.

Abscisic acid interacts antagonistically with classical defense pathways in rice–migratory nematode interaction

Studies involving plant-nematode interactions provide an opportunity to unravel plant defense signaling in root tissues. In this study, we have characterized the roles of salicylate (SA), jasmonate (JA), ethylene (ET) and abscisic acid (ABA) in plant defense against the migratory nematode Hirschmanniella oryzae in the monocot model plant rice (Oryza sativa). Experiments with exogenous hormone applications, biosynthesis inhibition and mutant/transgenic lines were executed to test the effect on H. oryzae parasitism in rice roots. Our results demonstrate that an intact ET, JA and SA biosynthesis pathway is a prerequisite for defense against H. oryzae. By contrast, exogenous ABA treatment drastically compromised the rice defense towards this nematode. Gene expression analyses using quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrate that the disease-inducing effect of ABA is likely to be the result of an antagonistic interaction between this hormone and the SA/JA/ET-dependent basal defense system. Collectively, in rice defense against H. oryzae, at least three pathways, namely SA, JA and ET, are important, while ABA plays a negative role in defense. Our results suggest that the balance of ABA and SA/JA/ET signaling is an important determinant for the outcome of the rice-H. oryzae interaction.

Induced resistance triggered by Piriformospora indica

The root endophytic Basidiomycete Piriformospora indica forms a specific type of mycorrhiza symbiosis with a broad spectrum of plant species, including the Brassicaceae. A recent report on the interaction of P. indica with Arabidopsis thaliana suggests that the fungus induces a mode of resistance to microbial pathogens reminiscent of Induced Systemic Resistance (ISR) first discovered with non-pathogenic rhizobacteria. The characteristics of P. indica mediated resistance are the dependency on JA-signalling and the cytosolic function of the master regulator protein Non-expressor-of-PR-genes 1 (NPR1), a low level of altered systemic gene expression in leaves before pathogen challenge, the induction of the JA-inducible marker gene vegetative storage protein 1 (VSP1) after pathogen challenge, and an independency of the resistance phenotype from salicylate biosynthesis and signalling. We discuss here two more factors regarding the P. indica-mediated ISR response: the role of the plant hormone ethylene as well as a possible contribution of the recently discovered close association of P. indica with the α-proteobacterium Rhizobium radiobacter.

Arabidopsis Isochorismate Synthase Functional in Pathogen-induced Salicylate Biosynthesis Exhibits Properties Consistent with a Role in Diverse Stress Responses

Salicylic acid (SA) is a phytohormone best known for its role in plant defense. It is synthesized in response to diverse pathogens and responsible for the large scale transcriptional induction of defense-related genes and the establishment of systemic acquired resistance. Surprisingly, given its importance in plant defense, an understanding of the underlying enzymology is lacking. In Arabidopsis thaliana, the pathogen-induced accumulation of SA requires isochorismate synthase (AtICS1). Here, we show that AtICS1 is a plastid-localized, stromal protein using chloroplast import assays and immunolocalization. AtICS1 acts as a monofunctional isochorismate synthase (ICS), catalyzing the conversion of chorismate to isochorismate (IC) in a reaction that operates near equilibrium (K(eq) = 0.89). It does not convert chorismate directly to SA (via an IC intermediate) as does Yersinia enterocolitica Irp9. Using an irreversible coupled spectrophotometric assay, we found that AtICS1 exhibits an apparent K(m) of 41.5 mum and k(cat) = 38.7 min(-1) for chorismate. This affinity for chorismate would allow it to successfully compete with other pathogen-induced, chorismate-utilizing enzymes. Furthermore, the biochemical properties of AtICS1 indicate its activity is not regulated by light-dependent changes in stromal pH, Mg(2+), or redox and that it is remarkably active at 4 degrees C consistent with a role for SA in cold-tolerant growth. Finally, our analyses support plastidic synthesis of stress-induced SA with the requirement for one or more additional enzymes responsible for the conversion of IC to SA, because non-enzymatic conversion of IC to SA under physiological conditions was negligible.

New concepts in collectin‐mediated host defense at the air–liquid interface of the lung

Surfactant proteins A (SP-A) and D are collectins that play key roles in innate immune defense of the lung. Recent studies suggest that these proteins have a direct effect on the growth and viability of Gram-negative bacteria, Mycoplasma pneumoniae and Histoplasma capsulatum. The researchers examined membrane permeabilization by BAL fluid of mice that were sufficient or deficient in surfactant proteins, of rough and smooth LPS-containing membranes, and of genetically altered bacteria. The permeability assay is based on the ability of agents to promote access of impermeable dyes propidium iodide and ELF97 to intracellular targets. The permeabilizing activity of concentrated BAL material from SP-A+/+ mice was substantially greater than that from SP-A-/- animals, and was sensitive to hyperoxic exposure. Oxidation of the collectins in vitro also blocked their antimicrobial effects. Rough but not smooth LPS conferred susceptibility of model bacterial membranes to permeabilization by the collectins. The screening of genetically engineered SP-A mice with a Pseudomonas aeruginosa signature tagged mutagenesis library has revealed several pathways which are required for resistance to the permeabilizing effects of SP-A, including those required for salicylate biosynthesis and flagellar function. The researchers conclude that the pulmonary collectins directly permeabilize bacteria in an LPS-dependent and rough LPS-specific manner. Oxidative damage blocks the permeabilizing activity of alveolar lining fluid and purified proteins. The mechanism(s) of permeabilization is not known, but the crystal structure of SP-A reveals a hydrophobic cleft lined by charged residues which may play a role in membrane perturbation.

Comparative Signature-Tagged Mutagenesis Identifies Pseudomonas Factors Conferring Resistance to the Pulmonary Collectin SP-A

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A+/+) and SP-A-deficient (SP-A−/−) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.

Salicylate Biosynthesis: Overexpression, Purification, and Characterization of Irp9, a Bifunctional Salicylate Synthase fromYersinia enterocolitica

In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.

Isochorismate Synthase (PchA), the First and Rate-limiting Enzyme in Salicylate Biosynthesis of Pseudomonas aeruginosa

In Pseudomonas aeruginosa the extracellular metabolite and siderophore pyochelin is synthesized from two major precursors, chorismate and l-cysteine via salicylate as an intermediate. The regulatory role of isochorismate synthase, the first enzyme in the pyochelin biosynthetic pathway, was studied. This enzyme is encoded by pchA, the last gene in the pchDCBA operon. The PchA protein was purified to apparent electrophoretic homogeneity from a PchA-overexpressing P. aeruginosa strain. The native enzyme was a 52-kDa monomer in solution, and its activity strictly depended on Mg(2+). At pH 7.0, the optimum, a K(m) = 4.5 microm and a k(cat) = 43.1 min(-1) were determined for chorismate. No feedback inhibitors or other allosteric effectors were found. The intracellular PchA concentration critically determined the rate of salicylate formation both in vitro and in vivo. In cultures grown in iron-limiting media to high cell densities, overexpression of the pchA gene resulted in overproduction of salicylate as well as in enhanced pyochelin formation. From this work and earlier studies, it is proposed that one important factor influencing the flux through the pyochelin biosynthetic pathway is the PchA concentration, which is determined at a transcriptional level, with pyochelin acting as a positive signal and iron as a negative signal.