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The microbial genome remains a huge treasure trove for the discovery of diverse natural products. Saccharopolyspora erythraea NRRL23338, the industry producer of erythromycin, has a dozen of biosynthetic gene clusters whose encoding products are unidentified. Heterologous expression of one of the polyketide clusters pks7 in Streptomyces albus B4 chassis resulted in the characterization of its function responsible for synthesizing both 6-methylsalicyclic acid and 6-ethylsalicyclic acid. Meanwhile, two new 6-ethylsalicyclic acid ester derivatives were isolated as shunt metabolites. Their structures were identified by comprehensive analysis of MS and NMR experiments. Putative functions of genes within the pks7 BGC were also discussed.

Abstract c-di-AMP is an essential and widespread nucleotide second messenger in bacterial signaling. For most c-di-AMP synthesizing organisms, c-di-AMP homeostasis and the molecular mechanisms pertaining to its signal transduction are of great concern. Here we show that c-di-AMP binds the N-acetylglucosamine (GlcNAc)-sensing regulator DasR, indicating a direct link between c-di-AMP and GlcNAc signaling. Beyond its foundational role in cell-surface structure, GlcNAc is attractive as a major nutrient and messenger molecule regulating multiple cellular processes from bacteria to humans. We show that increased c-di-AMP levels allosterically activate DasR as a master repressor of GlcNAc utilization, causing the shutdown of the DasR-mediated GlcNAc signaling cascade and leading to a consistent enhancement in the developmental transition and antibiotic production in Saccharopolyspora erythraea. The expression of disA, encoding diadenylate cyclase, is directly repressed by the regulator DasR in response to GlcNAc signaling, thus forming a self-sustaining transcriptional feedback loop for c-di-AMP synthesis. These findings shed light on the allosteric regulation by c-di-AMP, which appears to play a prominent role in global signal integration and c-di-AMP homeostasis in bacteria and is likely widespread in streptomycetes that produce c-di-AMP.

ABSTRACT Cyclic purine nucleotides are important signal transduction molecules across all domains of life. 3′,5′-cyclic di-adenosine monophosphate (c-di-AMP) has roles in both prokaryotes and eukaryotes, while the signals that adjust intracellular c-di-AMP and the molecular machinery enabling a network-wide homeostatic response remain largely unknown. Here, we present evidence for an acetyl phosphate (AcP)-governed network responsible for c-di-AMP homeostasis through two distinct substrates, the diadenylate cyclase DNA integrity scanning protein (DisA) and its newly identified transcriptional repressor, DasR. Correspondingly, we found that AcP-induced acetylation exerts these regulatory actions by disrupting protein multimerization, thus impairing c-di-AMP synthesis via K66 acetylation of DisA. Conversely, the transcriptional inhibition of disA was relieved during DasR acetylation at K78. These findings establish a pivotal physiological role for AcP as a mediator to balance c-di-AMP homeostasis. Further studies revealed that acetylated DisA and DasR undergo conformational changes that play crucial roles in differentiation. Considering the broad distribution of AcP-induced acetylation in response to environmental stress, as well as the high conservation of the identified key sites, we propose that this unique regulation of c-di-AMP homeostasis may constitute a fundamental property of central circuits in Actinobacteria and thus the global control of cellular physiology.IMPORTANCESince the identification of c-di-AMP is required for bacterial growth and cellular physiology, a major challenge is the cell signals and stimuli that feed into the decision-making process of c-di-AMP concentration and how that information is integrated into the regulatory pathways. Using the bacterium Saccharopolyspora erythraea as a model, we established that AcP-dependent acetylation of the diadenylate cyclase DisA and its newly identified transcriptional repressor DasR is involved in coordinating environmental and intracellular signals, which are crucial for c-di-AMP homeostasis. Specifically, DisA acetylated at K66 directly inactivates its diadenylate cyclase activity, hence the production of c-di-AMP, whereas DasR acetylation at K78 leads to increased disA expression and c-di-AMP levels. Thus, AcP represents an essential molecular switch in c-di-AMP maintenance, responding to environmental changes and possibly hampering efficient development. Therefore, AcP-mediated posttranslational processes constitute a network beyond the usual and well-characterized synthetase/hydrolase governing c-di-AMP homeostasis.

ABSTRACTInsulin resistance is the primary pathophysiological basis for metabolic syndrome and type 2 diabetes. Gut microbiota and microbiota-derived metabolites are pivotal in insulin resistance. However, identifying the specific microbes and key metabolites with causal roles is a challenging task, and the underlying mechanisms require further exploration. Here, we successfully constructed a model of insulin resistance in mice induced by a high-fat diet (HFD) and screened potential biomarkers associated with insulin resistance by integrating metagenomics and untargeted metabolomics. Our findings showed a significant increase in the abundance of 30 species of Alistipes in HFD mice compared to normal diet (ND) mice, while the abundance of Desulfovibrio and Candidatus Amulumruptor was significantly lower in HFD mice than in ND mice. Non-targeted metabolomics analysis identified 21 insulin resistance-associated metabolites, originating from the microbiota or co-metabolized by both the microbiota and the host. These metabolites were primarily enriched in aromatic amino acid metabolism (tryptophan metabolism, tyrosine metabolism, and phenylalanine metabolism) and arginine biosynthesis. Further analysis revealed a significant association between the three distinct genera and 21 differentiated metabolites in the HFD and ND mice. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of representative genomes from 12 species of the three distinct genera further revealed the functional potential in aromatic amino acid metabolism and arginine biosynthesis. This study lays the groundwork for future investigations into the mechanisms through which the gut microbiota and its metabolites impact insulin resistance.IMPORTANCEIn this study, we aim to identify the microbes and metabolites linked to insulin resistance, some of which have not been previously reported in insulin resistance-related studies. This adds a complementary dimension to existing research. Furthermore, we establish a correlation between alterations in the gut microbiota and metabolite levels. These findings serve as a foundation for identifying the causal bacterial species and metabolites. They also offer insights that guide further exploration into the mechanisms through which these factors influence host insulin resistance.

Adenosine-to-inosine (A-to-I) RNA editing is an important post-transcriptional modification mediated by the adenosine deaminases acting on RNA (ADAR) family of enzymes, expanding the transcriptome by altering selected nucleotides A to I in RNA molecules. Recently, A-to-I editing has been explored for correcting disease-causing mutations in RNA using therapeutic guide oligonucleotides to direct ADAR editing at specific sites. Humans have two active ADARs whose preferences and specificities are not well understood. To investigate their substrate specificity, we introduced hADAR1 and hADAR2, respectively, into Schizosaccharomyces pombe (S. pombe), which lacks endogenous ADARs, and evaluated their editing activities in vivo. Using transcriptome sequencing of S. pombe cultured at optimal growth temperature (30 °C), we identified 483 A-to-I high-confident editing sites for hADAR1 and 404 for hADAR2, compared with the non-editing wild-type control strain. However, these sites were mostly divergent between hADAR1 and hADAR2-expressing strains, sharing 33 common sites that are less than 9% for each strain. Their differential specificity for substrates was attributed to their differential preference for neighboring sequences of editing sites. We found that at the -3-position relative to the editing site, hADAR1 exhibits a tendency toward T, whereas hADAR2 leans toward A. Additionally, when varying the growth temperature for hADAR1- and hADAR2-expressing strains, we observed increased editing sites for them at both 20 and 35 °C, compared with them growing at 30 °C. However, we did not observe a significant shift in hADAR1 and hADAR2’s preference for neighboring sequences across three temperatures. The vast changes in RNA editing sites at lower and higher temperatures were also observed for hADAR2 previously in budding yeast, which was likely due to the influence of RNA folding at these different temperatures, among many other factors. We noticed examples of longer lengths of dsRNA around the editing sites that induced editing at 20 or 35 °C but were absent at the other two temperature conditions. We found genes’ functions can be greatly affected by editing of their transcripts, for which over 50% of RNA editing sites for both hADAR1 and hADAR2 in S. pombe were in coding sequences (CDS), with more than 60% of them resulting in amino acid changes in protein products. This study revealed the extensive differences in substrate selectivity between the two active human ADARS, i.e., ADAR1 and ADAR2, and provided novel insight when utilizing the two different enzymes for in vivo treatment of human genetic diseases using the RNA editing approach.

Abstract The di-2-ethylhexyl phthalate (DEHP) degrading strain LMB-7 was isolated from electronic waste soil. According to its biophysical/biochemical characteristics and 16S rRNA gene analysis, the strain was identified as Nocardia asteroides. Optimal pH and temperature for DEHP degradation were 8.0 and 30 °C, respectively, and DEHP removal reached 97.11% after cultivation for 24 h at an initial concentration of 400 mg/L. As degradation intermediates, di-butyl phthalates, mono-2-ethylhexyl phthalate and 2-ethylhexanol could be identified, and it could be confirmed that DEHP was completely degraded by strain LMB-7. To our knowledge, this is a new report of DEHP degradation by a strain of Nocardia asteroides, at rates higher than those reported to date. This finding provides a new way for DEHP elimination from environment.

RNA molecular chaperone S1 is acetylated and selectively recruits stress-responsive mRNAs to the ribosome during nitrogen starvation in E. coli, revealing a translation regulation mechanism for nutrient stress adaptation.

Abstract Background The disease caused by plant pathogenic bacteria in the production, transportation, and storage of many crops has brought huge losses to agricultural production. N-acylhomoserine lactonases (AHLases) can quench quorum-sensing (QS) by hydrolyzing acylhomoserine lactones (AHLs), which makes them the promising candidates for controlling infections of QS-dependent pathogenic bacteria. Although many AHLases have been isolated and considered as a potentially effective preventive and therapeutic agents for bacterial diseases, the intrinsically poor ambient stability has seriously restricted its application. Results Herein, we showed that a spheroid enzyme-based hybrid nanoflower (EHNF), AhlX@Ni3(PO4)2, can be easily synthesized, and it exhibited 10 times AHL (3OC8-HSL) degradation activity than that with free AhlX (a thermostable AHL lactonase). In addition, it showed intriguing stability even at the working concentration, and retained ~ 100% activity after incubation at room temperature (25 °C) for 40 days and approximately 80% activity after incubation at 60 °C for 48 h. Furthermore, it exhibited better organic solvent tolerance and long-term stability in a complicated ecological environment than that of AhlX. To reduce the cost and streamline production processes, CSA@Ni3(PO4)2, which was assembled from the crude supernatants of AhlX and Ni3(PO4)2, was synthesized. Both AhlX@Ni3(PO4)2 and CSA@Ni3(PO4)2 efficiently attenuated pathogenic bacterial infection. Conclusions In this study, we have developed N-acylhomoserine lactonase-based hybrid nanoflowers as a novel and efficient biocontrol reagent with significant control effect, outstanding environmental adaptability and tolerance. It was expected to overcome the bottlenecks of poor stability and limited environmental tolerance that have existed for over two decades and pioneered the practical application of EHNFs in the field of biological control. Graphical Abstract

Summary: Engineered microorganisms hold potential for disease diagnosis and treatment. Here, we present a protocol to engineer E. coli Nissle 1917 strain (iROBOT) using genome insertion and plasmid construction to diagnose, record, and ameliorate inflammatory bowel disease in mice. We describe steps for constructing and administering iROBOT, diagnosing and recording colitis, preparing samples, and analyzing fluorescence and base editing ratios of iROBOT. We detail a colitis ameliorating assay using the disease activity index, colon length, tissue pathological section, and cytokine analysis.For complete details of the use and execution of this protocol, please refer to Zou et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract Background Erythromycin A (Er A) has a broad antibacterial effect and is a source of erythromycin derivatives. Methylation of erythromycin C (Er C), catalyzed by S-adenosyl-methionine (SAM)-dependent O-methyltransferase EryG, is the key final step in Er A biosynthesis. Er A biosynthesis, including EryG production, is regulated by the phosphate response factor PhoP and the nitrogen response factor GlnR. However, the regulatory effect of these proteins upon S-adenosyl-methionine synthetase (MetK) production is unknown. Results In this study, we used bioinformatics approaches to identify metK (SACE_3900), which codes for S-adenosyl-methionine synthetase (MetK). Electrophoretic mobility shift assays (EMSAs) revealed that PhoP and GlnR directly interact with the promoter of metK, and quantitative PCR (RT-qPCR) confirmed that each protein positively regulated metK transcription. Moreover, intracellular SAM was increased upon overexpression of either phoP or glnR under phosphate or nitrogen limited conditions, respectively. Finally, both the production of Er A and the transformation ratio from Er C to Er A increased upon phoP overexpression, but surprisingly, not upon glnR overexpression. Conclusions Manipulating the phosphate and nitrogen response factors, PhoP and GlnR provides a novel strategy for increasing the yield of SAM and the production of Er A in Saccharopolyspora erythraea .

Summary: Because of the continued emergence of SARS-CoV-2 variants, there has been considerable interest in how to display multivalent antigens efficiently. Bacterial outer membrane vesicles (OMVs) can serve as an attractive vaccine delivery system because of their self-adjuvant properties and the ability to be decorated with antigens. Here we set up a bivalent antigen display platform based on engineered OMVs using mCherry and GFP and demonstrated that two different antigens of SARS-CoV-2 could be presented simultaneously in the lumen and on the surface of OMVs. Comparing immunogenicity, ClyA-NG06 fusion and the receptor-binding domain (RBD) of the spike protein in the OMV lumen elicited a stronger humoral response in mice than OMVs presenting either the ClyA-NG06 fusion or RBD alone. Taken together, we provided an efficient approach to display SARS-CoV-2 antigens in the lumen and on the surface of the same OMV and highlighted the potential of OMVs as general multi-antigen carriers.

Abstract Obesity and its related complications have become increasingly serious social problems that threaten people's lives. Lactic acid bacteria (LAB) have shown an effective influence on preventing obesity in animal experiments and human clinical trials, but its exact mechanism remains unclear. In this review, LAB and several of its metabolites are taken as the point of penetration. It is aimed to summarize the relationship between obesity and gut microbiota, the efficacy of LAB, and its potential mechanisms of preventing obesity and lipid metabolism. LAB intervention can regulate gut microbiota and improve intestinal permeability which is damaged by a high‐fat diet or lipopolysaccharide. Besides this, some metabolites produced by LAB can be involved in regulating host lipid metabolism‐related pathways. Short‐chain fatty acids, linoleic acid, and gamma‐aminobutyric acid, taken as representatives here, can participate in the regulation through AMP‐activated protein kinase or peroxisome proliferator‐activated receptor pathway and bowel–brain axis. Therefore, the ingestion of LAB may become a potential adjunctive treatment for obesity in future.

Arbutin is a hydroquinone glucoside and a natural product present in various plants. Arbutin potently inhibits melanin formation. This property has been exploited in whitening cosmetics and pharmaceuticals. Arbutin production relies mainly on chemical synthesis. The multi-step and complicated process can compromise product purity. With the increasing awareness of sustainable development, the current research direction prioritizes environment-friendly, biobased arbutin production. In this review, current strategies for arbutin production are critically reviewed, with a focus on plant extraction, chemical synthesis, biotransformation, and microbial fermentation. Furthermore, the bottlenecks and perspectives for future direction on arbutin biosynthesis are discussed.

Summary: Cyclic lipopeptide (CLP) antibiotics have a mechanism that causes membrane malfunction. Thus mechanisms of bacterial resistance to CLPs are thought to modify cell surfaces. However, we found that bacterial resistance to CLPs was strongly related to energy metabolism. Using polymyxin B (PB) as a model of CLPs, we showed that PB causes malfunction of respiration and serious depletion of ATP, contributing to PB-induced cell death and carbon starvation. Glucose addition could maintain the intracellular ATP level and reverse the carbon starvation response resulting from PB treatment. Another study revealed that glycolysis was stimulated by the presence of PB and glucose. The mechanism underlying glucose-enabled CLPs' resistance suggests that glucose could maintain the ATP level in PB-treated bacteria by enhancing glycolysis. Similar results were observed in Staphylococcus aureus, where daptomycin resistance was enhanced by glucose. These findings provide insight into the mode of action of CLPs and resistance to these antibiotics. : Multi-Drug Resistant Organisms; Molecular Mechanism of Behavior; Microbiology; Microbial Metabolism Subject Areas: Multi-Drug Resistant Organisms, Molecular Mechanism of Behavior, Microbiology, Microbial Metabolism

Abstract Background The choice of phosphate/nitrogen source and their concentrations have been shown to have great influences on antibiotic production. However, the underlying mechanisms responsible for this remain poorly understood. Results We show that nutrient-sensing regulator PhoP (phosphate regulator) binds to and upregulates most of genes (ery cluster genes) involved in erythromycin biosynthesis in Saccharopolyspora erythraea, resulting in increase of erythromycin yield. Furthermore, it was found that PhoP also directly interacted with the promoter region of bldD gene encoding an activator of erythromycin biosynthesis, and induced its transcription. Phosphate limitation and overexpression of phoP increased the transcript levels of ery genes to enhance the erythromycin production. The results are further supported by observation that an over-producing strain of S. erythraea expressed more PhoP than a wild-type strain. On the other hand, nitrogen signal exerts the regulatory effect on the erythromycin biosynthesis through GlnR negatively regulating the transcription of phoP gene. Conclusions These findings provide evidence that PhoP mediates the interplay between phosphate/nitrogen metabolism and secondary metabolism by integrating phosphate/nitrogen signals to modulate the erythromycin biosynthesis. Our study reveals a molecular mechanism underlying antibiotic production, and suggests new possibilities for designing metabolic engineering and fermentation optimization strategies for increasing antibiotics yield.

In this paper, an integrated system for specific adsorption of methyl red (MR) and sensitive detection of the residual amount of this dye in printing and dyeing wastewater is proposed. A molecularly imprinted polymer using MR as the template molecule (MR-MIP) was prepared for specific adsorption of MR from printing and dyeing wastewater. A solid-phase extraction column was filled with a paste of MR-MIP and cobalt-doped iron carbide nanoparticles as the working electrode to construct a highly sensitive sensor for MR detection. The distillation–precipitation approach was used to fabricate microspheres with molecularly imprinted surfaces using cellulose as the carbon source and methyl red (MR) as the template. Not only do the molecularly imprinted microspheres have a satisfactory adsorption performance for the template molecule MR, with a dynamic adsorption capacity up to 264.2 mg g−1, but also the electrochemical sensor built from the molecularly imprinted polymer and iron paste electrode, co-modified with nitrogen-doped carbonized iron nanoparticles, has great sensitivity for MR detection. It is very important to develop an integrated platform for dye treatment and residue detection in wastewater. This technique has a low detection limit and a broad linear range compared to other approaches.

Natural polyketides play important roles in clinical treatment, agriculture, and animal husbandry. Compared to natural hosts, heterologous chassis (especially Actinomycetes) have many advantages in production of polyketide compounds. As a widely studied model Actinomycete, Saccharopolyspora erythraea is an excellent host to produce valuable heterologous polyketide compounds. However, many host factors affect the expression efficiency of heterologous genes, and it is necessary to modify the host to adapt heterologous production. In this study, the CRISPR-Cas9 system was used to knock out the erythromycin biosynthesis gene cluster of Ab (erythromycin high producing stain). A fragment of 49491 bp in genome (from SACE_0715 to SACE_0733) was deleted, generating the recombinant strain AbΔery in which erythromycin synthesis was blocked and synthetic substrates methylmalonyl-CoA and propionyl-CoA accumulated enormously. Based on AbΔery as heterologous host, three genes, AsCHS, RgTAL, and Sc4CL, driven by strong promoters Pj23119, PermE, and PkasO, respectively, were introduced to produce novel polyketide by L-tyrosine and methylmalonyl-CoA. The product (E)-4-hydroxy-6-(4-hydroxystyryl)-3,5-dimethyl-2H-pyrone was identified in fermentation by LC-MS. High performance liquid chromatography analysis showed that knocking out ery BGC resulted in an increase of methylmalonyl-CoA by 142% and propionyl-CoA by 57.9% in AbΔery compared to WT, and the yield of heterologous product in AbΔery:AsCHS-RgTAL-Sc4CL was higher than WT:AsCHS-RgTAL-Sc4CL. In summary, this study showed that AbΔery could potentially serve as a precious heterologous host to boost the synthesis of other valuable polyketone compounds using methylmalonyl-CoA and propionyl-CoA in the future.

Abstract Many Lactobacillus plantarum strains can secrete some antimicrobial substances and be added to food as antimicrobial agents and preservatives. In this study, three L. plantarum strains (P1, S11, and M7) with strong antimicrobial activity against three pathogenic bacteria were isolated from Xinjiang traditional dairy products. Five common organic acids produced by fermentation of strains play a key role in inhibiting three pathogenic bacteria. At the same pH, the antimicrobial activity of the fermentation broth against Escherichia coli and Salmonella is stronger than that of the organic acid alone. Thus, three kinds of antimicrobial agents (P1‐1, M7‐1, and S11‐1) mixed with five common organic acids were produced. Moreover, the antimicrobial activity against Salmonella ASI.1174 of the antimicrobial agents was about 30% higher than that of the fermentation broth. In addition, organic acid antimicrobial agents combined in different proportions can inhibit different pathogenic bacteria. According to this result, it is a potential approach to develop novel antimicrobial agents used in food preservation by mixing different organic acids.

Mycobacterium tuberculosis is a global human pathogen that infects macrophages and can establish a latent infection. Emerging evidence has established the nutrients metabolism as a key point to study the pathogenesis of M. tuberculosis and host immunity. It was reported that fatty acids and cholesterol are the major nutrient sources of M. tuberculosis in the period of infection. However, the mechanism by which M. tuberculosis utilizes lipids for maintaining life activities in nutrient-deficiency macrophages is poorly understood. Mycobacterium smegmatis is fast-growing and generally used to study its pathogenic counterpart, M. tuberculosis. In this work, we found that the phosphate sensing regulator RegX3 of M. smegmatis is required for its growing on propionate and surviving in macrophages. We further demonstrated that the expression of prpR and related genes (prpDBC) in methylcitrate cycle could be enhanced by RegX3 in response to the phosphate-starvation condition. The binding sites of the promoter region of prpR for RegX3 and PrpR were investigated. In addition, cell morphology assay showed that RegX3 is responsible for cell morphological elongation, thus promoting the proliferation and survival of M. smegmatis in macrophages. Taken together, our findings revealed a novel transcriptional regulation mechanism of RegX3 on propionate metabolism, and uncovered that the nutrients-sensing regulatory system puts bacteria at metabolic steady state by altering cell morphology. More importantly, since we observed that M. tuberculosis RegX3 also binds to the prpR operon in vitro, the RegX3-mediated regulation might be general in M. tuberculosis and other mycobacteria for nutrient sensing and environmental adaptation.

Mycobacterium smegmatis (Msm), along with its pathogenic counterpart Mycobacterium tuberculosis (Mtb), utilizes fatty acids and cholesterol as important carbon and energy sources during the persistence within host cells. As a dual-functional enzyme in the glyoxylate cycle and the methylcitrate cycle, isocitrate lyase (ICL, encoded by icl or MSMEG_0911) is indispensable for the growth of Msm and Mtb on short-chain fatty acids. However, regulation of icl in mycobacteria in response to nutrient availability remains largely unknown. Here, we report that the global nitrogen metabolism regulator GlnR represses icl expression by binding to an atypical binding motif in the icl promoter region under nitrogen-limiting conditions. We further show that GlnR competes with PrpR, a transcriptional activator of icl, and dominantly occupies the co-binding motif in the icl promoter region. In the absence of GlnR or in response to the excess nitrogen condition, Msm cells elongate and exhibit robust growth on short-chain fatty acids due to the PrpR-mediated activation of icl, thereby inducing enhanced apoptosis in infected macrophages. Taken together, our findings reveal the GlnR-mediated repression of icl on fatty acid metabolism, which might be a general strategy of nutrient sensing and environmental adaptation employed by mycobacteria.

Gut microbiota is increasingly recognized as a metabolic organ essential for human health. Compelling evidences show a variety set of links between diets and gut microbial homeostasis. Changes in gut microbial flora would probably contribute to the development of certain diseases such as diabetes, heart disease, allergy, and psychiatric diseases. In addition to the composition of gut microbiota, the metabolites derived from gut microbiota have emerged as a pivotal regulator in diseases development. Since high-fat and high-protein diets substantially affect the gut microbial ecology and human health, the current review summarizes the gut microbiota-derived metabolites such as short-chain fatty acids (SCFAs), amino acids, and their derivatives and highlights the mechanisms underlying the host responses to these bioactive substances.

Summary: The monomer-to-filament transition of MAVS is essential for the RIG-I/MDA5-mediated antiviral signaling. In quiescent cells, monomeric MAVS is under strict regulation for preventing its spontaneous aggregation, which would result in dysregulated interferon (IFN-α/β) production and autoimmune diseases like systemic lupus erythematosus. However, the detailed mechanism by which MAVS is kept from spontaneous aggregation remains largely unclear. Here, we show that upstream open reading frames (uORFs) within the MAVS transcripts exert a post-transcriptional regulation for preventing MAVS spontaneous aggregation and auto-activation. Mechanistically, we demonstrate that uORFs are cis-acting elements initiating leaky ribosome scanning of the downstream ORF codons, thereby repressing the full-length MAVS translation. We further uncover that endogenous MAVS generated from the uORF-deprived transcript spontaneously aggregates, triggering the Nix-mediated mitophagic clearance of damaged mitochondria and aggregated MAVS. Our findings reveal the uORF-mediated quantity and quality control of MAVS, which prevents aberrant protein aggregation and maintains innate immune homeostasis.

ABSTRACT Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. During chronic infection within host lung tissues, cholesterol facilitates the phagocytosis of mycobacteria into macrophages. Cholesterol degradation leads to increased flux of acetyl-coenzyme A (CoA) and propionyl-CoA, providing energy and building blocks for virulence macromolecules as well as donors for global protein acylation. Potential functions of lysine acylation are gradually revealed in bacterial survival and pathogenesis. However, the mycobacterial proteome and posttranslational modification (PTM) changes involved in the cholesterol catabolism bioprocess remain unclear. Here, we used nonpathogenic Mycobacterium smegmatis as a model and simultaneously monitored mycobacterial proteome and acetylome changes in the presence of glucose and cholesterol. We discovered that cholesterol metabolic enzymes were upregulated with respect to both protein expression levels and lysine acylation levels during the metabolic shift from glucose to cholesterol. After that, adenylating enzymes related to cholesterol metabolism were proven to be precisely regulated at the propionylation level by mycobacterial acyltransferase M. smegmatis Kat (MsKat) in response to cellular propionyl-CoA accumulation. Furthermore, the kinase expression and phosphorylation levels were also changed along with fluctuations in cholesterol levels. Our results expanded current knowledge of acylation regulation in the cholesterol catabolism of mycobacteria and provided references for possible antimycobacterium strategy. IMPORTANCE Cholesterol assimilation is a critical step in mycobacterial chronic infection. However, knowledge from the dynamic characterization of cholesterol metabolism in mycobacteria at the protein expression and PTM levels remains limited. Our study uncovered the landscape of protein expression, lysine acetylation, lysine propionylation, and S/T/Y phosphorylation during the metabolic changes from glucose to cholesterol in mycobacteria. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol also promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. This study systematically characterized the regulation of cholesterol catabolism at several different levels, which provided the detailed references in mycobacterial proteome and potential antimycobacterial strategies.

L-ornithine, a valuable non-protein amino acid, has a wide range of applications in the pharmaceutical and food industries. Currently, microbial fermentation is a promising, sustainable, and environment-friendly method to produce L-ornithine. However, the industrial production capacity of L-ornithine by microbial fermentation is low and rarely meets the market demands. Various strategies have been employed to improve the L-ornithine production titers in the model strain, Corynebacterium glutamicum, which serves as a major indicator for improving the cost-effectiveness of L-ornithine production by microbial fermentation. This review focuses on the development of high L-ornithine-producing strains by metabolic engineering and reviews the recent advances in breeding strategies, such as reducing by-product formation, improving the supplementation of precursor glutamate, releasing negative regulation and negative feedback inhibition, increasing the supply of intracellular cofactors, modulating the central metabolic pathway, enhancing the transport system, and adaptive evolution for improving L-ornithine production.

Abstract Background ʟ-Ornithine is an important amino acid with broad applications in pharmaceutical and food industries. Despite lagging ʟ-ornithine productivity and cost reduction, microbial fermentation is a promising route for sustainable ʟ-ornithine production and thus development of robust microbial strains with high stability and productivity is essential. Results Previously, we systematically developed a new strain, SO1 originate from Corynebacterium glutamicum S9114, for ʟ-ornithine production. In this work, overexpression of cg3035 encoding N-acetylglutamate synthase (NAGS) using a plasmid or by inserting a strong P tac promoter into the chromosome was found to increase ʟ-ornithine production in the engineered C. glutamicum SO1. The genome-based cg3035 modulated strain was further engineered by attenuating the expression of pta and cat, inserting a strong P eftu promoter in the upstream region of glycolytic enzymes such as pfkA, gap, and pyk, and redirecting carbon flux to the pentose phosphate pathway. The final strain with all the exploratory metabolic engineering manipulations produced 32.3 g/L of ʟ-ornithine, a yield of 0.395 g ornithine per g glucose, which was 35.7% higher than that produced by the original strain (23.8 g/L). Conclusion These results clearly demonstrated that enhancing the expression of NAGS promoted ʟ-ornithine production and provide a promising alternative systematic blueprint for developing ʟ-ornithine-producing C. glutamicum strains.

Abstract l-Ornithine, a non-essential amino acid, has enormous industrial applications in food, pharmaceutical, and chemical industries. Currently, l-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high l-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. l-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-l-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable l-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved l-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of l-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of l-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.

Glycated haemoglobin (HbA1c) is a diagnostic biomarker for type 2 diabetes. Traditional analytical methods for haemoglobin (Hb) detection rely on chromatography, which requires significant instrumentation and is labour-intensive; consequently, miniaturized devices that can rapidly sense HbA1c are urgently required. With this research, we report on an aptamer-based sensor (aptasensor) for the rapid and selective electrochemical detection of HbA1c. Aptamers that specifically bind HbA1c and Hb were modified with a sulfhydryl and ferrocene group at the 3′ and 5′-end, respectively. The modified aptamers were coated through sulfhydryl-gold self-assembly onto screen printed electrodes, producing aptasensors with built in electroactivity. When haemoglobin was added to the electrodes, the current intensity of the ferrocene in the sensor system was reduced in a concentration-dependent manner as determined by differential pulse voltammetry. In addition, electrochemical impedance spectroscopy confirmed selective binding of the analytes to the aptamer-coated electrode. This research offers new insight into the development of portable electrochemical sensors for the detection of HbA1c

This paper presents the rapid synthesis of a corncob-based semi-interpenetrating polymer network (semi-IPN) slow-release nitrogen fertilizer (SRFs) with bentonite additives via microwave irradiation at 320 W for 4.5 min. The SRFs were based on urea incorporated in a polymer matrix composed of corncob-g-poly(acrylic acid)/bentonite network and linear polyvinylpyrrolidone. The structure and properties of the sample were characterized. Swelling measurements and water-retention studies indicated that the water absorbency of the SRFs was 1156 g/g in distilled water and that the water-retention capacity of the soil with 2% SRFs was 20.3% after 30 days. In addition, the SRFs possessed lower N leaching loss amount (13.2%) and N migrate-to-surface loss amount (6.8%) compared with urea. The SRFs could effectively reduce the N release rate (56.6% of N was released after 30 days) and consequently facilitate the growth of cotton plants. Thus, the high-performance SRFs capable of controlling water and N losses could be widely applied to agricultural fields, and microwave irradiation could be a significant strategy to produce SRFs.

Abstract Background l-Ornithine is a non-protein amino acid with extensive applications in medicine and the food industry. Currently, l-ornithine production is based on microbial fermentation, and few microbes are used for producing l-ornithine owing to unsatisfactory production titer. Results In this study, Corynebacterium glutamicum S9114, a high glutamate-producing strain, was developed for l-ornithine production by pathway engineering. First, argF was deleted to block l-ornithine to citrulline conversion. To improve l-ornithine production, ncgl1221 encoding glutamate transporter, argR encoding arginine repressor, and putP encoding proline transporter were disrupted. This base strain was further engineered by attenuating oxoglutarate dehydrogenase to increase l-ornithine production. Plasmid-based overexpression of argCJBD operon and lysine/arginine transport protein LysE was tested to strengthen l-ornithine synthesis and transportation. This resulted in efficient l-ornithine production at a titer of 18.4 g/L. Conclusion These results demonstrate the potential of Corynebacterium glutamicum S9114 for efficient l-ornithine production and provide new targets for strain development.

WhiB3 is a conserved cytoplasmic redox sensor which is required in the infection and lipid anabolism of Mycobacterium tuberculosis. The response of WhiB3 to environmental nutrient and its regulatory cascades are crucial during the persistent infection, while little is known about the relationship between WhiB3 and emergence of nutrient stress in this process. Here, we found that nitrogen regulator GlnR directly interacted with the WhiB3 promoter region and activated its transcription in response to nitrogen availability. In whiB3 promoter region, the typical GlnR-box was also identified. Moreover, GlnR controlled cell resistance to redox stress and SL-1 lipid anabolism by directly activating whiB3 expression. These results demonstrated that GlnR regulated redox sensor WhiB3 at the transcriptional level and mediated the interplay among nitrogen metabolism, redox sensing, and lipid anabolism.

Assimilation of short-chain fatty acids (SCFAs) plays an important role in the survival and lipid biosynthesis of Mycobacteria. However, regulation of this process has not been thoroughly described. In the present work, we demonstrate that GlnR as a well-known nitrogen-sensing regulator transcriptionally modulates the AMP-forming propionyl-CoA synthetase (MsPrpE), and acetyl-CoA synthetases (MsAcs) is associated with SCFAs assimilation in Mycobacterium smegmatis, a model Mycobacterium. GlnR can directly activate the expression of MsprpE and Msacs by binding to their promoter regions based upon sensed nitrogen starvation in the host. Moreover, GlnR can activate the expression of lysine acetyltransferase encoding Mspat, which significantly decreases the activity of MsPrpE and MsAcs through increased acylation. Next, growth curves and resazurin assay show that GlnR can further regulate the growth of M. smegmatis on different SCFAs to control the viability. These results demonstrate that GlnR-mediated regulation of SCFA assimilation in response to the change of nitrogen signal serves to control the survival of M. smegmatis. These findings provide insights into the survival and nutrient utilization mechanisms of Mycobacteria in their host, which may enable new strategies in drug discovery for the control of tuberculosis.

In order to enhance the photodegradation of methyl orange (MO) by ZnO under visible light irradiation, ZnO nanoparticles co-doped with Ag and N and supported on activated carbon (AC) with different properties were synthesized through the sol-gel method. The prepared photocatalysts were characterized in terms of the structure and properties through X-ray diffraction, N2 adsorption-desorption, ultraviolet-visible (UV-vis), diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy, photoluminescence, and electron spin resonance. The photocatalytic activities of these photocatalysts followed the order: Ag-N-ZnO/ACs > Ag-N-ZnO > N, or Ag single-doped ZnO > commercial ZnO. This result was attributed to the small particle size, large surface area, narrow band gap, and high charge separation of Ag-N-ZnO/ACs. The Ag-N-ZnO/coconut husk activated carbon (Ag-N-ZnO/CHAC) exhibited the highest degradation efficiency of 98.82% for MO under visible light irradiation. This outcome was due to the abundant pore structure of Ag-N-ZnO/CHAC, resulting in stronger adsorption than that of other Ag-N-ZnO/ACs. Moreover, the degradation of MO on photocatalysis followed first order kinetics. The reactive species ·OH and ·O2− played more important roles in the photocatalytic degradation of MO over composite photocatalyst. Ag-N-ZnO/CHAC photocatalyst exhibited higher photocatalytic activity than unsupported Ag-N-ZnO after five recycling runs.

Protein-protein interactions between sigma factor σ(R) and its corresponding zinc-binding anti-sigma (ZAS) protein RsrA trigger the thioredoxin system for maintaining cellular redox homeostasis in S. coelicolor. RsrA bound to zinc associates with σ(R), inhibiting its transcriptional activity in a reducing environment. During disulfide stress it forms intramolecular disulfide bonds, leading to zinc release and dissociation from σ(R), which initiates transcription to produce reductase and thioredoxin. We designed a fluorescence resonance energy transfer (FRET) based system for monitoring protein-protein interactions between σ(R) and RsrA to further understand how this redox switch regulates the thioredoxin system in S. coelicolor in response to its redox environment, especially various reactive oxygen species (ROS) derived from different metabolic pathways, and clarify the different response mechanisms between Zn-RsrA and apo-RsrA. By the use of the FRET approach described here, we showed that zinc protected thiols in RsrA and causes the σ(R)-RsrA complex to form a more compact structure. This system was also utilized to detect changes in redox status induced by ROS and diamide in real time in E. coli cells.

Saccharopolyspora erythraea produces a large number of secondary metabolites with biological activities, including erythromycin. Elucidation of the mechanisms through which the production of these secondary metabolites is regulated may help to identify new strategies for improved biosynthesis of erythromycin. In this paper, we describe the systematic prediction and analysis of small non-coding RNAs (sRNAs) in S. erythraea, with the aim to elucidate sRNA-mediated regulation of secondary metabolite biosynthesis. In silico and deep-sequencing technologies were applied to predict sRNAs in S. erythraea. Six hundred and forty-seven potential sRNA loci were identified, of which 382 cis-encoded antisense RNA are complementary to protein-coding regions and 265 predicted transcripts are located in intergenic regions. Six candidate sRNAs (sernc292, sernc293, sernc350, sernc351, sernc361, and sernc389) belong to four gene clusters (tpc3, pke, pks6, and nrps5) that are involved in secondary metabolite biosynthesis. Deep-sequencing data showed that the expression of all sRNAs in the strain HL3168 E3 (E3) was higher than that in NRRL23338 (M), except for sernc292 and sernc361 expression. The relative expression of six sRNAs in strain M and E3 were validated by qRT-PCR at three different time points (24, 48, and 72 h). The results showed that, at each time point, the transcription levels of sernc293, sernc350, sernc351, and sernc389 were higher in E3 than in M, with the largest difference observed at 72 h, whereas no signals for sernc292 and sernc361 were detected. sernc293, sernc350, sernc351, and sernc389 probably regulate iron transport, terpene metabolism, geosmin synthesis, and polyketide biosynthesis, respectively. The major significance of this study is the successful prediction and identification of sRNAs in genomic regions close to the secondary metabolism-related genes in S. erythraea. A better understanding of the sRNA-target interaction would help to elucidate the complete range of functions of sRNAs in S. erythraea, including sRNA-mediated regulation of erythromycin biosynthesis.

Long-term use of antibiotics has engendered a large number of resistant pathogens, which pose a serious threat to human health. Here, we investigated the mechanism of fusaricidin antibacterial activity toward Bacillus subtilis and characterized the pathways responsible for drug resistance. We found that σ(w), an extracytoplasmic function sigma factor, plays an important role in the resistance to fusaricidins during the initial 5 minutes of drug addition. Approximately 18 genes were induced more than 3-fold, of which 66.7% are known to be regulated by σ(w). Over the following 3 h, fusaricidins induced 194 genes more than three-fold, and most were associated with classes of antibiotic-responsive stimulons. Moreover, the fusaricidin treatment increased the catabolism of fatty and amino acids but strongly repressed glucose decomposition and gluconeogenesis. In summary, our data provide insight into the mechanism of fusaricidin activity, on which we based our suggested strategies for the development of novel antibiotic agents.

Dissolved oxygen (DO) is an important factor for adenosine fermentation. Our previous experiments have shown that low oxygen supply in the growth period was optimal for high adenosine yield. Herein, to better understand the link between oxygen supply and adenosine productivity in B. subtilis (ATCC21616), we sought to systematically explore the effect of DO on genetic regulation and metabolism through transcriptome analysis. The microarrays representing 4,106 genes were used to study temporal transcript profiles of B. subtilis fermentation in response to high oxygen supply (agitation 700 r/min) and low oxygen supply (agitation 450 r/min). The transcriptome data analysis revealed that low oxygen supply has three major effects on metabolism: enhance carbon metabolism (glucose metabolism, pyruvate metabolism and carbon overflow), inhibit degradation of nitrogen sources (glutamate family amino acids and xanthine) and purine synthesis. Inhibition of xanthine degradation was the reason that low oxygen supply enhanced adenosine production. These provide us with potential targets, which can be modified to achieve higher adenosine yield. Expression of genes involved in energy, cell type differentiation, protein synthesis was also influenced by oxygen supply. These results provided new insights into the relationship between oxygen supply and metabolism.

Microorganisms can restructure their transcriptional output to adapt to environmental conditions by sensing endogenous metabolite pools. In this paper, an Agilent customized microarray representing 4,106 genes was used to study temporal transcript profiles of Bacillus subtilis in response to valine, glutamate and glutamine pulses over 24 h. A total of 673, 835, and 1135 amino-acid-regulated genes were identified having significantly changed expression at one or more time points in response to valine, glutamate, and glutamine, respectively, including genes involved in cell wall, cellular import, metabolism of amino-acids and nucleotides, transcriptional regulation, flagellar motility, chemotaxis, phage proteins, sporulation, and many genes of unknown function. Different amino acid treatments were compared in terms of both the global temporal profiles and the 5-minute quick regulations, and between-experiment differential genes were identified. The highlighted genes were analyzed based on diverse sources of gene functions using a variety of computational tools, including T-profiler analysis, and hierarchical clustering. The results revealed the common and distinct modes of action of these three amino acids, and should help to elucidate the specific signaling mechanism of each amino acid as an effector.