Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Streptomyces acidiscabies

The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3' of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as (1)H- and (13)C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(L-4-nitrotryptophyl-L-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide.     

Effect of amino acids on thaxtomin A biosynthesis by Streptomyces scabies

The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.     

Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by Streptomyces scabies

Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.     

Specificity of acid proteinase A from Aspergillus niger var. macrosporus towards B-chain of performic acid oxidized bovine insulin.

1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.     

The so-called tRNAGlu1 of Escherichia coli is a stable denatured conformer of the major isoacceptor tRNAGlu2

A single peak of tRNAGlu is obtained upon chromatography of unfractionated tRNA from Escherichia coli on DEAE-Sephadex A-50 if this tRNA was previously renatured, whereas two peaks of tRNAGlu are resolved if the sample chromatographed is a mixture of native (renatured) and denatured tRNA. Higher resolution analysis of native E. coli tRNA by RPC-5 chromatography showed that most of the tRNAGlu is present in one peak, eluted shortly after a minor peak containing about or less than 5% of the total amount of tRNAGlu; these two peaks were also observed with commercially available tRNAGlu purified from E. coli. When denatured, the tRNAGlu present in each of these two peaks was eluted from the RPC-5 column at a much lower salt concentration. The properties of the denatured conformers obtained from native tRNAGlu present in the major and minor peaks, and the variation, with growth conditions of E. coli, in the relative amount of tRNAGlu in the minor peak suggest that the tRNAGlu present in the minor peak is an undermodified form of the tRNAGlu present in the major peak. This tRNAGluUUC (or tRNAGluSUC when modified in the anticodon) would then be the only tRNA species acceptor of glutamate in E. coli.     

Characterization of Streptomyces scabies mutants deficient in melanin biosynthesis

Streptomyces scabies, a causal agent of common scab, produces both melanin and a secondary metabolite called thaxtomin A. To establish a possible relation between melanin and thaxtomin A production in S. scabies, we carried out N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and isolated 11 melanin-negative mutants of S. scabies EF-35. These mutants were characterized for thaxtomin A production, pathogenicity, sporulation, and stress resistance. Nine of these mutants showed a significant reduction in thaxtomin A production when compared with the wild strain. However, only a few mutants exhibited a reduced level of virulence or a loss in their ability to induce common scab symptoms on potato tubers. Other pleiotrophic effects, such as higher sensitivity to heavy metals and incapacity to sporulate under certain stress conditions, were also associated with a deficiency in melanin production.     

Plant cell growth and ion flux responses to the streptomycete phytotoxin thaxtomin A: calcium and hydrogen flux patterns revealed by the non-invasive MIFE technique

Thaxtomin A, a key phytotoxin produced by plant pathogenic Streptomyces sp., is implicit in common scab disease expression in potato. Primary targets and modes of action of thaxtomin A toxicity in plant cells are not well understood. In this work, early signalling events associated with thaxtomin A toxicity were studied using the ion-selective microelectrode ion flux estimation (MIFE) technique. Thaxtomin A-induced changes in net ion fluxes were measured across the plasma membrane (PM) of root and pollen tube tissue in Arabidopsis thaliana and tomato. Within a minute after toxin application, a rapid and short-lived Ca2+ influx was observed. Well ahead of the marked inhibition of root growth, a significant shift towards net H+ efflux across the PM occurred in all tissues. Similar to root tissues, thaxtomin A significantly modified ion flux profiles from growing pollen tubes. Thaxtomin A was more effective in young, physiologically active tissues (root elongation zone or pollen tube apex), suggesting a higher density of thaxtomin A-binding sites in these regions. Overall, our data provide the first evidence that thaxtomin A triggers an early signalling cascade, which may be crucial in plant-pathogen interactions. It also suggests a possible interaction between thaxtomin A and PM auxin receptors, as revealed from experiments on the auxin-sensitive ucu2-2/gi2 A. thaliana mutant.     

TxtH is a key component of the thaxtomin biosynthetic machinery in the potato common scab pathogen Streptomyces scabies

Streptomyces scabies causes potato common scab disease, which reduces the quality and market value of affected tubers. The predominant pathogenicity determinant produced by S. scabies is the thaxtomin A phytotoxin, which is essential for common scab disease development. Production of thaxtomin A involves the nonribosomal peptide synthetases (NRPSs) TxtA and TxtB, both of which contain an adenylation (A-) domain for selecting and activating the appropriate amino acid during thaxtomin biosynthesis. The genome of S. scabies 87.22 contains three small MbtH-like protein (MLP)-coding genes, one of which (txtH) is present in the thaxtomin biosynthesis gene cluster. MLP family members are typically required for the proper folding of NRPS A-domains and/or stimulating their activities. This study investigated the importance of TxtH during thaxtomin biosynthesis in S. scabies. Biochemical studies showed that TxtH is required for promoting the soluble expression of both the TxtA and TxtB A-domains in Escherichia coli, and amino acid residues essential for this activity were identified. Deletion of txtH in S. scabies significantly reduced thaxtomin A production, and deletion of one of the two additional MLP homologues in S. scabies completely abolished production. Engineered expression of all three S. scabies MLPs could restore thaxtomin A production in a triple MLP-deficient strain, while engineered expression of MLPs from other Streptomyces spp. could not. Furthermore, the constructed MLP mutants were reduced in virulence compared to wild-type S. scabies. The results of our study confirm that TxtH plays a key role in thaxtomin A biosynthesis and plant pathogenicity in S. scabies.     

Characterization of a polyketide synthase in Aspergillus niger whose product is a precursor for both dihydroxynaphthalene (DHN) melanin and naphtho-γ-pyrone

The genome sequencing of the fungus Aspergillus niger uncovered a large cache of genes encoding enzymes thought to be involved in the production of secondary metabolites yet to be identified. Identification and structural characterization of many of these predicted secondary metabolites are hampered by their low concentration relative to the known A. niger metabolites such as the naphtho-γ-pyrone family of polyketides. We deleted a non-reducing PKS gene in A. niger strain ATCC 11414, a daughter strain of A. niger ATCC strain 1015 whose genome was sequenced by the DOE Joint Genome Institute. This PKS encoding gene we name albA is a predicted ortholog of alb1 from Aspergillus fumigatus which is responsible for production of the naphtho-γ-pyrone precursor for the 1,8-dihydroxynaphthalene (DHN) melanin/spore pigment. Our results show that the A. nigeralbA PKS is responsible for both the production of the spore pigment precursor and a family of naphtho-γ-pyrones commonly found in significant quantity in A. niger culture extracts. The generation of an A. niger strain devoid of naphtho-γ-pyrones will greatly facilitate the elucidation of cryptic biosynthetic pathways in this organism.     

More chemistry of the thaxtomin phytotoxins

Chemical and biochemical studies indicated the possible involvement of N-acetyltryptophan and 4-nitrotryptophan as intermediates in biosynthesis of the thaxtomin phytotoxins. A search for other potential pathways indirectly resulted in the identification of three unusual thaxtomin analogues derived from the o-thaxtomin A isomer. Investigations to resolve the identity of a previously described thaxtomin A di-glucoside were not supportive of the proposed structure.     

Studies on the metabolites of phylloquinone (vitamin K1) in the urine of man

The nature of the metabolites excreted in the urine was investigated up to 48 h after oral and intravenous administration of 0.3 to 1.3 mg [1′,2′-³H₂]phylloquinone. The metabolites were water-soluble of which the major fraction consisted of glucuronide conjugates. A chromatographic comparison of the aglycone fragments released by β-glucuronidase and by dilute HCl revealed the presence of at least three labelled aglycones. The major aglycones obtained by enzyme hydrolysis consisted of at least two closely related organic acids which were not separated by adsorption thin-layer chromatography but one of which on treatment with dilute acid yielded a neutral metabolite with the chromatographic properties of phylloquinone γ-lactone. The results suggest that phylloquinone γ-lactone, the only previously isolated urinary metabolite of phylloquinone, is an artifact produced by the conditions of acid hydrolysis. Although the acid labile aglycone was the minor component of the two acid metabolites, its proportion in urine extracts as measured by conversion to the lactone, increased with the time after administration of labelled phylloquinone.     

Production of thaxtomin a by two species of Streptomyces causing potato scab

A total of nine isolates of streptomycetes were isolated from scab lesions on potato tubers. Five out of them were pathogenic on potato minitubers and four of the pathogenic isolates produced thaxtomin A in infected tubers tissues. The lesion surface areas induced by thaxtomin A were highest in treatment of the minitubers with extract of OMB inoculated with S-6 and S-7, intermediate with that inoculated with S-4 and lowest with S-3. The pathogenic isolates were identified by their colour of aerial mycelia, melanin pigment productivity (+ or -), the type of spore chains morphology and carbon utilization as either S. scabies strains S-3, S-4 and S-8, or S. acidiscabies strains S-6 and S-7. S-3 and S-4 produced 0.65 and 1.60 micrograms thaxtomin A per milliliter of OMB, respectively, whereas S-6 and S-7 produced similar amounts of thaxtomin A, 2.36 and 2.10 micrograms per ml of OMB, respectively. The optimal temperature for production of thaxtomin A by S. scabies and S. acidiscabies was 28 degrees C. Production of thaxtomin A by S. scabies strain S-4 and S. acidiscabies strain S-6 was suppressed at least 50-fold at 0.5 and 0.3% of glucose, respectively. Fructose enhanced the production of thaxtomin A by both S. scabies and S. acidiscabies.     

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N -methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.     

Reconstruction of the central carbon metabolism of Aspergillus niger.

The topology of central carbon metabolism of Aspergillus niger was identified and the metabolic network reconstructed, by integrating genomic, biochemical and physiological information available for this microorganism and other related fungi. The reconstructed network may serve as a valuable database for annotation of genes identified in future genome sequencing projects on aspergilli. Based on the metabolic reconstruction, a stoichiometric model was set up that includes 284 metabolites and 335 reactions, of which 268 represent biochemical conversions and 67 represent transport processes between the different intracellular compartments and between the cell and the extracellular medium. The stoichiometry of the metabolic reactions was used in combination with biosynthetic requirements for growth and pseudo-steady state mass balances over intracellular metabolites for the quantification of metabolic fluxes using metabolite balancing. This framework was employed to perform an in silico characterisation of the phenotypic behaviour of A. niger grown on different carbon sources. The effects on growth of single reaction deletions were assessed and essential biochemical reactions were identified for different carbon sources. Furthermore, application of the stoichiometric model for assessing the metabolic capabilities of A. niger to produce metabolites was evaluated by using succinate production as a case study.     

Microbiological Transformation of Terpenes: II. Transformation of α-Pinene

Several strains of fungi were tested for their ability to metabolize alpha-pinene in shake cultures. A strain of Aspergillus niger showing marked efficiency in this respect was selected for further studies. The optimal conditions for fermentation were established with respect to substrate concentration, time, and temperature. From the fermentation products three major metabolites of alpha-pinene were isolated: a ketone, C(10)H(14)O, identified as d-verbenone; an alcohol, C(10)H(16)O, identified as d-cis-verbenol; and a crystalline diol, C(10)H(18)O(2), characterized as d-trans-sobrerol.     

Microbial transformation of dehydropinguisenol by Aspergillus sp

Two metabolites were obtained by microbial transformation of a furanosesquiterpene alcohol, dehydropinguisenol, using Aspergillus niger and Aspergillus cellulosae. Their structures were established as 10-oxo-lejeuneapinguisenol and lejeuneapinguisenol on the basis of their spectroscopic data. The latter compound was obtained after 4 and 9 days of incubation with A. cellulosae at 30 degrees C and 25 degrees C, respectively. Aspergillus niger produced both metabolites after 3 and 5 days incubation at 30 degrees C, respectively. A possible pathway for the formation of these compounds is discussed here together with their antimicrobial activity against A. niger and A. cellulosae.     

Evidence that thaxtomin C is a pathogenicity determinant of streptomyces ipomoeae, the causative agent of streptomyces soil rot disease of sweet potato

Streptomyces ipomoeae is the causal agent of Streptomyces soil rot of sweet potato, a disease marked by highly necrotic destruction of adventitious roots, including the development of necrotic lesions on the fleshy storage roots. Streptomyces potato scab pathogens produce a phytotoxin (thaxtomin A) that appears to facilitate their entrance into host plants. S. ipomoeae produces a less-modified thaxtomin derivative (thaxtomin C) whose role in pathogenicity has not been examined. Here, we cloned and sequenced the thaxtomin gene cluster (txt) of S. ipomoeae, and we then constructed targeted txt mutants that no longer produced thaxtomin C. The mutants were unable to penetrate intact adventitious roots but still caused necrosis on storage-root tissue. These results, taken in context with previous histopathological study of S. ipomoeae infection, suggest that thaxtomin C plays an essential role in inter- and intracellular penetration of adventitious sweet potato roots by S. ipomoeae. Once inside the plant host, the pathogen uses one or more yet-to-be-determined factors to necrotize root tissue, including that of any storage roots it encounters.     

<Emphasis Type="Italic">Streptomyces scabiei</Emphasis> and its toxin thaxtomin A induce scopoletin biosynthesis in tobacco and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Streptomyces scabiei is the predominant causal agent of common scab of potato in North America. The virulence of common scab-causing streptomycetes relies on their capacity to synthesize thaxtomins. In this study, the effects of S. scabiei infection and of thaxtomin A, the main toxin produced by S. scabiei, were tested for the elicitation of plant defense molecules in the model plants tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Tobacco leaves infected with spores of S. scabiei strain EF-35 or infiltrated with purified thaxtomin A produced a blue fluorescent compound that was not detected in leaves infiltrated with spores of a S. scabiei mutant deficient in thaxtomin A biosynthesis. Thin layer chromatography and high performance liquid chromatography identified this fluorescent compound as scopoletin, a plant defense phytoalexin. Arabidopsis seedlings grown in liquid medium also excreted scopoletin as a reaction to S. scabiei and thaxtomin A. The effects of the presence of scopoletin on S. scabiei were also investigated. The phytoalexin scopoletin caused a slight reduction of bacterial growth and a severe decrease of thaxtomin A production. Scopoletin was shown to inhibit thaxtomin A production by repression of a gene involved in the toxin biosynthesis.