A large, mobile pathogenicity island confers plant pathogenicity on Streptomyces species

Potato scab is a globally important disease caused by polyphyletic plant pathogenic Streptomyces species. Streptomyces acidiscabies, Streptomyces scabies and Streptomyces turgidiscabies possess a conserved biosynthetic pathway for the nitrated dipeptide phytotoxin thaxtomin. These pathogens also possess the nec1 gene which encodes a necrogenic protein that is an independent virulence factor. In this article we describe a large (325-660 kb) pathogenicity island (PAI) conserved among these three plant pathogenic Streptomyces species. A partial DNA sequence of this PAI revealed the thaxtomin biosynthetic pathway, nec1, a putative tomatinase gene, and many mobile genetic elements. In addition, the PAI from S. turgidiscabies contains a plant fasciation (fas) operon homologous to and colinear with the fas operon in the plant pathogen Rhodococcus fascians. The PAI was mobilized during mating from S. turgidiscabies to the non-pathogens Streptomyces coelicolor and Streptomyces diastatochromogenes on a 660 kb DNA element and integrated site-specifically into a putative integral membrane lipid kinase. Acquisition of the PAI conferred a pathogenic phenotype on S. diastatochromogenes but not on S. coelicolor. This PAI is the first to be described in a Gram-positive plant pathogenic bacterium and is responsible for the emergence of new plant pathogenic Streptomyces species in agricultural systems.     

Nitric oxide synthase inhibitors and nitric oxide donors modulate the biosynthesis of thaxtomin A, a nitrated phytotoxin produced by Streptomyces spp.

Evidence for the involvement of a bacterial nitric oxide synthase (NOS) in the biosynthesis of a phytotoxin is presented. Several species of Streptomyces bacteria produce secondary metabolites with unusual nitrogen groups, such as thaxtomin A (ThxA), which contains a nitroindole moiety. ThxA is a phytotoxin made by three pathogenic Streptomyces species that cause common scab of potato. All three species possess a gene homologous to the oxygenase domain of murine inducible NOS, and this gene, nos, is essential for normal levels of ThxA production. We grew Streptomyces turgidiscabies in the presence of several known NOS inhibitors and a nitric oxide (NO) scavenger to determine their effect on ThxA production. The NO scavenger (CPTIO) and four NOS inhibitors (NAME, NMMA, AG, and 7-NI) reduced ThxA production without affecting bacterial growth. A strain of S. turgidiscabies from which the nos gene had been deleted was grown in the presence of three NO donors (DEANO, SIN, and SNAP), and all three partially restored ThxA production. Our data suggest that bacterial nitric oxide synthases may, at least in part, produce NO for biosynthetic purposes, rather than for cellular signaling, as they do in mammals.     

Cloning and expression of a gene from Streptomyces scabies encoding a putative pathogenicity factor.

We cloned a 9.4-kb DNA fragment from Streptomyces scabies ATCC 41973 that allows the nonpathogen Streptomyces lividans 66 TK24 to necrotize and colonize potato tuber slices and produce scab-like symptoms on potato minitubers. Deletion analysis demonstrated that activity was conferred by a 1.6-kb DNA region. Sequence analysis of a 2.4-kb DNA fragment spanning the DNA region necessary for activity revealed three open reading frames (ORFs). The deduced amino acid sequence of ORF1, designated ORFtnp, showed high levels of identity with the first 233 amino acids of the putative transposases of the IS1164 elements from Rhodococcus rhodochrous (71%) and Mycobacterium bovis (68%), members of the Staphylococcus aureus IS256 family of transposases. No significant homologies to ORF2 and ORF3 were found in the nucleic acid and protein databases. ORFtnp is located 5' of ORF3. ORF2 is incomplete and is located 3' of ORF3. Subcloning of the individual ORFs demonstrated that ORF3, designated nec1, is sufficient for necrotizing activity in S. lividans 66 TK24. S. lividans 66 TK24 expressing nec1 does not produce thaxtomin A but produces an unidentified extracellular water-soluble compound that causes necrosis on potato tuber discs. The G+C content of nec1 suggests that it has moved horizontally from another genus. Southern analysis of ORFtnp and nec1 demonstrate that these genes are physically linked in Streptomyces strains, including S. scabies and Streptomyces acidiscabies strains, that are pathogenic on potato and that produce the phytotoxin thaxtomin A. These data suggest that nec1 may have been mobilized into S. scabies through a transposition event mediated by ORFtnp.     

High proportions of nonpathogenic Streptomyces are associated with common scab-resistant potato lines and less severe disease

Streptomyces isolates were obtained from potato tubers with common scab lesions from 2 fields over a 3 year period in Minnesota and a 5 year period in Maine. Isolates were obtained from different potato cultivars or breeding lines and types of scab lesions. A majority of isolates could be classified as putative pathogens based on the presence of genes for biosynthesis of the pathogenicity determinant, thaxtomin, but large numbers of streptomycetes lacking genes for thaxtomin biosynthesis (presumably nonpathogenic) were also recovered. Most Streptomyces isolates recovered from raised and pitted lesions were pathogens, whereas mostly nonpathogenic isolates were recovered from unblemished potato skin or nonscab lesions. Fewer pathogenic than nonpathogenic isolates were recovered from the most resistant potato lines. The proportion and diversity of nonpathogenic isolates recovered was higher in Maine than in Minnesota. The association between greater numbers of nonpathogenic Streptomyces and less severe common scab suggests that the interaction between plant genotype and Streptomyces microbial community is important in determining the severity of common scab on potato, and emphasizes the role of complex interactions between plants and microbial populations on and near plant roots in plant disease outcomes.     

Effect of carbohydrates on the production of thaxtomin A by <Emphasis Type="Italic">Streptomyces acidiscabies</Emphasis>

Several Streptomyces species cause plant diseases, including S. scabies, S. acidiscabies and S. turgidiscabies, which produce common scab of potato and similar diseases of root crops. These species produce thaxtomins, dipeptide phytotoxins that are responsible for disease symptoms. Thaxtomins are produced in vivo on diseased potato tissue and in vitro in oat-based culture media, but the regulation of thaxtomin biosynthesis is not understood. S. acidiscabies was grown in a variety of media to assess the impact of medium components on thaxtomin A (ThxA) production. ThxA biosynthesis was not correlated with bacterial biomass, nor was it stimulated by α-solanine or α-chaconine, the two most prevalent potato glycoalkaloids. ThxA production was stimulated by oat bran broth, even after exhaustive extraction, suggesting that specific carbohydrates may influence ThxA biosynthesis. Oat bran contains high levels of xylans and glucans, and both of these carbohydrates, as well as xylans from wheat and tamarind, stimulated ThxA production, but not to the same extent as oat bran. Starches and simple sugars did not induce ThxA production. The data indicate that complex carbohydrates may act as environmental signals to plant pathogenic Streptomyces, allowing production of thaxtomin and enabling bacteria to colonize its host.     

The ability of Pseudomonas sp. LBUM 223 to produce phenazine-1-carboxylic acid affects the growth of Streptomyces scabies, the expression of thaxtomin biosynthesis genes and the biological control potential against common scab of potato

Streptomyces scabies causes common scab, an economical disease affecting potato crops world-wide, for which no effective control measure exists. This pathogen produces the plant toxin thaxtomin A, which is involved in symptom development on potato tubers. A biological control approach that can limit S. scabies growth and repress thaxtomin production represents an attractive alternative to classical control strategies. Pseudomonas sp. LBUM 223 produces phenazine-1-carboxylic acid (PCA), an antibiotic that inhibits the growth of plant pathogens and contributes to the biological control of plant diseases. In this study, the involvement of LBUM 223's PCA-producing ability in the growth inhibition of S. scabies, repression of thaxtomin biosynthesis genes (txtA and txtC) and the biological control of common scab of potato was investigated using a mutant defective in PCA production (LBUM 223phzC(-) ). Streptomyces scabies growth was inhibited to a significantly lesser degree by LBUM 223phzC(-) than by the wild type. LBUM 223 also significantly repressed txtA and txtC expression in S. scabies and protected potato against disease, whereas LBUM 223phzC(-) did not. These results suggest that PCA production is central to the ability of LBUM 223 to limit pathogen growth, repress the expression of key pathogenicity genes and control common scab of potato.     

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.     

What does it take to be a plant pathogen: genomic insights from <Emphasis Type="Italic">Streptomyces</Emphasis> species

Plant pathogenicity is rare in the genus Streptomyces, with only a dozen or so species possessing this trait out of the more than 900 species described. Nevertheless, such species have had a significant impact on agricultural economies throughout the world due to their ability to cause important crop diseases such as potato common scab, which is characterized by lesions that form on the potato tuber surface. All pathogenic species that cause common scab produce a family of phytotoxins called the thaxtomins, which function as cellulose synthesis inhibitors. In addition, the nec1 and tomA genes are conserved in several pathogenic streptomycetes, the former of which is predicted to function in the suppression of plant defense responses. Streptomyces scabies is the oldest plant pathogen described and has a world-wide distribution, whereas species such as S. turgidiscabies and S. acidiscabies are believed to be newly emergent pathogens found in more limited geographical locations. The genome sequence of S. scabies 87-22 was recently completed, and comparative genomic analyses with other sequenced microbial pathogens have revealed the presence of additional genes that may play a role in plant pathogenicity, an idea that is supported by functional analysis of one such putative virulence locus. In addition, the availability of multiple genome sequences for both pathogenic and nonpathogenic streptomycetes has provided an opportunity for comparative genomic analyses to identify the Streptomyces pathogenome. Such genomic analyses will contribute to the fundamental understanding of the mechanisms and evolution of plant pathogenicity and plant-microbe biology within this genus.     

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.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.     

<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.     

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.     

Genetic background affects pathogenicity island function and pathogen emergence in Streptomyces

With few exceptions, thaxtomin A (ThxA), a nitrated diketopiperazine, is the pathogenicity determinant for plant‐pathogenic Streptomyces species. In Streptomyces scabiei (syn. S. scabies), the ThxA biosynthetic cluster is located within a 177‐kb mobile pathogenicity island (PAI), called the toxicogenic region (TR). In S. turgidiscabies, the ThxA biosynthetic cluster is located within a 674‐kb pathogenicity island (PAIst). The emergence of new plant pathogens occurs in this genus, but not frequently. This raises the question of whether the mobilization of these pathogenicity regions, through mating, is widespread and whether TR and PAIst can confer plant pathogenicity. We showed that ThxA biosynthetic clusters on TR and PAIst were transferred into strains from five non‐pathogenic Streptomyces species through mating with S. scabiei and S. turgidiscabies. However, not all of the transconjugants produced ThxA and exhibited the virulence phenotype, indicating that the genetic background of the recipient strains affects the functionality of the ThxA biosynthetic cluster and therefore would be expected to affect the emergence of novel pathogenic Streptomyces species. Thxs have been patented as natural herbicides, but have yet to be commercialized. Our results also demonstrated the potential of the heterologous production of ThxA as a natural and biodegradable herbicide in non‐pathogenic Streptomyces species.     

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.     

Hybrid antibiotics with the nikkomycin nucleoside and polyoxin peptidyl moieties

Acting as competitive inhibitors of chitin synthase, nikkomycins and polyoxins are potent antibiotics against pathogenic fungi. Taking advantage of the structural similarities between these two peptidyl nucleoside antibiotics, genes required for the biosynthesis of the dipeptidyl moiety of polyoxin from Streptomyces cacaoi were introduced into a Streptomyces ansochromogenes mutant producing the nucleoside moiety of nikkomycin X. Two hybrid antibiotics were generated. One of them was identified as polyoxin N, and the other, a novel compound, was named polynik A. The hybrid antibiotics exhibited merits from both parents: they had better inhibitory activity against phytopathogenic fungi than polyoxin B, and were more stable under different pH and temperature conditions than nikkomycin X. This study demonstrates the use of the combinatorial biosynthetic approach to produce valuable and novel hybrid antibiotics with improved properties.