The beta-glucosidase BglC fulfills multiple functions in both primary metabolism and induction of pathogenicity of Streptomyces scabiei, the causative agent of common scab in root and tuber crops. ...Indeed, this enzyme hydrolyzes cellobiose and cellotriose to feed glycolysis with glucose directly and modifies the intracellular concentration of these cello-oligosaccharides, which are the virulence elicitors. The inactivation of bglC led to unexpected phenotypes such as the constitutive overproduction of thaxtomin A, the main virulence determinant of S. scabiei. In this work, we reveal a new target substrate of BglC, the phytoalexin scopolin. Removal of the glucose moiety of scopolin generates scopoletin, a potent inhibitor of thaxtomin A production. The hydrolysis of scopolin by BglC displayed substrate inhibition kinetics, which contrasts with the typical Michaelis–Menten saturation curve previously observed for the degradation of its natural substrate cellobiose. Our work, therefore, reveals that BglC targets both cello-oligosaccharide elicitors emanating from the hosts of S. scabiei, and the scopolin phytoalexin generated by the host defense mechanisms, thereby occupying a key position to fine-tune the production of the main virulence determinant thaxtomin A.
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a label-free technique, producing images where pixels contain mass spectra. The technique allows the ...visualization of the spatial distribution of (bio)molecules from metabolites to proteins, on surfaces such as tissues sections or bacteria culture media. One particularly exciting example of MALDI-MSI use rests on its potential to localize ionized compounds produced during microbial interactions and chemical communication, offering a molecular snapshot of metabolomes at a given time. The huge size and the complexity of generated MSI data make the processing of the data challenging, which requires the use of computational methods. Despite recent advances, currently available commercial software relies mainly on statistical tools to identify patterns, similarities, and differences within data sets. However, grouping m/z values unique to a given data set according to microbiological contexts, such as coculture experiments, still requires tedious manual analysis. Here we propose a nontargeted method exploiting the differential signals between negative controls and tested experimental conditions, i.e., differential signal filtering (DSF), and a scoring of the ion images using image structure filtering (ISF) coupled with a fold change score between the controls and the conditions of interest. These methods were first applied to coculture experiments involving Escherichia coli and Streptomyces coelicolor, revealing specific MS signals during bacterial interaction. Two case studies were also investigated: (i) cellobiose-mediated induction for the pathogenicity of Streptomyces scabiei, the causative agent of common scab on root and tuber crops, and (ii) iron-repressed production of siderophores of S. scabiei. This report proposes guidelines for MALDI-MSI data treatment applied in the case of microbiology contexts, with enhanced ion peak annotation in specific culture conditions. The strengths and weaknesses of the methods are discussed.
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.
Members of the soil‐dwelling prokaryotic genus Streptomyces produce many secondary metabolites, including antibiotics and anti‐tumour agents. Their formation is coupled with the onset of development, ...which is triggered by the nutrient status of the habitat. We propose the first complete signalling cascade from nutrient sensing to development and antibiotic biosynthesis. We show that a high concentration of N‐acetylglucosamine—perhaps mimicking the accumulation of N‐acetylglucosamine after autolytic degradation of the vegetative mycelium—is a major checkpoint for the onset of secondary metabolism. The response is transmitted to antibiotic pathway‐specific activators through the pleiotropic transcriptional repressor DasR, the regulon of which also includes all N‐acetylglucosamine‐related catabolic genes. The results allowed us to devise a new strategy for activating pathways for secondary metabolite biosynthesis. Such ‘cryptic’ pathways are abundant in actinomycete genomes, thereby offering new prospects in the fight against multiple drug‐resistant pathogens and cancers.
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
-vinylphenol. Conversely, when iron is abundantly available, the biosynthetic pathway additionally produces a molecule based on
-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.
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.
Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO
) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building ...processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating
, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO
deposition. The metabolic activities involved in CaCO
transformation were furthermore assessed
among the collection of moonmilk
, which revealed that peptides/amino acids ammonification, and to a lesser extend ureolysis, could be privileged metabolic pathways participating in carbonate precipitation by increasing the pH of the bacterial environment. Additionally,
search for the genes involved in biomineralization processes including ureolysis, dissimilatory nitrate reduction to ammonia, active calcium ion transport, and reversible hydration of CO
allowed to identify genetic predispositions for carbonate precipitation in
. Finally, their biomineralization abilities were confirmed by environmental SEM, which allowed to visualize the formation of abundant mineral deposits under laboratory conditions. Overall, our study provides novel evidences that filamentous Actinobacteria could be key protagonists in the genesis of moonmilk through a wide spectrum of biomineralization processes.
A relatively small number of species in the large genus Streptomyces are pathogenic; the best characterized of these is Streptomyces scabies. The pathogenicity of S. scabies strains is dependent on ...the production of the nitrated diketopiperazine thaxtomin A, which is a potent plant cellulose synthesis inhibitor. Much is known about the genetic loci associated with plant virulence; however, the molecular mechanisms by which S. scabies triggers expression of thaxtomin biosynthetic genes, beyond the pathway-specific activator TxtR, are not well understood. In this study, we demonstrate that binding sites for the cellulose utilization repressor CebR occur and function within the thaxtomin biosynthetic cluster. This was an unexpected result, as CebR is devoted to primary metabolism and nutritive functions in nonpathogenic streptomycetes. In S. scabies, cellobiose and cellotriose inhibit the DNA-binding ability of CebR, leading to an increased expression of the thaxtomin biosynthetic and regulatory genes txtA, txtB, and txtR. Deletion of cebR results in constitutive thaxtomin A production and hypervirulence of S. scabies. The pathogenicity of S. scabies is thus under dual direct positive and negative transcriptional control where CebR is the cellobiose-sensing key that locks the expression of txtR, the key necessary to unlock the production of the phytotoxin. Interestingly, CebR-binding sites also lie upstream of and within the thaxtomin biosynthetic clusters in Streptomyces turgidiscabies and Streptomyces acidiscabies, suggesting that CebR is most likely an important regulator of virulence in these plant-pathogenic species as well.
What makes a microorganism pathogenic is not limited to the genes acquired for virulence. Using the main causative agent of scab lesions on root and tuber crops as an example, our work identified the subtle but essential genetic changes that generate the cis-acting elements necessary for proper timing of the expression of the cluster of genes responsible for the biosynthesis of thaxtomin A, the primary virulence factor in plant-pathogenic streptomycetes. These data illustrate a situation in which a regulator associated with primary metabolism in nonpathogens, CebR, has been coopted as a master regulator of virulence in pathogenic species. Furthermore, the manipulation of CebR-mediated control of thaxtomin production will facilitate overproduction of this natural and biodegradable herbicide for commercial purposes. Our work thus provides a concrete example of how a strictly theoretical and computational work was able to elucidate a regulatory mechanism associated with the virulence of a plant pathogen and to generate solutions to purely agro-industrial concerns.
Ferroverdins are ferrous iron (Fe2+)-nitrosophenolato complexes produced by a few Streptomyces species as a response to iron overload. Previously, three ferroverdins were identified: ferroverdin A, ...in which three molecules of p-vinylphenyl-3-nitroso-4-hydroxybenzoate (p-vinylphenyl-3,4-NHBA) are recruited to bind Fe2+, and Ferroverdin B and Ferroverdin C, in which one molecule of p-vinylphenyl-3,4-NHBA is substituted by hydroxy-p-vinylphenyl-3,4-NHBA, and by carboxy-p-vinylphenyl-3,4-NHBA, respectively. These molecules, especially ferroverdin B, are potent inhibitors of the human cholesteryl ester transfer protein (CETP) and therefore candidate hits for the development of drugs that increase the serum concentration of high-density lipoprotein cholesterol, thereby diminishing the risk of atherosclerotic cardiovascular disease. In this work, we used high-resolution mass spectrometry combined with tandem mass spectrometry to identify 43 novel ferroverdins from the cytosol of two Streptomyces lunaelactis species. For 13 of them (designated ferroverdins C2, C3, D, D2, D3, E, F, G, H, CD, DE, DF, and DG), we could elucidate their structure, and for the other 17 new ferroverdins, ambiguity remains for one of the three ligands. p-formylphenyl-3,4-NHBA, p-benzoic acid-3,4-NHBA, 3,4-NHBA, p-phenylpropionate-3,4-NHBA, and p-phenyacetate-3,4-NHBA were identified as new alternative chelators for Fe2+-binding, and two compounds (C3 and D3) are the first reported ferroverdins that do not recruit p-vinylphenyl-3,4-NHBA. Our work thus uncovered putative novel CETP inhibitors or ferroverdins with novel bioactivities.
Actinobacteria are prolific producers of antitumor antibiotics with antiproliferative activity, but why these bacteria synthetize metabolites with this bioactivity has so far remained a mystery. In ...this work we raised the hypothesis that under certain circumstances, production of antiproliferative agents could be part of a genetically programmed death of the producing organism. While programmed cell death (PCD) has been well documented when
species switch from vegetative (nutrition) to aerial (reproduction) growth, lethal determinants are yet to be discovered. Using DNA-damaging prodiginines of
as model system, we revealed that, under certain conditions, their biosynthesis is always triggered in the dying zone of the mycelial network prior to morphological differentiation, right after an initial round of cell death. The programmed massive death round of the vegetative mycelium is absent in a prodiginine non-producer (Δ
strain), and mutant complementation restored both prodiginine production and cell death. The
null mutant of
also showed increased DNA, RNA, and proteins synthesis when most of the mycelium of the wild-type strain was dead when prodiginines accumulated. Moreover, addition of the prodiginine synthesis inhibitors also resulted in enhanced accumulation of viable filaments. Overall, our data enable us to propose a model where the time-space production of prodiginines is programmed to be triggered by the perception of dead cells, and their biosynthesis further amplifies the PCD process. As prodiginine production coincides with the moment
undergoes morphogenesis, the production of these lethal compounds might be used to eradicate the obsolete part of the population in order to provide nutrients for development of the survivors. Hence, next to weapons in competition between organisms or signals in inter- and intra-species communications, we propose a third role for antibiotics (in the literal meaning of the word 'against life') i.e., elements involved in self-toxicity in order to control cell proliferation, and/or for PCD associated with developmental processes.
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