Secondary Metabolism Responses Research Articles

De novo assembly and Transcriptome Analysis of the Momordica charantia Seedlings Responding to methyl jasmonate using 454 pyrosequencing

Momordica charantia, a medicinal and edible species of the Cucurbitaceae family, has been widely used as a vegetable around the world. Hundreds of pharmacological compounds isolated from the M. charantia have been reported. However, the mechanism of action of the secondary metabolites has not been fully elucidated. In this study, 118,590 unigenes were gained by de novo assembly based on the raw data from high-throughput sequencing of mRNA (RNA-Sequencing) upon systemic analysis, among which, 51,860 (43.73%) could be annotated to the public sequence databases such as Nr, GO, Swiss-Prot, KEGG and KOG. The transcriptomic changes of M. charantia seedlings treated with or without methyl jasmonate (MeJA) were analyzed to identify key genes involved in MeJA treatment. Additionally, 554 differentially expressed genes (DEGs), including 328 up-regulated ones and 226 down-regulated genes, have been identified. Most DEGs were associated with secondary metabolism and stress responses. Meanwhile, six DEGs were further confirmed by quantitative real-time RT-PCR (qRT-PCR) analysis, resulting in similar expression patterns as compared to those of RNA-Sequencing. Nine significantly enriched pathways including 11 DEGs were identified to be possibly involved in the MeJA-responsive biosynthesis of secondary metabolites based on the transcriptome sequencing analysis. Among them, 4 DEGs, encoding two peroxidases, one cinnamyl alcohol dehydrogenase and one hypothetical protein Csa, might play important roles in the process of phenylpropanoid biosynthesis. In addition, 9 transcription factors (TFs) were also detected as DEGs from 1899 unigenes. Most of them up-regulated by MeJA treatment might be potentially involved in regulating secondary metabolites biosynthesis. This work is the first research on the large-scale assessment of M. charantia transcriptomic resources and the analysis of DEGs and TFs in secondary metabolites biosynthesis of M. charantia seedings treated with or without MeJA, which will be conducive to the further applications of M. charantia.

Photosynthetic signalling during high light stress and recovery: targets and dynamics.

The photosynthetic apparatus is one of the major primary sensors of the plant's external environment. Changes in environmental conditions affect the balance between harvested light energy and the capacity to deal with excited electrons in the stroma, which alters the redox homeostasis of the photosynthetic electron transport chain. Disturbances to redox balance activate photosynthetic regulation mechanisms and trigger signalling cascades that can modify the transcription of nuclear genes. H2O2 and oxylipins have been identified as especially prominent regulators of gene expression in response to excess light stress. This paper explores the hypothesis that photosynthetic imbalance triggers specific signals that target discrete gene profiles and biological processes. Analysis of the major retrograde signalling pathways engaged during high light stress and recovery demonstrates both specificity and overlap in gene targets. This work reveals distinct, time-resolved profiles of gene expression that suggest a regulatory interaction between rapidly activated abiotic stress response and induction of secondary metabolism and detoxification processes during recovery. The findings of this study show that photosynthetic electron transport provides a finely tuned sensor for detecting and responding to the environment through chloroplast retrograde signalling. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Cover Feature: Cryptic Chemical Communication: Secondary Metabolic Responses Revealed by Microbial Co‐culture (Chem. Asian J. 3/2020)

Cover Feature: Cryptic Chemical Communication: Secondary Metabolic Responses Revealed by Microbial Co‐culture (Chem. Asian J. 3/2020)

Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to Fusarium graminearum in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum-resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

Cryptic Chemical Communication: Secondary Metabolic Responses Revealed by Microbial Co-culture.

Microbial secondary metabolites (SMs) have long been viewed as a significant source of novel pharmaceutical and agrochemical molecules. With the increasing availability of genomic data, numerous biosynthetic gene clusters (BGCs) have been discovered. Despite the presence of tens of thousands of BGCs that can theoretically produce extremely diverse SMs, many gene clusters remain in a silent state under axenic culture conditions. Co-culture is a promising research approach as it stimulates the expression of cryptic BGCs to produce novel metabolites and also mimics natural interspecies interactions in a laboratory environment. In recent years, the roles of SMs in microbial communication have caught the attention of researchers and our understanding of microbes and their production of remarkable SMs has improved. SMs may be extensively involved in a variety of communication events among microorganisms. We herein summarize certain representative findings in the field of chemical communication involving SMs in co-culture systems.

Gene expression profile indicates involvement of uniconazole in Coix lachryma-jobi L. seedlings at low temperature.

Uniconazole (UNZ) can alleviate a variety of abiotic stresses such as low temperature. With application of UNZ on Coix lachryma‐jobi L. (coix) under low‐temperature stress, growth and physiological parameters were investigated in seedlings. Meanwhile, transcriptome profile in coix seedlings was characterized as well. The results showed an increase of 11.90%, 13.59%, and 10.98% in stem diameter, the aboveground and belowground biomass in 5 mg/L uniconazole application group (U3), compared with control check low‐temperature group (CKL). Some anti‐oxidase activities also show significant difference between CKL and U3 (p < .05). Transcriptome results showed that 3,901 and 1,040 genes had different expression level at control check (CK) and CKL, CKL and U3. A considerable number of different expressing genes (DEGs) related to the plant hormone signal transduction, photosynthesis, reactive oxygen species (ROS)‐related genes, and secondary metabolism in response to uniconazole application were identified in this study. The transcriptomic gene expression profiles present a valuable genomic tool to improve studying the molecular mechanisms underlying low‐temperature tolerance in coix. At the same time, it would provide a certain basis for the application of UNZ in the production of coix resistance under low temperature.

Response of Carbon and Nitrogen Metabolism and Secondary Metabolites to Drought Stress and Salt Stress in Plants

Carbon and nitrogen metabolism provide the main energy and basic nutrients for plants. However, environmental stress seriously affects carbon and nitrogen metabolism and thus hinders plant growth, especially drought stress and salt stress. Hence, numerous studies have been conducted to investigate the response of carbon and nitrogen metabolism to drought stress and salt stress by photosynthesis, sucrose and starch metabolism, nitrogen uptake and amino acids. Previous researchers also studied the response of secondary metabolism under both stresses on account of secondary metabolism may confer protection against environmental stresses. Our review highlights the diverse responses of carbon and nitrogen metabolism to drought stress and salt stress and the content changes of three secondary metabolites in plants under stresses.

The Regulation of Plant Secondary Metabolism in Response to Abiotic Stress: Interactions Between Heat Shock and Elevated CO2.

Future climate change is set to have an impact on the physiological performance of global vegetation. Increasing temperature and atmospheric CO2 concentration will affect plant growth, net primary productivity, photosynthetic capability, and other biochemical functions that are essential for normal metabolic function. Alongside the primary metabolic function effects of plant growth and development, the effect of stress on plant secondary metabolism from both biotic and abiotic sources will be impacted by changes in future climate. Using an untargeted metabolomic fingerprinting approach alongside emissions measurements, we investigate for the first time how elevated atmospheric CO2 and temperature both independently and interactively impact on plant secondary metabolism through resource allocation, with a resulting “trade-off” between secondary metabolic processes in Salix spp. and in particular, isoprene biosynthesis. Although it has been previously reported that isoprene is suppressed in times of elevated CO2, and that isoprene emissions increase as a response to short-term heat shock, no study has investigated the interactive effects at the metabolic level. We have demonstrated that at a metabolic level isoprene is still being produced during periods of both elevated CO2 and temperature, and that ultimately temperature has the greater effect. With global temperature and atmospheric CO2 concentrations rising as a result of anthropogenic activity, it is imperative to understand the interactions between atmospheric processes and global vegetation, especially given that global isoprene emissions have the potential to contribute to atmospheric warming mitigation.

Proteomics of Rice-Magnaporthe oryzae Interaction: What Have We Learned So Far?

Rice blast disease, caused by Magnaporthe oryzae, is one of the major constraints to rice production, which feeds half of the world’s population. Proteomic technologies have been used as effective tools in plant−pathogen interactions to study the biological pathways involved in pathogen infection, plant response, and disease progression. Advancements in mass spectrometry (MS) and apoplastic and plasma membrane protein isolation methods facilitated the identification and quantification of subcellular proteomes during plant-pathogen interaction. Proteomic studies conducted during rice−M. oryzae interaction have led to the identification of several proteins eminently involved in pathogen perception, signal transduction, and the adjustment of metabolism to prevent plant disease. Some of these proteins include receptor-like kinases (RLKs), mitogen-activated protein kinases (MAPKs), and proteins related to reactive oxygen species (ROS) signaling and scavenging, hormone signaling, photosynthesis, secondary metabolism, protein degradation, and other defense responses. Moreover, post−translational modifications (PTMs), such as phosphoproteomics and ubiquitin proteomics, during rice−M. oryzae interaction are also summarized in this review. In essence, proteomic studies carried out to date delineated the molecular mechanisms underlying rice-M. oryzae interactions and provided candidate proteins for the breeding of rice blast resistant cultivars.

Noninvasive Phenotyping of Plant-Pathogen Interaction: Consecutive In Situ Imaging of Fluorescing Pseudomonas syringae, Plant Phenolic Fluorescence, and Chlorophyll Fluorescence in Arabidopsis Leaves.

Plant–pathogen interactions have been widely studied, but mostly from the site of the plant secondary defense. Less is known about the effects of pathogen infection on plant primary metabolism. The possibility to transform a fluorescing protein into prokaryotes is a promising phenotyping tool to follow a bacterial infection in plants in a noninvasive manner. In the present study, virulent and avirulent Pseudomonas syringae strains were transformed with green fluorescent protein (GFP) to follow the spread of bacteria in vivo by imaging Pulse-Amplitude-Modulation (PAM) fluorescence and conventional binocular microscopy. The combination of various wavelengths and filters allowed simultaneous detection of GFP-transformed bacteria, PAM chlorophyll fluorescence, and phenolic fluorescence from pathogen-infected plant leaves. The results show that fluorescence imaging allows spatiotemporal monitoring of pathogen spread as well as phenolic and chlorophyll fluorescence in situ, thus providing a novel means to study complex plant–pathogen interactions and relate the responses of primary and secondary metabolism to pathogen spread and multiplication. The study establishes a deeper understanding of imaging data and their implementation into disease screening.

Functional activation of a novel R2R3-MYB protein gene, GmMYB68, confers salt-alkali resistance in soybean (Glycine max L.).

Soil salinity significantly reduces soybean (Glycine max L.) production worldwide. Plants resistance to stress conditions is a complex characteristic regulated by multiple signaling pathways. The v-Myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor (TF) plays a crucial role in plant development, secondary metabolism, and abiotic stress responses. GmMYB68-overexpression and RNA interference (RNAi) lines were established for examining the function of G. max GmMYB68 in plant responses to abiotic stresses. The predicted amino acid sequence of GmMYB68 was similar to that of R2R3-MYB proteins. Quantitative real-time PCR analysis revealed that GmMYB68 expression varied in response to abiotic stresses. GmMYB68-overexpression lines showed enhanced resistance to salt and alkali stresses and their osmotic adjustment and photosynthetic rates were also stronger than that of GmMYB68-RNAi and wild type plants. Although wild type and transgenic plants showed no significant differences in agronomic traits under normal conditions, the overexpression of GmMYB68 increased grain number and 100-grain weights under salt stress. Our study identified a valuable TF associated with stress response in soybean, as its overexpression might help improve salt and alkali tolerance in soybean and other crops.

Insights into VdCmr1‐mediated protection against high temperature stress and UV irradiation in Verticillium dahliae

The fungus Verticillium dahliae causes vascular wilt disease on more than 200 plant species worldwide. This fungus can survive for years in soil as melanized microsclerotia. We found that VdCmr1, a transcription factor, is required for the melanin production and increased survival following UV irradiation in V. dahliae but not for microsclerotia production or virulence. Here, we provided evidence how VdCmr1 protects against high temperature (HT) and UV irradiation in V. dahliae. The results indicate that VdCmr1 mediates entry to the diapause period in V. dahliae in response to HT and contributes to the expression of proteins to minimize protein misfolding and denaturation. VdCmr1 deletion results in the misregulation of DNA repair machinery, suggestive of reduced DNA repair capacity following UV irradiation and in correlation with the low survival rate of UV-treated VdCmr1 mutants. We discovered a putative VdCmr1-dependent gene cluster associated with secondary metabolism and stress responses. We also functionally characterized two VdCmr1-responsive genes participating in HT and UV response. These results shed further light on the roles of VdCmr1 in protection from HT or UV irradiation, and the additional insights into the mechanisms of this protection may be useful to exploit for more effective disease control.

The effects of a moderate grape temperature increase on berry secondary metabolites

Context and purpose of the study: Like in other wine producing regions around the world, Bordeaux vineyards already experience the effects of climate change. Recent trends as well as model outputs for the future strongly support an increase of average and extreme temperatures. For the maturation period, this increase will by far exceed mean atmospheric temperature increase, as the ripening period will occur earlier in hotter climatic conditions. Therefore, a detrimental secondary metabolism response is expected in grape berries, and of particular concern are the impacts on phenolics and aromas and aroma precursors. The effects of high temperatures on secondary metabolism control have been partly characterized for phenolics, however mostly in artificial growing conditions, while little is known with respect to aromas. A better understanding of how high temperatures influence grape berry secondary metabolites could help vineyard growers to adapt to climate change and maintain wine quality.Material and methods: A two-year field study was carried out in 2015 and 2016 in a vineyard in Bordeaux, France. Two treatments, heated (H) and control (C), were applied to two varieties, Cabernet-Sauvignon and Sauvignon blanc, from fruit-set to maturity. Field heating was achieved by a very local greenhouse effect applied to the bottom of the rows, by enclosing most of the underlying soil surface by polycarbonate shields. As the training system was vertically trellised, the heated volume surrounded most of the bunches but did not disturb most of the leaves in the canopy. This simple and robust setup allowed an increase of berry temperature by about +1.5°C in mean value, up to +5°C at times during clear sky days. This moderate increase of temperature was indicative of the predicted future climatic conditions for the mid-21st century. Berry samples were collected at 4 time points from bunch closure to maturity for each cultivar and treatment. Primary and secondary metabolites were measured in whole berries or skins.Results and conclusions: With this moderate temperature increase, primary metabolite content in berries did not change significantly. In H samples, anthocyanins were reduced and tannins increased before veraison, and both decreased thereafter. H samples also exhibited lower concentrations of some amino acids, especially alanine, serine and phenylalanine. IBMP (2-methoxy-3-isobutylpyrazine) concentrations were also reduced in H samples of Cabernet-Sauvignon, in both seasons, especially at bunch closure stage, but the differences diminished at full maturity. For thiol 3-sulfanyl hexanol precursors, H samples again exhibited much lower concentrations for both varieties, with weak differences at early stages that increased at later stages (up to -70% decline at maturity in 2015 for Sauvignon blanc). These results demonstrate the potential negative impact of elevated temperature on polyphenols and aroma quality of grape berries.Significance and impact of the study: For viticulture to adapt to new climatic conditions, the negative impacts of high temperature on secondary metabolites and aromas, and therefore on wine quality, need to be contemplated. Thus, already established or new vineyard plantings must prepare and consider practices able to mitigate these impacts, for instance practices that increase bunch shading.

GCN5 modulates trichome initiation in Arabidopsis by manipulating histone acetylation of core trichome initiation regulator genes.

Histone acetyltransferase GCN5 affects trichome initiation via mediating the expression of some core trichome initiation regulator genes in Arabidopsis. GENERAL CONTROL NON-REPRESSED PROTEIN5 (GCN5), a histone acetyltransferase involved in the regulation of cell differentiation, organ development, secondary metabolism, and plant responses to abiotic stresses, has recently been shown to modulate trichome branching in Arabidopsis. Here, we provide evidence that GCN5 is also involved in the regulation of trichome initiation. We found that mutation of GCN5 led to increased leaf trichome density in Arabidopsis. Quantitative RT-PCR results showed that the expression of CPC, GL1, GL2, and GL3, four well-known core trichome initiation regulator genes, was decreased in the gcn5 mutants. ChIP assays indicated that these four trichome initiation regulator genes are direct targets of GCN5. Consistent with these results, GCN5-mediated H3K14/K9 acetylation levels on the TSS regions of these genes were decreased. On the other hand, leaf trichome density was reduced in plants overexpressing GCN5, and both the transcript levels and GCN5-binding enrichments of CPC, GL1, GL2, and GL3 genes were elevated. Taken together, these data suggests that GCN5 affects trichome initiation by modulating the transcription activities of trichome initiation regulator genes via H3K9/14 acetylation.

Aspergillus nidulans in the post-genomic era: a top-model filamentous fungus for the study of signaling and homeostasis mechanisms

The accessibility to next-generation sequencing (NGS) techniques has enabled the sequencing of hundreds of genomes of species representing all kingdoms. In the case of fungi, genomes of more than a thousand of species are publicly available. This is far from covering the number of 2.2-3.8 million fungal species estimated to populate the world but has significantly improved the resolution of the fungal tree of life. Furthermore, it has boosted systematic evolutionary analyses, the development of faster and more accurate diagnostic analyses of pathogenic strains or the improvement of several biotechnological processes. Nevertheless, the diversification of the nature of fungal species used as model has also weakened research in other species that were traditionally used as reference in the pre-genomic era. In this context, and after more than 65years since the first works published by Pontecorvo, Aspergillus nidulans remains as one of the most referential model filamentous fungus in research fields such as hyphal morphogenesis, intracellular transport, developmental programs, secondary metabolism, or stress response. This mini-review summarizes how A. nidulans has contributed to the progress in these fields during the last years, and discusses how it could contribute in the future, assisted by NGS and new-generation molecular, microscopy, or cellular tools.

Climatic factors control the geospatial distribution of active ingredients in Salvia miltiorrhiza Bunge in China

Climate change profoundly influences the geospatial distribution of secondary metabolites and causes the geographical migration of plants. We planted seedlings of the same species in eighteen ecological regions along a latitudinal gradient in eastern and western China, in order to explore the regulation of multi-climatic factors on active ingredient accumulation in Salvia miltiorrhiza Bunge. The correlations between six active ingredient contents and ten climatic factors were investigated to clarify their relationships. We found that climatic factors not only regulated active ingredient contents but also markedly influenced their composition and led to a specific geospatial distribution of these active ingredients in China. The main climatic factors include the air temperature, precipitation, atmospheric vapour pressure and sunshine duration. Future warming in high-latitude regions could cause continued northward expansion of planting areas suitable for S. miltiorrhiza. The effect of extreme climatic conditions on active ingredients should not be overlooked. The findings of this study can help farmers scientifically choose suitable cultivation regions in the future. Furthermore, this study provides an innovative idea for the exploration of secondary metabolic responses to changing ecological factors in medicinal plants.

Effects of acute air exposure on the hematological characteristics and physiological stress response of olive flounder (Paralichthys olivaceus) and Japanese croaker (Nibea japonica)

We examined the effect of acute air exposure on the hematological characteristics and physiological stress responses of the olive flounder (Paralichthys olivaceus) and Japanese croaker (Nibea japonica). Fish were exposed to air for 30 s (30S), 90 s (90S), and 180 s (180S). The control group was not exposed to air after capture. Experiments were performed in triplicate. The water exchange rate was 32 times the water volume of the tank and dissolved oxygen was maintained at over 5 mg/L. After air exposure (AE), fish were kept in the experimental tanks for 24 h. Blood samples were taken from the fish at 0 h (before air exposure) and at 1, 6, and 24 h AE. We found that the olive flounders and the Japanese croakers differed in their primary (hormonal) and secondary (hydromineral and metabolic) responses to the air exposure. Cortisol levels were lower in the olive flounders than in the Japanese croakers. In the 180S group, the olive flounder cortisol levels were increased by 62-fold whereas the Japanese croaker cortisol levels were increased by 75-fold. The cortisol levels increases were associated with elevated glucose levels in both olive flounders and Japanese croakers. Olive flounders and Japanese croakers (except in 180S) had increased glucose levels that recovered to a stable level or showed a decreasing trend until 24 h AE. Several studies have reported on the resilience of flounders to short-term stress. This was confirmed in the present study by the fast recovery of elevated cortisol, glucose, and lactic acid levels that were induced by the stressor. Compared to the stress response in the olive flounder, the Japanese croaker exhibited a stronger response associated with the cortisol, glucose, and lactic acid levels

IdeR, a DtxR Family Iron Response Regulator, Controls Iron Homeostasis, Morphological Differentiation, Secondary Metabolism, and the Oxidative Stress Response in Streptomyces avermitilis.

Iron, an essential element for microorganisms, functions as a vital cofactor in a wide variety of key metabolic processes. On the other hand, excess iron may have toxic effects on bacteria by catalyzing the formation of reactive oxygen species through the Fenton reaction. The prevention of iron toxicity requires the precise control of intracellular iron levels in bacteria. Mechanisms of iron homeostasis in the genus Streptomyces (the producers of various antibiotics) are poorly understood. Streptomyces avermitilis is the industrial producer of avermectins, which are potent anthelmintic agents widely used in medicine, agriculture, and animal husbandry. We investigated the regulatory role of IdeR, a DtxR family regulator, in S. avermitilis In the presence of iron, IdeR binds to a specific palindromic consensus sequence in promoters and regulates 14 targets involved in iron metabolism (e.g., iron acquisition, iron storage, heme metabolism, and Fe-S assembly). IdeR also directly regulates 12 targets involved in other biological processes, including morphological differentiation, secondary metabolism, carbohydrate metabolism, and the tricarboxylic acid (TCA) cycle. ideR transcription is positively regulated by the peroxide-sensing transcriptional regulator OxyR. A newly constructed ideR deletion mutant (DideR) was found to be less responsive to iron levels and more sensitive to H2O2 treatment than the wild-type strain, indicating that ideR is essential for oxidative stress responses. Our findings, taken together, demonstrate that IdeR plays a pleiotropic role in the overall coordination of metabolism in Streptomyces spp. in response to iron levels.IMPORTANCE Iron is essential to almost all organisms, but in the presence of oxygen, iron is both poorly available and potentially toxic. Streptomyces species are predominantly present in soil where the environment is complex and fluctuating. So far, the mechanism of iron homeostasis in Streptomyces spp. remains to be elucidated. Here, we characterized the regulatory role of IdeR in the avermectin-producing organism S. avermitilis IdeR maintains intracellular iron levels by regulating genes involved in iron absorption and storage. IdeR also directly regulates morphological differentiation, secondary metabolism, and central metabolism. ideR is under the positive control of OxyR and is indispensable for an efficient response to oxidative stress. This investigation uncovered that IdeR acts as a global regulator coordinating iron homeostasis, morphological differentiation, secondary metabolism, and oxidative stress response in Streptomyces species. Elucidation of the pleiotropic regulation function of IdeR provides new insights into the mechanisms of how Streptomyces spp. adapt to the complex environment.

Lysine propionylation modulates the transcriptional activity of phosphate regulator PhoP in Saccharopolyspora erythraea.

Phosphate concentration extensively modulates the central physiological processes mediated by the two-component system PhoR-PhoP in actinobacteria. The system serves a role beyond phosphate metabolism, mediating crucial functions in nitrogen and carbon metabolism, and secondary metabolism in response to the nutritional states. Here, we found that the phosphate-sensing regulator PhoP was propionylated, and thus lost its DNA-binding activity in vivo and in vitro in Saccharopolyspora erythraea. Two key conserved lysine residues 198 and 203 (K198 and K203) in winged HTH motif at the C-terminal domain of PhoP are propionylated by protein acyltransferase AcuA (encoding by sace_5148). Single amino acid mutation of these two lysine residues resulted in severely impaired binding of PhoP to PHO box. The addition of propionate (to supply precursors for erythromycin biosynthesis) increases the intracellular propionylation level of PhoP, resulting in the loss of response to phosphate availability. Furthermore, simultaneous mutation of K198 and K203 of PhoP to arginine, mimicking the non-propionylated form, promotes the expression of the PhoP regulon under the condition of propionate addition. Together, these findings present a common regulatory mechanism of genes' expression mediated by posttranslational regulation of OmpR family transcriptional regulator PhoP and provide new insights into the multifaceted regulation of metabolism in response to nutritional signals.

Response of Secondary Metabolism of Hypogean Actinobacterial Genera to Chemical and Biological Stimuli.

Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses under different stimulus conditions. Here, we surveyed the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics, with over 30% of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via nuclear magnetic resonance (NMR), revealing several known secondary metabolite families, including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate of Streptosporangium when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with Bacillus species. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, Streptosporangium sp. strain KDCAGE35, which facilitated a deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies.IMPORTANCE Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Here, we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low-abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.