RESUMO
Streptomyces have a uniquely complex developmental life cycle that involves the coordination of morphological differentiation with the production of numerous bioactive specialized metabolites. The majority of Streptomyces spp. are soil-dwelling saprophytes, while plant pathogenicity is a rare attribute among members of this genus. Phytopathogenic Streptomyces are responsible for economically important diseases such as common scab, which affects potato and other root crops. Following the acquisition of genes encoding virulence factors, Streptomyces pathogens are expected to have specifically adapted their regulatory pathways to enable transition from a primarily saprophytic to a pathogenic lifestyle. Investigations of the regulation of pathogenesis have primarily focused on Streptomyces scabiei and the principal pathogenicity determinant thaxtomin A. The coordination of growth and thaxtomin A production in this species is controlled in a hierarchical manner by cluster-situated regulators, pleiotropic regulators, signalling and plant-derived molecules, and nutrients. Although the majority of phytopathogenic Streptomyces produce thaxtomins, many also produce additional virulence factors, and there are scab-causing pathogens that do not produce thaxtomins. The development of effective control strategies for common scab and other Streptomyces plant diseases requires a more in-depth understanding of the genetic and environmental factors that modulate the plant pathogenic lifestyle of these organisms.
Assuntos
Regulação Bacteriana da Expressão Gênica , Doenças das Plantas , Streptomyces , Fatores de Virulência , Streptomyces/patogenicidade , Streptomyces/genética , Streptomyces/metabolismo , Streptomyces/fisiologia , Doenças das Plantas/microbiologia , Virulência , Fatores de Virulência/metabolismo , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Plantas/microbiologia , Solanum tuberosum/microbiologia , Indóis , PiperazinasRESUMO
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.
Assuntos
Toxinas Bacterianas , Solanum tuberosum , Streptomyces , Toxinas Bacterianas/genética , Regulação Bacteriana da Expressão Gênica , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/genética , Streptomyces/patogenicidadeRESUMO
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.
Assuntos
Doenças das Plantas/microbiologia , Streptomyces/patogenicidade , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/genética , Reguladores de Crescimento de Plantas/metabolismo , Tubérculos/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/genética , Streptomyces/metabolismo , VirulênciaRESUMO
Phytotoxins are secondary metabolites that contribute to the development and/or severity of diseases caused by various plant pathogenic microorganisms. The coronafacoyl phytotoxins are an important family of plant toxins that are known or suspected to be produced by several phylogenetically distinct plant pathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabies. At least seven different family members have been identified, of which coronatine was the first to be described and is the best-characterized. Though nonessential for disease development, coronafacoyl phytotoxins appear to enhance the severity of disease symptoms induced by pathogenic microbes during host infection. In addition, the identification of coronafacoyl phytotoxin biosynthetic genes in organisms not known to be plant pathogens suggests that these metabolites may have additional roles other than as virulence factors. This review focuses on our current understanding of the structures, biosynthesis, regulation, biological activities and evolution of coronafacoyl phytotoxins as well as the different methods that are used to detect these metabolites and the organisms that produce them.
Assuntos
Toxinas Bacterianas , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Plantas/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Bactérias/patogenicidade , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Evolução Biológica , Indenos/química , Indenos/metabolismo , Estrutura Molecular , Família Multigênica , Doenças das Plantas/microbiologia , Fatores de Virulência/biossíntese , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Ácido Graxo Sintases/metabolismo , Indenos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Streptomyces/metabolismo , Aminoácidos/metabolismo , Vias Biossintéticas , Ácido Graxo Sintases/genética , Deleção de Genes , Genes Bacterianos , Interações Hospedeiro-Patógeno , Família Multigênica , NADH NADPH Oxirredutases/genética , Doenças das Plantas/microbiologia , Homologia de Sequência de Aminoácidos , Solanum tuberosum/microbiologia , Streptomyces/enzimologia , Streptomyces/genéticaRESUMO
Coronafacoyl phytotoxins are an important family of plant toxins that are produced by several different phytopathogenic bacteria, including the gammaproteobacterium Pseudomonas syringae and the actinobacterium Streptomyces scabiei (formerly Streptomyces scabies). The phytotoxins consist of coronafacic acid (CFA) linked via an amide bond to different amino acids or amino acid derivatives. Previous work suggested that S. scabiei and P. syringae use distinct biosynthetic pathways for producing CFA, which is subsequently linked to its amino acid partner to form the complete phytotoxin. Here, we provide further evidence that the S. scabiei CFA biosynthetic pathway is novel by characterizing the role of CYP107AK1, a predicted cytochrome P450 that has no homologue in P. syringae Deletion of the CYP107AK1 gene abolished production of coronafacoyl-isoleucine (CFA-Ile), the primary coronafacoyl phytotoxin produced by S. scabiei Structural elucidation of accumulated biosynthetic intermediates in the ΔCYP107AK1 mutant indicated that CYP107AK1 is required for introducing the oxygen atom that ultimately forms the carbonyl group in the CFA backbone. The CYP107AK1 gene along with two additional genes involved in CFA-Ile biosynthesis in S. scabiei were found to be associated with putative CFA biosynthetic genes in other actinobacteria but not in other organisms. Analysis of the overall genetic content and organization of known and putative CFA biosynthetic gene clusters, together with phylogenetic analysis of the core biosynthetic genes, indicates that horizontal gene transfer has played an important role in the dissemination of the gene cluster and that rearrangement, insertion, and/or deletion events have likely contributed to the divergent biosynthetic evolution of coronafacoyl phytotoxins in bacteria.IMPORTANCE The ability of plants to defend themselves against invading pathogens relies on complex signaling pathways that are controlled by key phytohormones such as jasmonic acid (JA). Some phytopathogenic bacteria have evolved the ability to manipulate JA signaling in order to overcome host defenses by producing coronatine (COR), which functions as a potent JA mimic. COR and COR-like molecules, collectively referred to as coronafacoyl phytotoxins, are produced by several different plant-pathogenic bacteria, and this study provides supporting evidence that different biosynthetic pathways are utilized by different bacteria for production of these phytotoxins. In addition, our study provides a greater understanding of how coronafacoyl phytotoxin biosynthesis may have evolved in phylogenetically distinct bacteria, and we demonstrate that production of these compounds may be more widespread than previously recognized and that their role for the producing organism may not be limited to host-pathogen interactions.
RESUMO
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.
Assuntos
Ilhas Genômicas/genética , Indóis/metabolismo , Piperazinas/metabolismo , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/patogenicidade , Evolução Biológica , Filogenia , Streptomyces/genética , VirulênciaRESUMO
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.
Assuntos
Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/isolamento & purificação , Proteínas de Bactérias/genética , Sequência de Bases , Genótipo , Indóis/metabolismo , Dados de Sequência Molecular , Terra Nova e Labrador , Fenótipo , Filogenia , Piperazinas/metabolismo , Folhas de Planta/microbiologia , Tubérculos/microbiologia , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Deleção de Sequência , Streptomyces/genética , Streptomyces/patogenicidade , Streptomyces/fisiologia , VirulênciaRESUMO
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 ß-lactamase inhibitor clavulanic acid, which is used in combination with ß-lactam antibiotics to treat certain ß-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.
Assuntos
Ácido Clavulânico/biossíntese , Regulação Bacteriana da Expressão Gênica , Proteômica , Streptomyces/crescimento & desenvolvimento , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Códon/genética , Proteoma/genética , Proteoma/metabolismo , Streptomyces/genética , Inibidores de beta-Lactamases/metabolismoRESUMO
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.
Assuntos
Aminoácidos/química , Toxinas Bacterianas/química , Indenos/química , Doenças das Plantas/microbiologia , Streptomyces/metabolismo , Aminoácidos/isolamento & purificação , Toxinas Bacterianas/isolamento & purificação , Técnicas de Cultura de Células , Meios de Cultura/química , Meios de Cultura/metabolismo , Genes Bacterianos/genética , Teste de Complementação Genética , Indenos/isolamento & purificação , Indenos/metabolismo , Família Multigênica/genética , Mutação , Streptomyces/química , Streptomyces/patogenicidadeRESUMO
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.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Indóis/metabolismo , Piperazinas/metabolismo , Doenças das Plantas/microbiologia , Solanum tuberosum/microbiologia , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Regulação para Baixo , Deleção de Genes , Teste de Complementação Genética , Indóis/análise , Família Multigênica , Fenótipo , Piperazinas/análise , Raphanus/microbiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/microbiologia , Streptomyces/patogenicidade , Streptomyces/fisiologia , VirulênciaRESUMO
Streptomyces bacteria are notable for producing chemically diverse specialized metabolites that exhibit various bioactivities and mediate interactions with different organisms. Streptomyces sp. 11-1-2 is a plant pathogen that produces nigericin and geldanamycin, both of which display toxic effects against various plants. Here, the 'One Strain Many Compounds' approach was used to characterize the metabolic potential of Streptomyces sp. 11-1-2. Organic extracts were prepared from 11-1-2 cultures grown on six different agar media, and the extracts were tested in antimicrobial and plant bioassays and were subjected to untargeted metabolomics and molecular networking. Most extracts displayed strong bioactivity against Gram-positive bacteria and yeast, and they exhibited phytotoxic activity against potato tuber tissue and radish seedlings. Several known specialized metabolites, including musacin D, galbonolide B, guanidylfungin A, meridamycins and elaiophylin, were predicted to be present in the extracts along with closely related compounds with unknown structure and bioactivity. Targeted detection confirmed the presence of elaiophylin in the extracts, and bioassays using pure elaiophylin revealed that it enhances the phytotoxic effects of geldanamycin and nigericin on potato tuber tissue. Overall, this study reveals novel insights into the specialized metabolites that may mediate interactions between Streptomyces sp. 11-1-2 and other bacteria and eukaryotic organisms.
Assuntos
Metaboloma , Streptomyces , Streptomyces/metabolismo , Raphanus/efeitos dos fármacos , Raphanus/metabolismo , Raphanus/microbiologia , Doenças das Plantas/microbiologia , Metabolômica , Solanum tuberosum/metabolismo , Solanum tuberosum/microbiologia , Antibacterianos/farmacologiaRESUMO
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.
Assuntos
Solanum tuberosum , Streptomyces , Benzoquinonas , Lactamas Macrocíclicas , Nigericina/metabolismo , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Solanum tuberosum/microbiologia , Streptomyces/genética , Streptomyces/metabolismoRESUMO
Streptomyces scabiei is a key causative agent of common scab disease, which causes significant economic losses to potato growers worldwide. This organism produces several phytotoxins that are known or suspected to contribute to host-pathogen interactions and disease development; however, the full metabolic potential of S. scabiei has not been previously investigated. In this study, we used a combined metabolomic and genomic approach to investigate the metabolites that are produced by S. scabiei. The genome sequence was analyzed using antiSMASH and DeepBGC to identify specialized metabolite biosynthetic gene clusters. Using untargeted liquid chromatography-coupled tandem mass spectrometry (LC-MS2), the metabolic profile of S. scabiei was compared after cultivation on three different growth media. MS2 data were analyzed using Feature-Based Molecular Networking and hierarchical clustering in BioDendro. Metabolites were annotated by performing a Global Natural Products Social Molecular Networking (GNPS) spectral library search or using Network Annotation Propagation, SIRIUS, MetWork, or Competitive Fragmentation Modeling for Metabolite Identification. Using this approach, we were able to putatively identify new analogues of known metabolites as well as molecules that were not previously known to be produced by S. scabiei. To our knowledge, this study represents the first global analysis of specialized metabolites that are produced by this important plant pathogen.
RESUMO
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.
Assuntos
Genes Bacterianos , Interações Hospedeiro-Patógeno/fisiologia , Indenos/metabolismo , Família Multigênica , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Plântula/microbiologia , Streptomyces/fisiologia , Aminoácidos/biossíntese , Aminoácidos/genética , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Loci Gênicos , Doenças das Plantas/genética , Streptomyces/patogenicidadeRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Indóis/metabolismo , Peptídeo Sintases/metabolismo , Piperazinas/metabolismo , Streptomyces/metabolismo , Triptofano/análogos & derivados , Proteínas de Bactérias/genética , Peptídeo Sintases/genética , RNA Bacteriano/genética , Streptomyces/genética , Especificidade por Substrato , Triptofano/metabolismoRESUMO
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.
Assuntos
Genômica , Doenças das Plantas/microbiologia , Streptomyces/genética , Streptomyces/patogenicidade , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Dados de Sequência Molecular , Filogenia , Plantas/microbiologia , Alinhamento de Sequência , Streptomyces/classificação , Streptomyces/metabolismo , Fatores de Virulência/química , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Thaxtomin A is a potent phytotoxin that serves as the principle pathogenicity determinant of the common scab pathogen, Streptomyces scabiei, and is also a promising natural herbicide for agricultural applications. The biosynthesis of thaxtomin A involves the non-ribosomal peptide synthetases (NRPSs) TxtA and TxtB, and an MbtH-like protein (MLP), TxtH, which may function as a chaperone by promoting the proper folding of the two NRPS enzymes in S. scabiei. MLPs are required for the proper function of many NRPS enzymes in bacteria, and they are often capable of interacting with NRPSs from different biosynthetic pathways, though the mechanism by which this occurs is still poorly understood. To gain additional insights into MLP functional cross-talk, we conducted a broad survey of MLPs from diverse phylogenetic lineages to determine if they could functionally replace TxtH. The MLPs were assessed using a protein solubility assay to determine whether they could promote the soluble expression of the TxtA and TxtB adenylation domains. In addition, the MLPs were tested for their ability to restore thaxtomin production in a S. scabiei mutant that lacked TxtH and other endogenous MLPs. Our results showed that the MLPs investigated vary in their ability to exhibit functional cross-talk with TxtH, with two of the MLPs being unable to compensate for the loss of TxtH in the assays performed. The ability of an MLP to serve as a functional partner for the thaxtomin NRPS was not correlated with its overall amino acid similarity with TxtH, but instead with the presence of highly conserved residues. In silico structural analysis of TxtH in association with the TxtA and TxtB adenylation domains revealed that several such residues are situated at the predicted interaction interface, suggesting that they might be critical for promoting functional interactions between MLPs and the thaxtomin NRPS enzymes. Overall, our study provides additional insights into the mechanism of MLP cross-talk, and it enhances our understanding of the thaxtomin biosynthetic machinery. It is anticipated that our findings will have useful applications for both the control of common scab disease and the commercial production of thaxtomin A for agricultural use.
RESUMO
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.
Assuntos
Antibacterianos/biossíntese , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Mapeamento de Interação de Proteínas , Streptomyces coelicolor/citologia , Streptomyces coelicolor/fisiologia , Proteínas de Bactérias/antagonistas & inibidores , Far-Western Blotting , Cromatografia de Afinidade , Teste de Complementação Genética , Ligação Proteica , Streptomyces coelicolor/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.