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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.

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., (© 2016 John Wiley & Sons Ltd.)

The genus Streptomyces comprises bacteria that undergo a complex developmental life cycle and produce many metabolites of importance to industry and medicine. Streptomyces clavuligerus produces the β-lactamase inhibitor clavulanic acid, which is used in combination with β-lactam antibiotics to treat certain β-lactam resistant bacterial infections. Many aspects of how clavulanic acid production is globally regulated in S. clavuligerus still remains unknown. We conducted comparative proteomics analysis using the wild type strain of S. clavuligerus and two mutants (ΔbldA and ΔbldG), which are defective in global regulators and vary in their ability to produce clavulanic acid. Approximately 33.5 % of the predicted S. clavuligerus proteome was detected and 192 known or putative regulatory proteins showed statistically differential expression levels in pairwise comparisons. Interestingly, the expression of many proteins whose corresponding genes contain TTA codons (predicted to require the bldA tRNA for translation) was unaffected in the bldA mutant.

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.

Streptomyces scabies is an important causative agent of common scab disease of potato tubers and other root crops. The primary virulence factor produced by this pathogen is a phytotoxic secondary metabolite called thaxtomin A, which is essential for disease development. In addition, the genome of S. scabies harbors a virulence-associated biosynthetic gene cluster called the coronafacic acid (CFA)-like gene cluster, which was previously predicted to produce metabolites that resemble the Pseudomonas syringae coronatine (COR) phytotoxin. COR consists of CFA linked to an ethylcyclopropyl amino acid called coronamic acid, which is derived from L-allo-isoleucine. Using a combination of genetic and chemical analyses, we show that the S. scabies CFA-like gene cluster is responsible for producing CFA-L-isoleucine as the major product as well as other minor COR-like metabolites. Production of the metabolites was shown to require the cfl gene, which is located within the CFA-like gene cluster and encodes an enzyme involved in ligating CFA to its amino acid partner. CFA-L-isoleucine purified from S. scabies cultures was shown to exhibit bioactivity similar to that of COR, though it was found to be less toxic than COR. This is the first report demonstrating the production of coronafacoyl phytotoxins by S. scabies, which is the most prevalent scab-causing pathogen in North America.

Potato common scab is an economically important crop disease that is characterized by the formation of superficial, raised or pitted lesions on the potato tuber surface. The most widely distributed causative agent of the disease is Streptomyces scabies, which produces the phytotoxic secondary metabolite thaxtomin A that serves as a key virulence factor for the organism. Recently, it was demonstrated that S. scabies can also produce the phytotoxic secondary metabolite coronafacoyl-L-isoleucine (CFA-L-Ile) as well as other related metabolites in minor amounts. The expression of the biosynthetic genes for CFA-L-Ile production is dependent on a PAS-LuxR family transcriptional regulator, CfaR, which is encoded within the phytotoxin biosynthetic gene cluster in S. scabies. In this study, we show that CfaR activates coronafacoyl phytotoxin production by binding to a single site located immediately upstream of the putative -35 hexanucleotide box within the promoter region for the biosynthetic genes. The binding activity of CfaR was shown to require both the LuxR and PAS domains, the latter of which is involved in protein homodimer formation. We also show that CFA-L-Ile production is greatly enhanced in S. scabies by overexpression of both cfaR and a downstream co-transcribed gene, orf1. Our results provide important insight into the regulation of coronafacoyl phytotoxin production, which is thought to contribute to the virulence phenotype of S. scabies. Furthermore, we provide evidence that CfaR is a novel member of the PAS-LuxR family of regulators, members of which are widely distributed among actinomycete bacteria.

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.

PAISt is a large genomic island located in the chromosome of the plant pathogen Streptomyces turgidiscabies Car8. The island carries clustered virulence genes, transfers to other Streptomyces species, and integrates by site-specific recombination at the 8 bp palindrome TTCATGAA. The palindrome is located at the 3' end of the bacitracin resistance gene (bacA). We demonstrate that PAISt is able to excise in modules by recombination of one internal and two flanking palindromic direct repeats. The gene intSt located at the 3( end of PAISt encodes a tyrosine recombinase. Site-specific recombination activity of intSt was tested and confirmed by heterologous expression in Streptomyces coelicolor. Comparative analysis of PAISt homologues in Streptomyces scabies 87-22 and Streptomyces acidiscabies 84-104 indicates that these islands have been fixed by sequence erosion of intSt and the recombination sites.

Streptomyces is a large genus consisting of soil-dwelling, filamentous bacteria that are best known for their capability of producing a vast array of medically and agriculturally useful secondary metabolites. In addition, a small number of Streptomyces spp. are capable of colonizing and infecting the underground portions of living plants and causing economically important crop diseases such as potato common scab (CS). Research into the mechanisms of Streptomyces plant pathogenicity has led to the identification and characterization of several phytotoxic secondary metabolites that are known or suspected of contributing to diseases in various plants. The best characterized are the thaxtomin phytotoxins, which play a critical role in the development of CS, acid scab and soil rot of sweet potato. In addition, the best-characterized CS-causing pathogen, Streptomyces scabies, produces a molecule that is predicted to resemble the Pseudomonas syringae coronatine phytotoxin and which contributes to seedling disease symptom development. Other Streptomyces phytotoxic secondary metabolites that have been identified include concanamycins, FD-891 and borrelidin. Furthermore, there is evidence that additional, unknown metabolites may participate in Streptomyces plant pathogenicity. Such revelations have implications for the rational development of better management procedures for controlling CS and other Streptomyces plant diseases., (© 2013 The Society for Applied Microbiology.)

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.

Plant-pathogenic Streptomyces spp. cause scab disease on economically important root and tuber crops, the most important of which is potato. Key virulence determinants produced by these species include the cellulose synthesis inhibitor, thaxtomin A, and the secreted Nec1 protein that is required for colonization of the plant host. Recently, the genome sequence of Streptomyces scabies 87-22 was completed, and a biosynthetic cluster was identified that is predicted to synthesize a novel compound similar to coronafacic acid (CFA), a component of the virulence-associated coronatine phytotoxin produced by the plant-pathogenic bacterium Pseudomonas syringae. Southern analysis indicated that the cfa-like cluster in S. scabies 87-22 is likely conserved in other strains of S. scabies but is absent from two other pathogenic streptomycetes, S. turgidiscabies and S. acidiscabies. Transcriptional analyses demonstrated that the cluster is expressed during plant-microbe interactions and that expression requires a transcriptional regulator embedded in the cluster as well as the bldA tRNA. A knockout strain of the biosynthetic cluster displayed a reduced virulence phenotype on tobacco seedlings compared with the wild-type strain. Thus, the cfa-like biosynthetic cluster is a newly discovered locus in S. scabies that contributes to host-pathogen interactions.

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.

The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout 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.

Streptomyces scabies is the best studied of those streptomycetes that cause an economically important disease known as potato scab. The phytotoxin thaxtomin is made exclusively by these pathogens and is required for virulence. Here we describe regulation of thaxtomin biosynthesis by TxtR, a member of the AraC/XylS family of transcriptional regulators. The txtR gene is imbedded in the thaxtomin biosynthetic pathway and is located on a conserved pathogenicity island in S. scabies, S. turgidiscabies and S. acidiscabies. Thaxtomin biosynthesis was abolished and virulence was almost eliminated in the txtR deletion mutant of S. scabies 87.22. Accumulation of thaxtomin biosynthetic gene (txtA, txtB, txtC, nos) transcripts was reduced compared with the wild-type S. scabies 87.22. NOS-dependent nitric oxide production by S. scabies was also reduced in the mutant. The TxtR protein bound cellobiose, an inducer of thaxtomin production, and transcription of txtR and thaxtomin biosynthetic genes was upregulated in response to cellobiose. TxtR is the first example of an AraC/XylS family protein regulated by cellobiose. Together, these data suggest that cellobiose, the smallest oligomer of cellulose, may signal the availability of expanding plant tissue, which is the site of action of thaxtomin.

Gamma-butyrolactones regulate secondary metabolism and, sometimes, sporulation in actinomycetes by binding to specific receptor proteins, causing their dissociation from DNA targets and releasing the latter from transcriptional repression. Previously, in engineered strains of Streptomyces lividans, we showed that TylP, a deduced gamma-butyrolactone receptor, downregulated reporter gene expression driven by tylP, tylQ or tylS promoter DNA. These genes all control tylosin production in Streptomyces fradiae. Thus, at early stages of fermentation, TylQ represses tylR whereas TylS is needed for transcriptional activation of tylR. Importantly, TylR is the key activator of tylosin-biosynthetic genes. Here, we show that HIS-tagged TylP binds to specific DNA sequences, similar to the targets for authentic gamma-butyrolactone receptors, in the promoters of tylP, tylQ and tylS. Moreover, such binding is disrupted by material produced in S. fradiae and extractable by organic solvent. That putative gamma-butyrolactone material was not produced when orf18 * was disrupted within the S. fradiae genome and only about 1% of that activity survived inactivation of orf16 *, suggesting roles for the respective gene products in gamma-butyrolactone synthesis. Continued synthesis of tylosin by the disrupted strains contrasts with other reports that loss of gamma-butyrolactones abolishes antibiotic production.

Tylosin production in Streptomyces fradiae is regulated via interplay between a repressor, TylQ, and an activator of the SARP family, TylS, during regulation of tylR. The latter encodes the pathway-specific activator of the tylosin-biosynthetic (tyl) genes. Also controlled by TylS is a hitherto unassigned gene, tylU, whose product is shown here to be important for tylosin production. Thus, targeted disruption of tylU reduced tylosin yields by about 80% and bioconversion analysis with the resultant strain revealed defects in both polyketide metabolism and deoxyhexose biosynthesis. Such defects were completely eliminated by engineered overexpression of tylR (but not tylS) and Western analysis revealed significantly reduced levels of TylR in the tylU-disrupted strain. These results are consistent with a model in which TylS and TylU act in concert to facilitate expression of tylR, for which TylU (but not TylS) is nonessential. Activator proteins of the SARP family, such as TylS, are widespread among Streptomyces spp. and are important regulators of antibiotic production. Their action has been widely studied with no prior indication of associated 'helper' activity, the prevalence of which now remains to be established.

In Streptomyces coelicolor, bldG encodes a putative anti-anti-sigma factor that regulates both aerial hypha formation and antibiotic production, and a downstream transcriptionally linked open reading frame (orf3) encodes a putative anti-sigma factor protein. A cloned DNA fragment from Streptomyces clavuligerus contained an open reading frame that encoded a protein showing 92% identity to the S. coelicolor BldG protein and 91% identity to the BldG ortholog in Streptomyces avermitilis. Sequencing of the region downstream of bldG in S. clavuligerus revealed the presence of an open reading frame encoding a protein showing 72 and 69% identity to the ORF3 proteins in S. coelicolor and S. avermitilis, respectively. Northern analysis indicated that, as in S. coelicolor, the S. clavuligerus bldG gene is expressed as both a monocistronic and a polycistronic transcript, the latter including the downstream orf3 gene. High-resolution S1 nuclease mapping of S. clavuligerus bldG transcripts revealed the presence of three bldG-specific promoters, and analysis of expression of a bldGp-egfp reporter indicated that the bldG promoter is active at various stages of development and in both substrate and aerial hyphae. A bldG null mutant was defective in both morphological differentiation and in the production of secondary metabolites, such as cephamycin C, clavulanic acid, and the 5S clavams. This inability to produce cephamycin C and clavulanic acid was due to the absence of the CcaR transcriptional regulator, which controls the expression of biosynthetic genes for both secondary metabolites as well as the expression of a second regulator of clavulanic acid biosynthesis, ClaR. This makes bldG the first regulatory protein identified in S. clavuligerus that functions upstream of CcaR and ClaR in a regulatory cascade to control secondary metabolite production.

The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.