A Novel Pathway for Biosynthesis of the Herbicidal Phosphonate Natural Product Phosphonothrixin Is Widespread in Actinobacteria.

Phosphonothrixin is an herbicidal phosphonate natural product with an unusual, branched carbon skeleton. Bioinformatic analyses of the ftx gene cluster, which is responsible for synthesis of the compound, suggest that early steps of the biosynthetic pathway, up to production of the intermediate 2,3-dihydroxypropylphosphonic acid (DHPPA) are identical to those of the unrelated phosphonate natural product valinophos. This conclusion was strongly supported by the observation of biosynthetic intermediates from the shared pathway in spent media from two phosphonothrixin producing strains. Biochemical characterization of ftx-encoded proteins confirmed these early steps, as well as subsequent steps involving the oxidation of DHPPA to 3-hydroxy-2-oxopropylphosphonate and its conversion to phosphonothrixin by the combined action of an unusual heterodimeric, thiamine-pyrophosphate (TPP)-dependent ketotransferase and a TPP-dependent acetolactate synthase. The frequent observation of ftx-like gene clusters within actinobacteria suggests that production of compounds related to phosphonothrixin is common within these bacteria. IMPORTANCE Phosphonic acid natural products, such as phosphonothrixin, have great potential for biomedical and agricultural applications; however, discovery and development of these compounds requires detailed knowledge of the metabolism involved in their biosynthesis. The studies reported here reveal the biochemical pathway phosphonothrixin production, which enhances our ability to design strains that overproduce this potentially useful herbicide. This knowledge also improves our ability to predict the products of related biosynthetic gene clusters and the functions of homologous enzymes.

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.

Mycobacterial Membrane Proteins QcrB and AtpE: Roles in Energetics, Antibiotic Targets, and Associated Mechanisms of Resistance.

Infections caused by mycobacteria are difficult to treat due to their inherent physiology, cellular structure, and intracellular lifestyle. Mycobacterium tuberculosis is a pathogen of global concern as it causes tuberculosis (TB) in humans, which requires 6-9 months of chemotherapy. The situation is further exacerbated in the case of infections caused by drug-resistant strains, which necessitate the prolonged use of agents associated with increased host toxicities. Great effort has been invested into the development of new agents for the treatment of drug-resistant infections, in addition to novel strategies to reduce treatment time. Energy production using oxidative phosphorylation is essential for the survival of M. tuberculosis, even under conditions of dormancy. Many compounds have been recently discovered that inhibit different aspects of energy metabolism in mycobacteria, some of which have been approved for human use or are currently undergoing development. The most successful examples include inhibitors of QcrB and AtpE, which are part of the cytochrome bc 1 complex and FoF1-ATP synthase, respectively. In addition, many of the discovered inhibitors are active against drug-resistant strains of M. tuberculosis, inhibit nonreplicating cells, and also show potential for the treatment of other mycobacterial infections. In the current review, we focus on the discovery of mycobacterial QcrB and AtpE inhibitors, their modes of action, and the associated mechanisms of resistance observed to date.

The coronafacoyl phytotoxins: structure, biosynthesis, regulation and biological activities.

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.

Functional and Evolutionary Characterization of a Gene Transfer Agent's Multilocus "Genome".

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality.

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.

Coronafacoyl Phytotoxin Biosynthesis and Evolution in the Common Scab Pathogen Streptomyces scabiei.

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.

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.

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.

Draft Genome Sequence of the Plant Pathogen Streptomyces sp. Strain 11-1-2.

Streptomyces sp. strain 11-1-2 is a Gram-positive filamentous bacterium that was isolated from a common scab lesion on a potato tuber. The strain is highly pathogenic to plants but does not produce the virulence-associated Streptomyces phytotoxin thaxtomin A. Here, we report the draft genome sequence of Streptomyces sp. 11-1-2.

Purification of N-coronafacoyl Phytotoxins from Streptomyces scabies.

This procedure is used for large-scale purification of N-coronafacoyl phytotoxins that are produced by the potato common scab pathogen Streptomyces scabies. The procedure employs organic extraction of S. scabies culture supernatants under alternating basic and acidic conditions in order to preferentially isolate the phytotoxin - containing carboxylic acid fraction of the supernatant. Preparative thin layer chromatography and semi-preparative reverse phase - high performance liquid chromatography are then used to further purify the individual N-coronafacoyl phytotoxins of interest.

Regulation of coronafacoyl phytotoxin production by the PAS-LuxR family regulator CfaR in the common scab pathogen Streptomyces scabies.

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.