Nigericin and Geldanamycin Are Phytotoxic Specialized Metabolites Produced by the Plant Pathogen Streptomyces sp. 11-1-2.

Streptomyces bacteria are a key source of microbial specialized metabolites with useful applications in medicine and agriculture. In addition, some species are important plant pathogens and cause diseases such as potato scab, which reduces the quality and market value of affected potato crops. Most scab-associated Streptomyces spp. produce the phytotoxic metabolite thaxtomin A as the principal pathogenicity factor. However, recent reports have described scab-causing strains that do not produce thaxtomin A, but instead produce other phytotoxins that are thought to contribute to plant host infection and symptom development. Streptomyces sp. 11-1-2 is a highly pathogenic strain that was originally isolated from a scab symptomatic potato tuber in Newfoundland, Canada. The strain secretes one or more phytotoxic compounds of unknown identity, and it is hypothesized that these compounds serve as virulence factors for this organism. We analyzed the genome sequence of Streptomyces sp. 11-1-2 and found biosynthetic gene clusters for producing the known herbicidal compounds nigericin and geldanamycin. Phytotoxic culture extracts were analyzed using liquid chromatography-coupled tandem mass spectrometry and molecular networking, and this confirmed the production of both compounds by Streptomyces sp. 11-1-2 along with other, potentially related metabolites. The biosynthesis of both metabolites was found to be suppressed by the addition of N-acetylglucosamine to the culture medium, and pure nigericin and geldanamycin were able to exhibit phytotoxic effects against both radish seedlings and potato tuber tissue. Furthermore, the coadministration of the two compounds produced greater phytotoxic effects against potato tuber tissue than administration of each compound alone. IMPORTANCE Plant pathogens use a variety of mechanisms, including the production of phytotoxic specialized metabolites, to establish an infection of host tissue. Although thaxtomin A is considered the key phytotoxin involved in the development of potato scab disease, there is increasing evidence that other phytotoxins can play a role in disease development in some instances. In this study, we show that the highly pathogenic Streptomyces sp. 11-1-2 is capable of producing nigericin and geldanamycin, which individually and combined can cause significant damage to potato tuber tissue and radish seedlings. Our results suggest that the pathogenic phenotype of Streptomyces sp. 11-1-2 is due in part to the production of these specialized metabolites. As the biological activity of nigericin and geldanamycin is vastly different from the proposed activity of thaxtomin A against plants, the secretion of these compounds may represent a novel mechanism of plant pathogenicity exhibited by some Streptomyces species.

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.

Virulence mechanisms of plant-pathogenic Streptomyces species: an updated review.

Gram-positive Actinobacteria from the genus Streptomyces are best known for their morphological complexity and for their ability to produce numerous bioactive specialized metabolites with useful applications in human and veterinary medicine and in agriculture. In contrast, the ability to infect living plant tissues and to cause diseases of root and tuber crops such as potato common scab (CS) is a rare attribute among members of this genus. Research on the virulence mechanisms of plant-pathogenic Streptomyces spp. has revealed the importance of the thaxtomin phytotoxins as key pathogenicity determinants produced by several species. In addition, other phytotoxic specialized metabolites may contribute to the development or severity of disease caused by Streptomyces spp., along with the production of phytohormones and secreted proteins. A thorough understanding of the molecular mechanisms of plant pathogenicity will enable the development of better management procedures for controlling CS and other plant diseases caused by the Streptomyces.

Positive and Negative Regulation of the Virulence-Associated Coronafacoyl Phytotoxin in the Potato Common Scab Pathogen Streptomyces scabies.

The potato common scab pathogen Streptomyces scabies produces N-coronafacoyl-l-isoleucine (CFA-Ile), which is a member of the coronafacoyl family of phytotoxins that are synthesized by multiple plant pathogenic bacteria. The CFA-Ile biosynthetic gene cluster contains a regulatory gene, cfaR, which directly controls the expression of the phytotoxin structural genes. In addition, a gene designated orf1 encodes a predicted ThiF family protein and is cotranscribed with cfaR, suggesting that it also plays a role in the regulation of CFA-Ile production. In this study, we demonstrated that CfaR is an essential activator of coronafacoyl phytotoxin production, while ORF1 is dispensable for phytotoxin production and may function as a helper protein for CfaR. We also showed that CFA-Ile inhibits the ability of CfaR to bind to the promoter region driving expression of the phytotoxin biosynthetic genes and that elevated CFA-Ile production by overexpression of both cfaR and orf1 in S. scabies increases the severity of disease symptoms induced by the pathogen during colonization of potato tuber tissue. Overall, our study reveals novel insights into the regulatory mechanisms controlling CFA-Ile production in S. scabies and it provides further evidence that CFA-Ile is an important virulence factor for this organism.

Promiscuous Pathogenicity Islands and Phylogeny of Pathogenic Streptomyces spp.

Approximately 10 Streptomyces species cause disease on underground plant structures. The most economically important of these is potato scab, and the most studied of these pathogens is Streptomyces scabiei (syn. S. scabies). The main pathogenicity determinant of scab-causing Streptomyces species is a nitrated diketopiperazine, known as thaxtomin A (ThxA). In the pathogenic species Streptomyces turgidiscabies, ThxA biosynthetic genes reside on a mobile pathogenicity island (PAI). However, the mobilization of PAIs in other Streptomyces species remains uncharacterized. Here, we investigated the mobilization of the PAI of S. scabiei 87-22. Based on whole genome sequences, we inferred the evolutionary relationships of pathogenic Streptomyces species and discovered that Streptomyces sp. strain 96-12, a novel pathogenic species isolated from potatoes in Egypt, was phylogenetically grouped with nonpathogenic species rather than with known pathogenic species. We also found that Streptomyces sp. strain 96-12 contains a PAI that is almost identical to the PAI in S. scabiei 87-22, despite significant differences in their genome sequences. This suggested direct or indirect in vivo mobilization of the PAI between S. scabiei and nonpathogenic Streptomyces species. To test whether the S. scabiei 87-22 PAI could, indeed, be mobilized, S. scabiei 87-22 deletion mutants containing antibiotic resistance markers in the PAI were mated with Streptomyces diastatochromogenes, a nonpathogenic species. The PAI of S. scabiei was site-specifically inserted into the aviX1 gene of S. diastatochromogenes and conferred pathogenicity in radish seedling assays. Our results demonstrated that S. scabiei, the earliest described Streptomyces pathogen, could be the source of a PAI responsible for the emergence of novel pathogenic species.

Production of the Streptomyces scabies coronafacoyl phytotoxins involves a novel biosynthetic pathway with an F420 -dependent oxidoreductase and a short-chain dehydrogenase/reductase.

Coronafacoyl phytotoxins are secondary metabolites that are produced by various phytopathogenic bacteria, including several pathovars of the Gram-negative bacterium Pseudomonas syringae as well as the Gram-positive potato scab pathogen Streptomyces scabies. The phytotoxins are composed of the polyketide coronafacic acid (CFA) linked via an amide bond to amino acids or amino acid derivatives, and their biosynthesis involves the cfa and cfa-like gene clusters that are found in P. syringae and S. scabies, respectively. The S. scabies cfa-like gene cluster was previously reported to contain several genes that are absent from the P. syringae cfa gene cluster, including one (oxr) encoding a putative F420 -dependent oxidoreductase, and another (sdr) encoding a predicted short-chain dehydrogenase/reductase. Using gene deletion analysis, we demonstrated that both oxr and sdr are required for normal production of the S. scabies coronafacoyl phytotoxins, and structural analysis of metabolites that accumulated in the Δsdr mutant cultures revealed that Sdr is directly involved in the biosynthesis of the CFA moiety. Our results suggest that S. scabies and P. syringae use distinct biosynthetic pathways for producing coronafacoyl phytotoxins, which are important mediators of host-pathogen interactions in various plant pathosystems.

Isolation and Characterization of Plant-Pathogenic Streptomyces Species Associated with Common Scab-Infected Potato Tubers in Newfoundland.

Potato common scab (CS) is an economically important crop disease that is caused by several members of the genus Streptomyces. In this study, we characterized the plant-pathogenic Streptomyces spp. associated with CS-infected potato tubers harvested in Newfoundland, Canada. A total of 17 pathogenic Streptomyces isolates were recovered from potato scab lesions, of which eight were determined to be most similar to the known CS pathogen S. europaeiscabiei. All eight S. europaeiscabiei isolates were found to produce the thaxtomin A phytotoxin and to harbor the nec1 virulence gene, and most also carry the putative virulence gene tomA. The remaining isolates appear to be novel pathogenic species that do not produce thaxtomin A, and only two of these isolates were determined to harbor the nec1 or tomA genes. Of the non-thaxtomin-producing isolates, strain 11-1-2 was shown to exhibit a severe pathogenic phenotype against different plant hosts and to produce a novel, secreted phytotoxic substance. This is the first report documenting the plant-pathogenic Streptomyces spp. associated with CS disease in Newfoundland. Furthermore, our findings provide further evidence that phytotoxins other than thaxtomin A may also contribute to the development of CS by Streptomyces spp.

Thaxtomin A production and virulence are controlled by several bld gene global regulators in Streptomyces scabies.

Streptomyces scabies is the main causative agent of common scab disease, which leads to significant annual losses to potato growers worldwide. The main virulence factor produced by S. scabies is a phytotoxic secondary metabolite called thaxtomin A, which functions as a cellulose synthesis inhibitor. Thaxtomin A production is controlled by the cluster-situated regulator TxtR, which activates expression of the thaxtomin biosynthetic genes in response to cello-oligosaccharides. Here, we demonstrate that at least five additional regulatory genes are required for wild-type levels of thaxtomin A production and plant pathogenicity in S. scabies. These regulatory genes belong to the bld gene family of global regulators that control secondary metabolism or morphological differentiation in Streptomyces spp. Quantitative reverse-transcriptase polymerase chain reaction showed that expression of the thaxtomin biosynthetic genes was significantly downregulated in all five bld mutants and, in four of these mutants, this downregulation was attributed to the reduction in expression of txtR. Furthermore, all of the mutants displayed reduced expression of other known or predicted virulence genes, suggesting that the bld genes may function as global regulators of virulence gene expression in S. scabies.

Thaxtomin biosynthesis: the path to plant pathogenicity in the genus Streptomyces.

Streptomyces species are best known for their ability to produce a wide array of medically and agriculturally important secondary metabolites. However, there is a growing number of species which, like Streptomyces scabies, can function as plant pathogens and cause scab disease on economically important crops such as potato. All of these species produce the phytotoxin thaxtomin, a nitrated dipeptide which inhibits cellulose synthesis in expanding plant tissue. The biosynthesis of thaxtomin involves conserved non-ribosomal peptide synthetases, P450 monooxygenases, and a nitric oxide synthase, the latter being required for nitration of the toxin. This nitric oxide synthase is also responsible for the production of diffusible nitric oxide by scab-causing streptomycetes at the host-pathogen interface, suggesting that nitric oxide production might play an additional role during the infection process. The thaxtomin biosynthetic genes are transcriptionally regulated by an AraC/XylS family regulator, TxtR, which is conserved in pathogenic streptomycetes and is encoded within the thaxtomin biosynthetic gene cluster. The TxtR protein specifically binds cellobiose, a known inducer of thaxtomin biosynthesis, and cellobiose is required for expression of the biosynthetic genes. A second virulence gene in pathogenic Streptomyces species, nec1, encodes a novel secreted protein that may suppress plant defence responses. The thaxtomin biosynthetic genes and nec1 are contained on a large mobilizable pathogenicity island; the transfer of this island to recipient streptomycetes likely explains the rapid emergence of new pathogenic species. The newly available genome sequence of S. scabies will provide further insight into the mechanisms utilized by pathogenic streptomycetes during plant-microbe interactions.