Nitrogen sources enhance siderophore-mediated competition for iron between potato common scab and late blight causative agents.

How do pathogens affecting the same host interact with each other? We evaluated here the types of microbe-microbe interactions taking place between Streptomyces scabiei and Phytophthora infestans, the causative agents of common scab and late blight diseases in potato crops, respectively. Under most laboratory culture conditions tested, S. scabiei impaired or completely inhibited the growth of P. infestans by producing either soluble and/or volatile compounds. Increasing peptone levels correlated with increased inhibition of P. infestans. Comparative metabolomics showed that production of S. scabiei siderophores (desferrioxamines, pyochelin, scabichelin, and turgichelin) increased with the quantity of peptone, thereby suggesting that they participate in the inhibition of the oomycete growth. Mass spectrometry imaging further uncovered that the zones of secreted siderophores and of P. infestans growth inhibition coincided. Moreover, either the repression of siderophore production or the neutralization of their iron-chelating activity led to a resumption of P. infestans growth. Replacement of peptone by natural nitrogen sources such as ammonium nitrate, sodium nitrate, ammonium sulfate, and urea also triggered siderophore production in S. scabiei. Interestingly, nitrogen source-induced siderophore production also inhibited the growth of Alternaria solani, the causative agent of the potato early blight. Overall, our work further emphasizes the importance of competition for iron between microorganisms that colonize the same niche. As common scab never alters the vegetative propagation of tubers, we propose that S. scabiei, under certain conditions, could play a protective role for its hosts against much more destructive pathogens through exploitative iron competition and volatile compound production.

Common scab disease: structural basis of elicitor recognition in pathogenic Streptomyces species.

IMPORTANCE: Common scab is a disease caused by a few Streptomyces species that affects important root and tuber crops including potato, beet, radish, and parsnip, resulting in major economic losses worldwide. In this work, we unveiled the molecular basis of host recognition by these pathogens by solving the structure of the sugar-binding protein CebE of Streptomyces scabiei in complex with cellotriose, the main elicitor of the pathogenic lifestyle of these bacteria. We further revealed that the signaling pathway from CebE-mediated transport of cellotriose is conserved in all pathogenic species except Streptomyces ipomoeae, which causes soft rot disease in sweet potatoes. Our work also provides the structural basis of the uptake of cellobiose and cellotriose in saprophytic Streptomyces species, the first step activating the expression of the enzymatic system degrading the most abundant polysaccharide on earth, cellulose.

Role of Alternative Elicitor Transporters in the Onset of Plant Host Colonization by Streptomyces scabiei 87-22.

Plant colonization by Streptomyces scabiei, the main cause of common scab disease on root and tuber crops, is triggered by cello-oligosaccharides, cellotriose being the most efficient elicitor. The import of cello-oligosaccharides via the ATP-binding cassette (ABC) transporter CebEFG-MsiK induces the production of thaxtomin phytotoxins, the central virulence determinants of this species, as well as many other metabolites that compose the 'virulome' of S. scabiei. Homology searches revealed paralogues of the CebEFG proteins, encoded by the cebEFG2 cluster, while another ABC-type transporter, PitEFG, is encoded on the pathogenicity island (PAI). We investigated the gene expression of these candidate alternative elicitor importers in S. scabiei 87-22 upon cello-oligosaccharide supply by transcriptomic analysis, which revealed that cebEFG2 expression is highly activated by both cellobiose and cellotriose, while pitEFG expression was barely induced. Accordingly, deletion of pitE had no impact on virulence and thaxtomin production under the conditions tested, while the deletion of cebEFG2 reduced virulence and thaxtomin production, though not as strong as the mutants of the main cello-oligosaccharide transporter cebEFG1. Our results thus suggest that both ceb clusters participate, at different levels, in importing the virulence elicitors, while PitEFG plays no role in this process under the conditions tested. Interestingly, under more complex culture conditions, the addition of cellobiose restored thaxtomin production when both ceb clusters were disabled, suggesting the existence of an additional mechanism that is involved in sensing or importing the elicitor of the onset of the pathogenic lifestyle of S. scabiei.

Structure and Function of BcpE2, the Most Promiscuous GH3-Family Glucose Scavenging Beta-Glucosidase.

Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl ß-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.

The virulome of Streptomyces scabiei in response to cello-oligosaccharide elicitors.

The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the 'green light' to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs' expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei.

Identification of Novel Rotihibin Analogues in Streptomyces scabies, Including Discovery of Its Biosynthetic Gene Cluster.

Streptomyces scabies is a phytopathogen associated with common scab disease. This is mainly attributed to its ability to produce the phytotoxin thaxtomin A, the biosynthesis of which is triggered by cellobiose. During a survey of other metabolites released in the presence of cellobiose, we discovered additional compounds in the thaxtomin-containing extract from Streptomyces scabies. Structural analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) revealed that these compounds are amino acid sequence variants of the TOR (target of rapamycin) kinase (TORK) pathway-inhibitory lipopeptide rotihibin A, and the main compounds were named rotihibins C and D. In contrast to thaxtomin, the production of rotihibins C and D was also elicited in the presence of glucose, indicating different regulation of their biosynthesis. Through a combination of shotgun and targeted proteomics, the putative rotihibin biosynthetic gene cluster rth was identified in the publicly available genome of S. scabies 87-22. This cluster spans 33 kbp and encodes 2 different nonribosomal peptide synthetases (NRPSs) and 12 additional enzymes. Homologous rth biosynthetic gene clusters were found in other publicly available and complete actinomycete genomes. Rotihibins C and D display herbicidal activity against Lemna minor and Arabidopsis thaliana at low concentrations, shown by monitoring the effects on growth and the maximal photochemistry efficiency of photosystem II. IMPORTANCE Rotihibins A and B are plant growth inhibitors acting on the TORK pathway. We report the isolation and characterization of new sequence analogues of rotihibin from Streptomyces scabies, a major cause of common scab in potato and other tuber and root vegetables. By combining proteomics data with genomic analysis, we found a cryptic biosynthetic gene cluster coding for enzyme machinery capable of rotihibin production. This work may lead to the biotechnological production of variants of this lipopeptide to investigate the exact mechanism by which it can target the plant TORK pathway in Arabidopsis thaliana. In addition, bioinformatics revealed the existence of other variants in plant-associated Streptomyces strains, both pathogenic and nonpathogenic species, raising new questions about the actual function of this lipopeptide. The discovery of a module in the nonribosomal peptide synthetase (NRPS) that incorporates the unusual citrulline residue may improve the prediction of peptides encoded by cryptic NRPS gene clusters.

Deletion of bglC triggers a genetic compensation response by awakening the expression of alternative beta-glucosidase.

In the plant pathogen Streptomyces scabies, the gene bglC encodes a GH1 family cellobiose beta-glucosidase that is both required for primary metabolism and for inducing virulence of the bacterium. Deletion of bglC (strain ΔbglC) surprisingly resulted in the augmentation of the global beta-glucosidase activity of S. scabies. This paradoxical phenotype is highly robust as it has been observed in all bglC deletion mutants independently generated, thereby highlighting a phenomenon of genetic compensation. Comparative proteomics allowed to identify two glycosyl hydrolases - named BcpE1 and BcpE2 - of which peptide levels were significantly increased in strain ΔbglC. Quantitative RT-PCR revealed that the higher abundance of BcpE1 and BcpE2 is triggered at the transcriptional level, the expression of their respective gene being 100 and 15 times upregulated. Enzymatic studies with pure BcpE proteins showed that they both possess beta-glucosidase activity thereby explaining the genotypic-phenotypic discrepancy of the bglC deletion mutant. The GH1 family BcpE1 could hydrolyze cellobiose and generate glucose similarly to BglC itself thereby mainly contributing to the survival of strain ΔbglC when cellobiose is provided as sole nutrient source. The low affinity of BcpE2 for cellobiose suggests that this GH3 family beta-glucosidase would instead primarily target another and yet unknown glucose-beta-1,4-linked substrate. These results make S. scabies a new model system to study genetic compensation. Discovering how, either the bglC DNA locus, its mRNA, the BglC protein, or either its enzymatic activity controls bcpE genes' expression, will unveil new mechanisms directing transcriptional repression.

A Single Biosynthetic Gene Cluster Is Responsible for the Production of Bagremycin Antibiotics and Ferroverdin Iron Chelators.

Biosynthetic gene clusters (BGCs) are organized groups of genes involved in the production of specialized metabolites. Typically, one BGC is responsible for the production of one or several similar compounds with bioactivities that usually only vary in terms of strength and/or specificity. Here we show that the previously described ferroverdins and bagremycins, which are families of metabolites with different bioactivities, are produced from the same BGC, whereby the fate of the biosynthetic pathway depends on iron availability. Under conditions of iron depletion, the monomeric bagremycins are formed, representing amino-aromatic antibiotics resulting from the condensation of 3-amino-4-hydroxybenzoic acid with p-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on p-vinylphenyl-3-nitroso-4-hydroxybenzoate, which complexes iron to form the trimeric ferroverdins that have anticholesterol activity. Thus, our work shows a unique exception to the concept that BGCs should only produce a single family of molecules with one type of bioactivity and that in fact different bioactive molecules may be produced depending on the environmental conditions.IMPORTANCE Access to whole-genome sequences has exposed the general incidence of the so-called cryptic biosynthetic gene clusters (BGCs), thereby renewing their interest for natural product discovery. As a consequence, genome mining is the often first approach implemented to assess the potential of a microorganism for producing novel bioactive metabolites. By revealing a new level of complexity of natural product biosynthesis, we further illustrate the difficulty of estimation of the panel of molecules associated with a BGC based on genomic information alone. Indeed, we found that the same gene cluster is responsible for the production of compounds which differ in terms of structure and bioactivity. The production of these different compounds responds to different environmental triggers, which suggests that multiplication of culture conditions is essential for revealing the entire panel of molecules made by a single BGC.

Proteomic Response to Thaxtomin Phytotoxin Elicitor Cellobiose and to Deletion of Cellulose Utilization Regulator CebR in Streptomyces scabies.

Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies.

Complete Genome Sequence of Streptomyces lunaelactis MM109T, Isolated from Cave Moonmilk Deposits.

Streptomyces lunaelactis MM109T is a ferroverdin A (anticholesterol) producer isolated from cave moonmilk deposits. The complete genome sequence of MM109T was obtained by combining Oxford Nanopore MinION and Illumina HiSeq and MiSeq technologies, revealing an 8.4-Mb linear chromosome and two plasmids, pSLUN1 (127,264 bp, linear) and pSLUN2 (46,827 bp, circular).

Contribution of the ß-glucosidase BglC to the onset of the pathogenic lifestyle of Streptomyces scabies.

Common scab disease on root and tuber plants is caused by Streptomyces scabies and related species which use the cellulose synthase inhibitor thaxtomin A as the main phytotoxin. Thaxtomin production is primarily triggered by the import of cello-oligosaccharides. Once inside the cell, the fate of the cello-oligosaccharides is dichotomized: (i) the fuelling of glycolysis with glucose for the saprophytic lifestyle through the action of ß-glucosidase(s) (BGs); and (ii) elicitation of the pathogenic lifestyle by the inhibition of CebR-mediated transcriptional repression of thaxtomin biosynthetic genes. Here, we investigated the role of scab57721, encoding a putative BG (BglC), in the onset of the pathogenicity of S. scabies. Enzymatic assays showed that BglC was able to release glucose from cellobiose, cellotriose and all other cello-oligosaccharides tested. Its inactivation resulted in a phenotype opposite to that expected, as reduced production of thaxtomin was monitored when the mutant was cultivated on medium containing cello-oligosaccharides as unique carbon source. This unexpected phenotype could be attributed to the highly increased activity of alternative intracellular BGs, probably as a compensation for bglC inactivation, which then prevented cellobiose and cellotriose accumulation to reduce the activity of CebR. In contrast, when the bglC null mutant was cultivated on medium devoid of cello-oligosaccharides, it instead constitutively produced thaxtomin. This observed hypervirulent phenotype does not fit with the proposed model of the cello-oligosaccharide-mediated induction of thaxtomin production, and suggests that the role of BglC in the route to the pathogenic lifestyle of S. scabies is more complex than currently presented.

Tracking the Subtle Mutations Driving Host Sensing by the Plant Pathogen Streptomyces scabies.

The acquisition of genetic material conferring the arsenal necessary for host virulence is a prerequisite on the path to becoming a plant pathogen. More subtle mutations are also required for the perception of cues signifying the presence of the target host and optimal conditions for colonization. The decision to activate the pathogenic lifestyle is not "taken lightly" and involves efficient systems monitoring environmental conditions. But how can a pathogen trigger the expression of virulence genes in a timely manner if the main signal inducing its pathogenic behavior originates from cellulose, the most abundant polysaccharide on earth? This situation is encountered by Streptomyces scabies, which is responsible for common scab disease on tuber and root crops. We propose here a series of hypotheses of how S. scabies could optimally distinguish whether cello-oligosaccharides originate from decomposing lignocellulose (nutrient sources, saprophyte) or, instead, emanate from living and expanding plant tissue (virulence signals, pathogen) and accordingly adapt its physiological response.