scpA, a new salicylate hydroxylase gene localized in salicylate/caprolactam degradation plasmids

Both caprolactams and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders are controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids have been assigned to the P-7 incompatibility group. The new salicylate 1-hydroxylase gene (scpA) has been detected in SAL/CAP plasmids and partially sequenced. The scpA gene was equally related to the closest homolog genes nahG (NAH7), salA (P. reinekei MT1), and nahU (pND6-1); however, the identity rate did not exceed 72–74%. The synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited the highest specific activity toward 4-methylsalicylate and nonsubstituted salicylate substrates. Furthermore, conjugative pseudomonads’ plasmids of salicylate degradation without the classical nah2 operon, which harbors only salicylate 1-hydroxylase gene nahU have been described for the first time.     

Gastric cytoprotection by sodium salicylate

Sodium salicylate (SA), contrary to acetylsalicylic acid (ASA, aspirin), was not ulcerogenic in rats. SA was also found to be cytoprotective: it prevented formation of gastric mucosal necrosis produced by either absolute ethanol or 0.6 M HCl, and formation of gastric ulcers produced by acidified ASA. The degree of protection was dose dependent. The mechanism of this cytoprotection is unknown, but unlike cytoprotection elicited by mild irritants, e.g., 20% ethanol or 0.35 M HCl, whose effects appear to be due to endogenous formation of PG by the stomach, SA acts through a different mechanism, since its protective effect was not blocked by indomethacin.     

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.     

The effects of salicylate on bacteria

Salicylate and related compounds, such as aspirin, have a variety of effects in eucaryotic systems and are well known for their medicinal properties. Salicylate also has numerous effects on bacteria, yet only a handful of individuals within the scientific community appreciate these findings. From a bacterial viewpoint, growth in the presence of salicylate can be both beneficial and detrimental. On one hand, growth of certain bacteria in the presence of salicylate can induce an intrinsic multiple antibiotic resistance phenotype. On the other hand, growth in the presence of salicylate can reduce the resistance to some antibiotics and affect virulence factor production in some bacteria. This review provides an overview of the effects salicylate has on various bacterial species.     

Rhizobacterial salicylate production provokes headaches!

Background

Salicylic acid (SA) is produced in significant amounts by certain plant growth promoting rhizosphere bacteria, and some of these rhizobacteria have the ability to induce systemic resistance against diseases in plants. Exogenous application of SA to plants has long been known to lead to protection against a range of plant pathogens through the elicitation of systemic acquired resistance. Thus, it is reasonable to assume that the SA producing plant beneficial rhizobacteria elicit induced resistance through the production of SA.

Scope and conclusions

However, we discuss here that bacterial secretion of SA in vitro appears to be an artifact and that the bacteria will normally incorporate SA into SA-containing metabolites, mainly SA-based siderophores, under environmental conditions. Therefore, we argue that rhizobacteria do not likely excrete free SA into the rhizosphere thereby not inducing resistance in plants through this metabolite. SA detected in the rhizosphere is most likely produced by the plant and we discuss the impact of this phenolic compound on microbial interactions.     

Crystal structures of Yersinia enterocolitica salicylate synthase and its complex with the reaction products salicylate and pyruvate

The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.     

Studies of electron-transfer properties of salicylate hydroxylase from Pseudomonas cepacia and effects of salicylate and benzoate binding

The pH dependence of the redox behavior of salicylate hydroxylase from Pseudomonas cepacia as well as the effects of salicylate, benzoate, and chloride binding is described. At pH 7.6 in 0.02 M potassium phosphate buffer E1(0')(EFl ox/EFl.-) is -0.150 V and E2(0')(EFl.-/EFl red H-) is -0.040 V versus the standard hydrogen electrode (SHE). A maximum of 5% of FAD anion semiquinone is thermodynamically stabilized under these conditions. However, in coulometric and dithionite titrations more semiquinone is kinetically formed, indicating slow transfer of the second electron. The potential/pH dependence is consistent with a two-electron, one-proton transfer. Upon salicylate binding the midpoint potential is shifted 0.020 V negative from -0.094 to -0.114 V vs SHE at pH 7.6. A maximum of 7% of the neutral semiquinone is stabilized both in potentiometric and coulometric titrations. This small potential shift indicates that the substrate is bound nearly to the same extent to all three oxidation states of the enzyme. It is clear that the substrate binding does not make the reduction of the flavin thermodynamically more favorable. In contrast to salicylate, the potential shift caused by the effector, benzoate, is much more significant. (A maximum potential shift of -0.07 V is calculated.) Benzoate binds most tightly to the oxidized form and is least tightly bound to the two-electron-reduced form of the enzyme. For the reduction of the free enzyme the transfer of the second electron or the transfer of the proton is rate limiting, as is shown by the kinetic formation of the anionic semiquinone.(ABSTRACT TRUNCATED AT 250 WORDS)