salicylates and pyochelin

Pseudomonas aeruginosa is an increasingly antibiotic-resistant pathogen that causes severe lung infections, burn wound infections, and diabetic foot infections. P. aeruginosa produces the siderophore pyochelin through the use of a non-ribosomal peptide synthetase (NRPS) biosynthetic pathway. Targeting members of siderophore NRPS proteins is one avenue currently under investigation for the development of new antibiotics against antibiotic-resistant organisms. Here, the crystal structure of the pyochelin adenylation domain PchD is reported. The structure was solved to 2.11 Å when co-crystallized with the adenylation inhibitor 5'-O-(N-salicylsulfamoyl)adenosine (salicyl-AMS) and to 1.69 Å with a modified version of salicyl-AMS designed to target an active site cysteine (4-cyano-salicyl-AMS). In the structures, PchD adopts the adenylation conformation, similar to that reported for AB3403 from Acinetobacter baumannii.

Topics: Anti-Bacterial Agents; Phenols; Pseudomonas aeruginosa; Salicylates; Siderophores; Thiazoles

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.

Topics: Amino Acid Sequence; Arabidopsis; Base Sequence; Caulimovirus; Codon; Intramolecular Transferases; Lyases; Molecular Sequence Data; Phenols; Plant Leaves; Plants, Genetically Modified; Pseudomonas aeruginosa; Recombinant Fusion Proteins; Restriction Mapping; Salicylates; Thiazoles

The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.

Topics: ATP-Binding Cassette Transporters; Bacterial Proteins; Chorismic Acid; Gene Expression Regulation, Bacterial; Genes, Bacterial; Iron Chelating Agents; Molecular Sequence Data; Oxidoreductases Acting on CH-NH Group Donors; Phenols; Pseudomonas aeruginosa; Repressor Proteins; Salicylates; Siderophores; Thiazoles

Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming with phosphopantetheine to enable covalent tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates. Two cyclization domains (Cy1 in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.

Topics: Adenosine Triphosphate; Bacterial Proteins; Catalysis; Chromatography, High Pressure Liquid; Cysteine; Diphosphates; Iron Chelating Agents; Pantothenic Acid; Peptide Synthases; Phenols; Protein Processing, Post-Translational; Pseudomonas aeruginosa; Salicylates; Siderophores; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thiazoles

Sixty-one Burkholderia cepacia isolates from patients with cystic fibrosis (CF) and four plant isolates were screened for production of the siderophores salicylic acid (SA), pyochelin, cepabactin, and ornibactins and fingerprinted by a PCR-based randomly amplified polymorphic DNA (RAPD) method. Of the 24 RAPD types determined, 22 (92%) were associated with isolates that produced SA, 21 (87%) were associated with isolates that produced ornibactins, 15 (60%) were associated with isolates that produced pyochelin, and 3 (12%) were associated with isolates that produced cepabactin. Of the 24 RAPD types plus 2 phenotypic variants of types 1 and 9, 3 were associated with isolates that produced all four siderophores, 8 were associated with isolates that produced three siderophores, 12 were associated with isolates that produced two siderophores, and 3 were associated with isolates that produced only one siderophore. These results suggest that the numbers and types of siderophores produced by CF isolates of B. cepacia correlate with RAPD type and that SA and ornibactins are the most prevalent siderophores produced.

Topics: Burkholderia cepacia; Cystic Fibrosis; Humans; Phenols; Pyridones; Random Amplified Polymorphic DNA Technique; Salicylates; Salicylic Acid; Siderophores; Thiazoles

The siderophore pyochelin of Pseudomonas aeruginosa is derived from one molecule of salicylate and two molecules of cysteine. Two cotranscribed genes, pchEF, encoding peptide synthetases have been identified and characterized. pchE was required for the conversion of salicylate to dihydroaeruginoate (Dha), the condensation product of salicylate and one cysteine residue and pchF was essential for the synthesis of pyochelin from Dha. The deduced PchE (156 kDa) and PchF (197 kDa) proteins had adenylation, thiolation and condensation/cyclization motifs arranged as modules which are typical of those peptide synthetases forming thiazoline rings. The pchEF genes were coregulated with the pchDCBA operon, which provides enzymes for the synthesis (PchBA) and activation (PchD) of salicylate as well as a putative thioesterase (PchC). Expression of a translational pchE'-'lacZ fusion was strictly dependent on the PchR regulator and was induced by extracellular pyochelin, the end product of the pathway. Iron replete conditions led to Fur (ferric uptake regulator)-dependent repression of the pchE'-'lacZ fusion. A translational pchD'-'lacZ fusion was also positively regulated by PchR and pyochelin and repressed by Fur and iron. Thus, autoinduction by pyochelin (or ferric pyochelin) and repression by iron ensure a sensitive control of the pyochelin pathway in P. aeruginosa.

Topics: Amino Acid Sequence; Bacterial Outer Membrane Proteins; Bacterial Proteins; Base Sequence; beta-Galactosidase; Enzyme Induction; Genes, Bacterial; Iron; Molecular Sequence Data; Operon; Peptide Synthases; Phenols; Physical Chromosome Mapping; Pseudomonas aeruginosa; Receptors, Cell Surface; Repressor Proteins; Salicylates; Thiazoles; Transcription, Genetic

The high-affinity siderophore salicylate is an intermediate in the biosynthetic pathway of pyochelin, another siderophore and chelator of transition metal ions, in Pseudomonas aeruginosa. The 2.5-kb region upstream of the salicylate biosynthetic genes pchBA was sequenced and found to contain two additional, contiguous genes, pchD and pchC, having the same orientation. The deduced amino acid sequence of the 60-kDa PchD protein was similar to those of the EntE protein (2,3-dihydroxybenzoate-AMP ligase) of Escherichia coli and other adenylate-forming enzymes, suggesting that salicylate might be adenylated at the carboxyl group by PchD. The 28-kDa PchC protein showed similarities to thioesterases of prokaryotic and eukaryotic origin and might participate in the release of the product(s) formed from activated salicylate. One potential product, dihydroaeruginoate (Dha), was identified in culture supernatants of iron-limited P. aeruginosa cells. The antifungal antibiotic Dha is thought to arise from the reaction of salicylate with cysteine, followed by cyclization of cysteine. Inactivation of the chromosomal pchD gene by insertion of the transcription and translation stop element omega Sm/Sp abolished the production of Dha and pyochelin, implying that PchD-mediated activation of salicylate may be a common first step in the synthesis of both metabolites. Furthermore, the pchD::omega Sm/Sp mutation had a strong polar effect on the expression of the pchBA genes, i.e., on salicylate synthesis, indicating that the pchDCBA genes constitute a transcriptional unit. A full-length pchDCBA transcript of ca. 4.4 kb could be detected in iron-deprived, growing cells of P. aeruginosa. Transcription of pchD started at tandemly arranged promoters, which overlapped with two Fur boxes (binding sites for the ferric uptake regulator) and the promoter of the divergently transcribed pchR gene encoding an activator of pyochelin biosynthesis. This promoter arrangement allows tight iron-mediated repression of the pchDCBA operon.

Topics: Amino Acid Sequence; Bacterial Proteins; Base Composition; Base Sequence; Genes, Bacterial; Iron; Models, Chemical; Molecular Sequence Data; Operon; Phenols; Pseudomonas aeruginosa; Restriction Mapping; RNA, Bacterial; RNA, Messenger; Salicylates; Salicylic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Siderophores; Thiazoles; Transcription, Genetic

The ferripyochelin receptor A (fptA) gene codes for the transport of the ferrisiderophore ferripyochelin in Pseudomonas aeruginosa. A P. aeruginosa fptA internal fragment was used to probe chromosomal DNA from P. aureofaciens, B. cepacia, P. fluorescens, P. putida, and five strains of P.aeruginosa. These bacteria all contained DNA that hybridized to the fptA fragment. Four of the five P. aeruginosa strains displayed marked and identical patterns, indicating a high degree of sequence similarities among these strains. DNA from the non-P. aeruginosa bacteria, in contrast, hybridized less to the fptA fragment. Pseudomonas aeruginosa and B. cepacia synthesize pyochelin. Experiments were performed to confirm P. fluorescens pyochelin synthesis and to determine if pyochelin, cepabactin or salicylic acid were made by P. aureofaciens, P. putida, and P. fluorescens. Only pyochelin was isolated and identified from P. fluorescens. P. aureofaciens and P. putida produced none of these compounds. While all of these bacteria contain chromosomal DNA that hybridized to the fptA fragment probe, pyochelin synthesis did not occur in all, indicating that fptA fragment hybridization cannot always be correlated with pyochelin biosynthesis.

Topics: Bacterial Outer Membrane Proteins; Blotting, Southern; Cloning, Molecular; DNA Probes; Escherichia coli; Nucleic Acid Hybridization; Phenols; Polymorphism, Restriction Fragment Length; Pseudomonas; Pyridones; Receptors, Cell Surface; Salicylates; Salicylic Acid; Sequence Homology, Nucleic Acid; Thiazoles

Two iron-regulated compounds have been found in acidified ethyl acetate extracts from culture supernatants of Pseudomonas aeruginosa and Pseudomonas cepacia type-strains. Synthesis of both compounds paralleled iron-deficient growth, and was repressed in the presence of 100 microM-FeCl3. Yields of these substances varied among different strains and attained maximum levels during stationary phase. Thin layer chromatographic analysis in five different solvent systems revealed that the slower-moving compound chromatographed as two distinct bands, and showed RF values and spectral properties similar to pyochelin. The faster-moving compound co-migrated as a single band with a standard of commercial salicylic acid in each of the chromatographic systems tested. Moreover, a molecule with an identical RF was also produced by Pseudomonas fluorescens CHA401, which is known to synthesize salicylic acid as the only siderophore during iron-limited growth. Spectrophotometric and spectrofluorometric titrations led to the identification of this iron-regulated compound as salicylic acid, in agreement with the structure deduced from 1H-NMR and mass spectroscopy. The identity of the P. cepacia siderophore azurechelin as salicylic acid was also conclusively demonstrated. Salicylic acid, like pyochelin and pyoverdin, promoted P. aeruginosa growth in an iron-depleted medium. These results are consistent with a putative siderophore activity for salicylic acid, i.e. azurechelin, as has been demonstrated for P. aeruginosa, P. fluorescens and P. cepacia. Thus, salicylic acid is likely to act as a siderophore in more than one species belonging to the genus Pseudomonas.

Topics: Burkholderia cepacia; Iron; Phenols; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas fluorescens; Salicylates; Salicylic Acid; Siderophores; Species Specificity; Thiazoles

Under iron-starvation conditions of growth, Pseudomonas fluorescens CHA0, a soil isolate involved in phytopathogenic fungi antagonisms, produced, together with pyoverdine, a second iron-chelating compound which was purified and identified by spectroscopy, HPLC and 1H-NMR to be salicylic acid. Mutants unable to synthesize pyoverdine overproduced this compound by a factor of 9-14. The biosynthesis of salicylic acid was under iron control; it was fully inhibited by 5 microM added iron in the growth medium. In contrast, salicylic acid of either bacterial or commercial origin facilitated labeled iron incorporation in iron-starved cells. Based on these two relationships observed with bacterial iron metabolism it is concluded that salicylic acid has a siderophore function for this strain.

Topics: Iron; Oligopeptides; Phenols; Pigments, Biological; Pseudomonas fluorescens; Salicylates; Salicylic Acid; Siderophores; Thiazoles

A novel iron-binding compound was identified in ethyl acetate extracts of the supernates from Pseudomonas cepacia cultures. This compound, named azurechelin, was produced by 88% of P. cepacia strains isolated from the respiratory tract. Production of azurechelin was regulated by the iron concentration in the culture medium. Azurechelin enhanced the growth of P. cepacia in a medium containing transferrin 200 mg/L. Azurechelin released iron from transferrin in an equilibrium dialysis assay, suggesting that it could complete with transferrin for iron. Azurechelin could also stimulate iron uptake by P. cepacia. This siderophore appeared to have a novel structure with neither the typical characteristics of catechol nor of hydroxamate compounds.

Topics: Biological Transport; Burkholderia cepacia; Chromatography, Gel; Chromatography, Thin Layer; Culture Media; Humans; Ionophores; Iron; Iron Chelating Agents; Phenols; Salicylates; Siderophores; Thiazoles

Pseudomonas aeruginosa mutants requiring salicylic acid for pyochelin biosynthesis were isolated after chemical mutagenesis by plating on a siderophore detection medium. Like the wild type, these mutants incorporated 7-[14C]salicylic acid into pyochelin, demonstrating that salicylic acid is an intermediate in the biosynthesis pathway of pyochelin.

Topics: Carbon Radioisotopes; Iron Chelating Agents; Kinetics; Mutation; Phenols; Pseudomonas aeruginosa; Salicylates; Salicylic Acid; Thiazoles