Antibiotic Biosynthesis Research Articles

New Insights into Pseudomonas spp.-Produced Antibiotics: Genetic Regulation of Biosynthesis and Implementation in Biotechnology.

Pseudomonas bacteria are renowned for their remarkable capacity to synthesize antibiotics, namely mupirocin, gluconic acid, pyrrolnitrin, and 2,4-diacetylphloroglucinol (DAPG). While these substances are extensively employed in agricultural biotechnology to safeguard plants against harmful bacteria and fungi, their potential for human medicine and healthcare remains highly promising for common science. However, the challenge of obtaining stable producers that yield higher quantities of these antibiotics continues to be a pertinent concern in modern biotechnology. Although the interest in antibiotics of Pseudomonas bacteria has persisted over the past century, many uncertainties still surround the regulation of the biosynthetic pathways of these compounds. Thus, the present review comprehensively studies the genetic organization and regulation of the biosynthesis of these antibiotics and provides a comprehensive summary of the genetic organization of antibiotic biosynthesis pathways in pseudomonas strains, appealing to both molecular biologists and biotechnologists. In addition, attention is also paid to the application of antibiotics in plant protection.

Transcriptome Analysis of Juvenile Black Rockfish Sebastes schlegelii under Air Exposure Stress

The study aimed to uncover the molecular response of juvenile Sebastes schlegelii to air exposure stress by identifying differentially expressed genes (DEGs) that may underlie their anti-stress mechanisms. Juvenile Sebastes schlegelii were subjected to varying durations of air exposure stress. The total RNA was extracted from whole tissues and sequenced using the Illumina NovaSeq 6000 platform. The transcriptome data were analyzed to identify DEGs through pairwise comparisons across a control group and two experimental groups exposed to air for 40 s and 2 min 30 s, respectively. The comparative DEG analysis revealed a significant number of transcripts responding to air exposure stress. Specifically, 5173 DEGs were identified in the 40 s exposure group (BS) compared to the control (BC), 6742 DEGs in the 2 min 30 s exposure group (BD) compared to the control (BC), and 2653 DEGs when comparing the BD to the BS group. Notably, Gene Ontology (GO) analysis showed an enrichment of DEGs associated with peptidase activity and extracellular regions, suggesting a role in the organism’s stress response. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis pointed to the involvement of metabolic pathways, which are crucial for energy management under stress. The upregulation of protein digestion and absorption pathways may indicate a physiological adaptation to nutrient scarcity during stress. Additionally, the identification of antibiotic biosynthesis pathways implies a potential role in combating stress-induced infections or damage. The identified DEGs and enriched pathways provide insights into the complex anti-stress response mechanisms in juvenile Sebastes schlegelii. The enrichment of peptidase activity and extracellular region-related genes may reflect the initiation of tissue repair and immune response following air exposure. The connection between protein digestion and absorption pathways and anti-stress capabilities could be interpreted as a metabolic readjustment to prioritize energy-efficient processes and nutrient assimilation during stress. The role of antibiotic biosynthesis pathways suggests a defensive mechanism against oxidative stress or microbial invasion that might occur with air exposure.

Enhanced triacylglycerol metabolism contributes to the efficient biosynthesis of spinosad in Saccharopolyspora spinosa

Triacylglycerol (TAG) is crucial for antibiotic biosynthesis derived from Streptomyces, as it serves as an important carbon source. In this study, the supplementation of exogenous TAG led to a 3.92-fold augmentation in spinosad production. The impact of exogenous TAG on the metabolic network of Saccharopolyspora spinosa were deeply analyzed through comparative proteomics. To optimize TAG metabolism and enhance spinosad biosynthesis, the lipase-encoding genes lip886 and lip385 were overexpressed or co-expressed. The results shown that the yield of spinosad was increased by 0.8-fold and 0.4-fold when lip886 and lip385 genes were overexpressed, respectively. Synergistic co-expression of these genes resulted in a 2.29-fold increase in the yield of spinosad. Remarkably, the combined overexpression of lip886 and lip385 in the presence of exogenous TAG elevated spinosad yields by 5.5-fold, led to a drastic increase in spinosad production from 0.036 g/L to 0.234 g/L. This study underscores the modification of intracellular concentrations of free fatty acids (FFAs), short-chain acyl-CoAs, ATP, and NADPH as mechanisms by which exogenous TAG modulates spinosad biosynthesis. Overall, the findings validate the enhancement of TAG catabolism as a beneficial strategy for optimizing spinosad production and provide foundational insights for engineering secondary metabolite biosynthesis pathways in another Streptomyces.

Structure classification of the proteins from Salmonella enterica pangenome revealed novel potential pathogenicity islands

Salmonella enterica is a pathogenic bacterium known for causing severe typhoid fever in humans, making it important to study due to its potential health risks and significant impact on public health. This study provides evolutionary classification of proteins from Salmonella enterica pangenome. We classified 17,238 domains from 13,147 proteins from 79,758 Salmonella enterica strains and studied in detail domains of 272 proteins from 14 characterized Salmonella pathogenicity islands (SPIs). Among SPIs-related proteins, 90 proteins function in the secretion machinery. 41% domains of SPI proteins have no previous sequence annotation. By comparing clinical and environmental isolates, we identified 3682 proteins that are overrepresented in clinical group that we consider as potentially pathogenic. Among domains of potentially pathogenic proteins only 50% domains were annotated by sequence methods previously. Moreover, 36% (1330 out of 3682) of potentially pathogenic proteins cannot be classified into Evolutionary Classification of Protein Domains database (ECOD). Among classified domains of potentially pathogenic proteins the most populated homology groups include helix-turn-helix (HTH), Immunoglobulin-related, and P-loop domains-related. Functional analysis revealed overrepresentation of these protein in biological processes related to viral entry into host cell, antibiotic biosynthesis, DNA metabolism and conformation change, and underrepresentation in translational processes. Analysis of the potentially pathogenic proteins indicates that they form 119 clusters or novel potential pathogenicity islands (NPPIs) within the Salmonella genome, suggesting their potential contribution to the bacterium’s virulence. One of the NPPIs revealed significant overrepresentation of potentially pathogenic proteins. Overall, our analysis revealed that identified potentially pathogenic proteins are poorly studied.

Integrative Genomics and Bioactivity-Guided Isolation of Novel Antimicrobial Compounds from Streptomyces sp. KN37 in Agricultural Applications.

Actinomycetes have long been recognized as an important source of antibacterial natural products. In recent years, actinomycetes in extreme environments have become one of the main research directions. Streptomyces sp. KN37 was isolated from the cold region of Kanas in Xinjiang. It demonstrated potent antimicrobial activity, but the primary active compounds remained unclear. Therefore, we aimed to combine genomics with traditional isolation methods to obtain bioactive compounds from the strain KN37. Whole-genome sequencing and KEGG enrichment analysis indicated that KN37 possesses the potential for synthesizing secondary metabolites, and 41 biosynthetic gene clusters were predicted, some of which showed high similarity to known gene clusters responsible for the biosynthesis of antimicrobial antibiotics. The traditional isolation methods and activity-guided fractionation were employed to isolate and purify seven compounds with strong bioactivity from the fermentation broth of the strain KN37. These compounds were identified as 4-(Diethylamino)salicylaldehyde (1), 4-Nitrosodiphenylamine (2), N-(2,4-Dimethylphenyl)formamide (3), 4-Nitrocatechol (4), Methylsuccinic acid (5), Phenyllactic acid (6) and 5,6-Dimethylbenzimidazole (7). Moreover, 4-(Diethylamino)salicylaldehyde exhibited the most potent inhibitory effect against Rhizoctonia solani, with an EC50 value of 14.487 mg/L, while 4-Nitrosodiphenylamine showed great antibacterial activity against Erwinia amylovora, with an EC50 value of 5.715 mg/L. This study successfully isolated several highly active antimicrobial compounds from the metabolites of the strain KN37, which could contribute as scaffolds for subsequent chemical synthesis. On the other hand, the newly predicted antibiotic-like substances have not yet been isolated, but they still hold significant research value. They are instructive in the study of active natural product biosynthetic pathways, activation of silent gene clusters, and engineering bacteria construction.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein–protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor.Key points• GarR/GarS is a TCS with domains for signal transduction and response regulation• GarR/GarS is an essential negative regulator of the ACT and RED production• GarR/GarS putatively interacts with and regulates activators of ACT and RED

Improving the production of carbamoyltobramycin by an industrial Streptoalloteichus tenebrarius through metabolic engineering

Tobramycin is an essential and extensively used broad-spectrum aminoglycoside antibiotic obtained through alkaline hydrolysis of carbamoyltobramycin, one of the fermentation products of Streptoalloteichus tenebrarius. To simplify the composition of fermentation products from industrial strain, the main byproduct apramycin was blocked by gene disruption and constructed a mutant mainly producing carbamoyltobramycin. The generation of antibiotics is significantly affected by the secondary metabolism of actinomycetes which could be controlled by modifying the pathway-specific regulatory proteins within the cluster. Within the tobramycin biosynthesis cluster, a transcriptional regulatory factor TobR belonging to the Lrp/AsnC family was identified. Based on the sequence and structural characteristics, tobR might encode a pathway-specific transcriptional regulatory factor during biosynthesis. Knockout and overexpression strains of tobR were constructed to investigate its role in carbamoyltobramycin production. Results showed that knockout of TobR increased carbamoyltobramycin biosynthesis by 22.35%, whereas its overexpression decreased carbamoyltobramycin production by 10.23%. In vitro electrophoretic mobility shift assay (EMSA) experiments confirmed that TobR interacts with DNA at the adjacent tobO promoter position. Strains overexpressing tobO with ermEp* promoter exhibited 36.36% increase, and tobO with kasOp* promoter exhibited 22.84% increase in carbamoyltobramycin titer. When the overexpressing of tobO and the knockout of tobR were combined, the production of carbamoyltobramycin was further enhanced. In the shake-flask fermentation, the titer reached 3.76 g/L, which was 42.42% higher than that of starting strain. Understanding the role of Lrp/AsnC family transcription regulators would be useful for other antibiotic biosynthesis in other actinomycetes.Key points• The transcriptional regulator TobR belonging to the Lrp/AsnC family was identified. • An oxygenase TobO was identified within the tobramycin biosynthesis cluster.• TobO and TobR have significant effects on the synthesis of carbamoyltobramycin.

Metagenomic Analysis Reveals A Gut Microbiota Structure and Function Alteration between Healthy and Diarrheic Juvenile Yaks.

Diarrhea-induced mortality among juvenile yaks is highly prevalent in the pastoral areas of the Qinghai-Tibet plateau. Although numerous diseases have been linked to the gut microbial community, little is known about how diarrhea affects the gut microbiota in yaks. In this work, we investigated and compared changes in the gut microbiota of juvenile yaks with diarrhea. The results demonstrated a considerable drop in the alpha diversity of the gut microbiota in diarrheic yaks, accompanied by Eysipelatoclostridium, Parabacteroides, and Escherichia-Shigella, which significantly increased during diarrhea. Furthermore, a PICRust analysis verified the elevation of the gut-microbial metabolic pathways in diarrhea groups, including glycine, serine, and threonine metabolism, alanine, aspartate, oxidative phosphorylation, glutamate metabolism, antibiotic biosynthesis, and secondary metabolite biosynthesis. Taken together, our study showed that the harmful bacteria increased, and beneficial bacteria decreased significantly in the gut microbiota of yaks with diarrhea. Moreover, these results also indicated that the dysbiosis of the gut microbiota may be a significant driving factor of diarrhea in yaks.

Anthracene degradation involved by antibiotic biosynthesis monooxygenase (ABM) in Comamonas testosteroni

It is well known that anthracene can cause serious health problems, which is why anthracene biodegradation as a method to reduce health risks has drawn the interest of researchers. However, antibiotic contamination in the environment can seriously affect the biodegradation of anthracene. In the present study, Comamonas testosteroni (CT1) had the highest degradation efficiency of anthracene (88.1%), and was still 46.6% when erythromycin concentration was 1/4MIC (8 μg mL−1). Also, compared to CK, the prokaryotic transcriptome analysis of CT1 in anthracene degradation revealed an up-regulated gene that encodes antibiotic biosynthesis monooxygenase (ABM) in both anthracene and anthracene-erythromycin groups. In addition, compared to strain CT1, the CtABM knockout mutant (CT-M) showed a significant decrease in anthracene degradation efficiency. In contrast, Escherichia coli (E.coli) DH5α transformed with CtABM (EM1) exhibited a faster degradation efficiency than DH5α. Furthermore, the antimicrobial susceptibility test showed that compared to DH5α, EM1 had significant resistance to erythromycin. And the purified recombinant CtABM (rABM) had a specific activity of 2.53 μmol min−1·mg−1 protein based on the oxidation of anthracene at pH 7.5 and 35 °C. Additionally, compositional analysis identified 4-benzyloxy-3-methoxybenzyl alcohol and 4-methylphthalaldehyde as anthracene metabolites by EM1, suggesting a novel anthracene degradation pathway.

Root rot induces a core assemblage of bacterial microbiome to prevent disease infection in Sanqi ginseng

Plants rely on rhizosphere microbes as the first line of defense against pathogen invasion and for maintaining the health of itself. Increasing evidence suggests that plants recruit beneficial microbes to the roots, strengthening their resistance to pathogens. However, the response of the rhizosphere core bacterial microbiome to root rot infection remains largely unclear. In this study, we investigated the rhizosphere and bulk soil core bacterial microbiome of healthy and root rot-diseased Sanqi ginseng in genuine production areas in China. We utilized 16S amplicon sequencing, co-occurrence network analysis, and strain culture approaches to explore whether root rot-induced plant rhizosphere alterations in the bacterial core microbiome could improve disease suppression. Root rot infection had a significant impact on the rhizosphere core bacterial microbiome of Sanqi ginseng. Root rot led to a reduction in the diversity and evenness of the core bacterial microbiome in the rhizosphere, while the variability of the core bacterial community in the rhizosphere increased. In response to fungal root rot pathogen infestation, Sanqi ginseng recruited potentially beneficial bacteria with disease-suppressive functions to the rhizosphere, such as Sphingobium, Pseudoxanthomonas, Pseudomonas, Stenotrophomonas, and Flavobacterium. Function prediction analysis demonstrated a remarkable enrichment of several functional genes involved in antibiotic biosynthesis in the rhizosphere that was infected with root rot. Strain isolation and pot addition experiments further confirmed the effectiveness of root rot rhizosphere-specific enrichment of Operational Taxonomic Units (OTUs) for controlling root rot. Furthermore, co-occurrence network analysis indicated that root rot activated the disease-suppressive functions of the rhizosphere core bacterial microbiome by enhancing complexity and connectivity at the expense of network stability. These findings expand our knowledge of plant-microbe interactions and provides new evidence supporting the “cry for help” strategy for rhizosphere responses to root rot.

Magnesium oxide nanoparticles reduce clubroot by regulating plant defense response and rhizosphere microbial community of tumorous stem mustard (Brassica juncea var. tumida).

Clubroot, caused by Plasmodiophora brassicae, is a major disease that significantly impairs the yield of cruciferous crops and causes significant economic losses across the globe. The prevention of clubroot, especially in tumorous stem mustard (without resistant varieties), are is limited and primarily relies on fungicides. Engineered nanoparticles have opened up new avenues for the management of plant diseases, but there is no report on their application in the prevention of clubroot. The results showed that the control efficacy of 500 mg/L MgO NPs against clubroot was 54.92%. However, when the concentration was increased to 1,500 and 2,500 mg/L, there was no significant change in the control effect. Compared with CK, the average fresh and dry weight of the aerial part of plants treated with MgO NPs increased by 392.83 and 240.81%, respectively. Compared with the F1000 treatment, increases were observed in the content of soil available phosphorus (+16.72%), potassium (+9.82%), exchangeable magnesium (+24.20%), and water-soluble magnesium (+20.64%) in the 1,500 mg/L MgO NPs treatment. The enzyme-linked immune sorbent assay (ELISA) results showed that the application of MgO NPs significantly increased soil peroxidase (POD, +52.69%), alkaline protease (AP, +41.21%), alkaline phosphatase (ALP, +79.26%), urease (+52.69%), and sucrase (+56.88%) activities; And also increased plant L-phenylalanine ammonla-lyase (PAL, +70.49%), polyphenol oxidase (PPO, +36.77%), POD (+38.30%), guaiacol peroxidase (POX, +55.46%) activities and salicylic acid (SA, +59.86%) content. However, soil and plant catalase (CAT, -27.22 and - 19.89%, respectively), and plant super oxidase dismutase (SOD, -36.33%) activities were significantly decreased after the application of MgO NPs. The metagenomic sequencing analysis showed that the MgO NPs treatments significantly improved the α-diversity of the rhizosphere soil microbial community. The relative abundance of beneficial bacteria genera in the rhizosphere soil, including Pseudomonas, Sphingopyxis, Acidovorax, Variovorax, and Bosea, was significantly increased. Soil metabolic functions, such as oxidative phosphorylation (ko00190), carbon fixation pathways in prokaryotes (ko00720), indole alkaloid biosynthesis (ko00901), and biosynthesis of various antibiotics (ko00998) were significantly enriched. These results suggested that MgO NPs might control clubroot by promoting the transformation and utilization of soil nutrients, stimulating plant defense responses, and enriching soil beneficial bacteria.

De Novo Transcriptome Analysis by PacBio SMRT-Seq and Illumina RNA-Seq Provides New Insights into Polyphenol Biosynthesis in Chinese Olive Fruit

Polyphenols play a crucial role in fruit flavor. To elucidate the mechanism of fruit polyphenol metabolism, we constructed a transcriptome atlas through PacBio single-molecule real-time (SMRT) sequencing and Illumina next-generation sequencing (NGS) using Canarium album (Lour.) Raeusch., which is a fantastic fruit rich in polyphenolic compounds. In this work, PacBio full-length transcriptome assembly generated 135,439 isoforms with an average length of all isoforms of 2687.94 bp and an N50 length of 3224 bp. To gain deeper insights into the molecular mechanisms of polyphenol biosynthesis in C. album, we constructed twelve RNA-Seq libraries from four developmental stages of the fruits. We identified a total of 28,658 differentially expressed genes (DEGs). We found that many DEGs were involved in metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, starch and sucrose metabolism, and plant hormone signal transduction. Here, we report the expression profiles of 215 DEGs encoding 27 enzymes involved in the polyphenol biosynthesis pathway in C. album. In addition, 285 differentially expressed transcription factors (TFs) continuously down-regulated in four developmental periods of C. album fruit, which may indicate their potential role in the response to polyphenol metabolism and phenylpropanoid biosynthesis pathways. This report will help us understand polyphenol biosynthesis’s functions and metabolic mechanism in C. album. The transcriptome data provide a valuable resource for genetic and genomics research. They will facilitate future work exploiting C. album and other fruits used as medicine and food.

Effect of Precursors and Their Regulators on the Biosynthesis of Antibiotics in Actinomycetes.

During the life activities of microorganisms, a variety of secondary metabolites are produced, including antimicrobials and antitumor drugs, which are widely used in clinical practice. In addition to exploring new antibiotics, this makes it one of the research priorities of Actinomycetes to effectively increase the yield of antibiotics in production strains by various means. Most antibiotic-producing strains have a variety of functional regulatory factors that regulate their growth, development, and secondary metabolite biosynthesis processes. Through the study of precursor substances in antibiotic biosynthesis, researchers have revealed the precursor biosynthesis process and the mechanism by which precursor synthesis regulators affect the biosynthesis of secondary metabolites, which can be used to obtain engineered strains with high antibiotic production. This paper summarizes the supply of antibiotic biosynthesis precursors and the progress of research on the role of regulators in the process of precursors in biosynthesis. This lays the foundation for the establishment of effective breeding methods to improve antibiotic yields through the manipulation of precursor synthesis genes and related regulators.

Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

Unraveling the antimicrobial potential of Lactiplantibacillus plantarum strains TE0907 and TE1809 sourced from Bufo gargarizans: advancing the frontier of probiotic-based therapeutics.

In an era increasingly defined by the challenge of antibiotic resistance, this study offers groundbreaking insights into the antibacterial properties of two distinct Lactiplantibacillus plantarum strains, TE0907 and TE1809, hailing from the unique ecosystem of Bufo gargarizans. It uniquely focuses on elucidating the intricate components and mechanisms that empower these strains with their notable antibacterial capabilities. The research employs a multi-omics approach, including agar diffusion tests to assess antibacterial efficacy and adhesion assays with HT-29 cells to understand the preliminary mechanisms. Additionally, gas chromatography-mass spectrometry (GC-MS) is employed to analyze the production of organic acids, notably acetic acid, and whole-genome sequencing is utilized to identify genes linked to the biosynthesis of antibiotics and bacteriocin-coding domains. The comparative analysis highlighted the exceptional antibacterial efficacy of strains TE0907 and TE1809, with mean inhibitory zones measured at 14.97 and 15.98 mm, respectively. A pivotal discovery was the significant synthesis of acetic acid in both strains, demonstrated by a robust correlation coefficient (cor ≥ 0.943), linking its abundance to their antimicrobial efficiency. Genomic exploration uncovered a diverse range of elements involved in the biosynthesis of antibiotics similar to tetracycline and vancomycin and potential regions encoding bacteriocins, including Enterolysin and Plantaricin. This research illuminates the remarkable antibacterial efficacy and mechanisms intrinsic to L. plantarum strains TE0907 and TE1809, sourced from B. gargarizans. The findings underscore the strains' extensive biochemical and enzymatic armamentarium, offering valuable insights into their role in antagonizing enteric pathogens. These results lay down a comprehensive analytical foundation for the potential clinical deployment of these strains in safeguarding animal gut health, thereby enriching our understanding of the role of probiotic bacteria in the realm of antimicrobial interventions.

Microbiome composition and function within the Kellet's whelk perivitelline fluid.

Microbiomes have gained significant attention in ecological research, owing to their diverse interactions and essential roles within different organismal ecosystems. Microorganisms, such as bacteria, archaea, and viruses, have profound impact on host health, influencing digestion, metabolism, immune function, tissue development, and behavior. This study investigates the microbiome diversity and function of Kellet's whelk (Kelletia kelletii) perivitelline fluid (PVF), which sustains thousands of developing K. kelletii embryos within a polysaccharide and protein matrix. Our core microbiome analysis reveals a diverse range of bacteria, with the Roseobacter genus being the most abundant. Additionally, genes related to host-microbe interactions, symbiosis, and quorum sensing were detected, indicating a potential symbiotic relationship between the microbiome and Kellet's whelk embryos. Furthermore, the microbiome exhibits gene expression related to antibiotic biosynthesis, suggesting a defensive role against pathogenic bacteria and potential discovery of novel antibiotics. Overall, this study sheds light on the microbiome's role in Kellet's whelk development, emphasizing the significance of host-microbe interactions in vulnerable life history stages. To our knowledge, ours is the first study to use 16S sequencing coupled with RNA sequencing (RNA-seq) to profile the microbiome of an invertebrate PVF.IMPORTANCEThis study provides novel insight to an encapsulated system with strong evidence of symbiosis between the microbial inhabitants and developing host embryos. The Kellet's whelk perivitelline fluid (PVF) contains microbial organisms of interest that may be providing symbiotic functions and potential antimicrobial properties during this vulnerable life history stage. This study, the first to utilize a comprehensive approach to investigating Kellet's whelk PVF microbiome, couples 16S rRNA gene long-read sequencing with RNA-seq. This research contributes to and expands our knowledge on the roles of beneficial host-associated microbes.

Catalytic divergencies in the mechanism of L-arginine hydroxylating nonheme iron enzymes.

Many enzymes in nature utilize a free arginine (L-Arg) amino acid to initiate the biosynthesis of natural products. Examples include nitric oxide synthases, which generate NO from L-Arg for blood pressure control, and various arginine hydroxylases involved in antibiotic biosynthesis. Among the groups of arginine hydroxylases, several enzymes utilize a nonheme iron(II) active site and let L-Arg react with dioxygen and α-ketoglutarate to perform either C3-hydroxylation, C4-hydroxylation, C5-hydroxylation, or C4-C5-desaturation. How these seemingly similar enzymes can react with high specificity and selectivity to form different products remains unknown. Over the past few years, our groups have investigated the mechanisms of L-Arg-activating nonheme iron dioxygenases, including the viomycin biosynthesis enzyme VioC, the naphthyridinomycin biosynthesis enzyme NapI, and the streptothricin biosynthesis enzyme OrfP, using computational approaches and applied molecular dynamics, quantum mechanics on cluster models, and quantum mechanics/molecular mechanics (QM/MM) approaches. These studies not only highlight the differences in substrate and oxidant binding and positioning but also emphasize on electronic and electrostatic differences in the substrate-binding pockets of the enzymes. In particular, due to charge differences in the active site structures, there are changes in the local electric field and electric dipole moment orientations that either strengthen or weaken specific substrate C-H bonds. The local field effects, therefore, influence and guide reaction selectivity and specificity and give the enzymes their unique reactivity patterns. Computational work using either QM/MM or density functional theory (DFT) on cluster models can provide valuable insights into catalytic reaction mechanisms and produce accurate and reliable data that can be used to engineer proteins and synthetic catalysts to perform novel reaction pathways.

Comparing the gut microbiota of Sichuan golden monkeys across multiple captive and wild settings: roles of anthropogenic activities and host factors

BackgroundCaptivity and artificial food provision are common conservation strategies for the endangered golden snub-nosed monkey (Rhinopithecus roxellana). Anthropogenic activities have been reported to impact the fitness of R. roxellana by altering their gut microbiota, a crucial indicator of animal health. Nevertheless, the degree of divergence in gut microbiota between different anthropogenically-disturbed (AD) R. roxellana and their counterparts in the wild has yet to be elucidated. Here, we conducted a comparative analysis of the gut microbiota across nine populations of R. roxellana spanning China, which included seven captive populations, one wild population, and another wild population subject to artificial food provision.ResultsBoth captivity and food provision significantly altered the gut microbiota. AD populations exhibited common variations, such as increased Bacteroidetes and decreased Firmicutes (e.g., Ruminococcus), Actinobacteria (e.g., Parvibacter), Verrucomicrobia (e.g., Akkermansia), and Tenericutes. Additionally, a reduced Firmicutes/Bacteroidetes ratiosuggested diminished capacity for complex carbohydrate degradation in captive individuals. The results of microbial functional prediction suggested that AD populations displayed heightened microbial genes linked to vitamin and amino acid metabolism, alongside decreased genes associated antibiotics biosynthesis (e.g., penicillin, cephalosporin, macrolides, and clavulanic acid) and secondary metabolite degradation (e.g., naphthalene and atrazine). These microbial alterations implied potential disparities in the health status between AD and wild individuals. AD populations exhibited varying degrees of microbial changes compared to the wild group, implying that the extent of these variations might serve as a metric for assessing the health status of AD populations. Furthermore, utilizing the individual information of captive individuals, we identified associations between variations in the gut microbiota of R. roxellana and host age, as well as pedigree. Older individuals exhibited higher microbial diversity, while a closer genetic relatedness reflected a more similar gut microbiota.ConclusionsOur aim was to assess how anthropogenic activities and host factors influence the gut microbiota of R. roxellana. Anthropogenic activities led to consistent changes in gut microbial diversity and function, while host age and genetic relatedness contributed to interindividual variations in the gut microbiota. These findings may contribute to the establishment of health assessment standards and the optimization of breeding conditions for captive R. roxellana populations.

Natural Products Biosynthesis by Streptomyces netropsis IMV Ac-5025 under Exogenous Sterol Action.

Streptomycetes are known as producers of bioactive substances, particularly antibiotics. Streptomyces netropsis IMV Ac-5025 simultaneously produces different classes of antibiotics, including polyene compounds, phytohormones, and sterols, but the metabolic pathways involved in their biosynthesis are largely understudied. The aim of this work was to explore the biosynthesis of polyene antibiotics, sterols, and phytohormones when the producer is cultivated in a nutrient medium supplemented with exogenous β-sitosterol. Gas chromatography and high-performance liquid chromatography were applied to analyze the spectrum of bioactive compounds. The obtained results demonstrated not only an increase in the accumulation of biomass but also polyene antibiotics, intracellular sterols, auxins, and cytokinins, when cultivating S. netropsis IMV Ac-5025 in a liquid medium with the addition of β-sitosterol. The amount of biomass raised 1.5-2-fold, whilst the sum of polyene antibiotics increased 4.5-fold, sterols' sum (ergosterol, cholesterol, stigmasterol, β-sitosterol, and 24-epibrassinolide) by 2.9-fold, auxins' sum (indole-3-acetic acid, indole-3-acetic acid hydrazide, indole-3-carbinol, indole-3-butyric acid, indole-3-carboxaldehyde, and indole-3-carboxylic acid) by 6-fold, and cytokinins' sum (zeatin, isopentyladenine, zeatin riboside, and isopentenyladenosine) by 11-fold. Thus, we put forward the hypothesis that β-sitosterol plays a regulatory role in the network of biosynthetic reactions of S. netropsis IMV Ac-5025.

The Impact of Heterologous Regulatory Genes from Lipodepsipeptide Biosynthetic Gene Clusters on the Production of Teicoplanin and A40926.

StrR-like pathway-specific transcriptional regulators (PSRs) function as activators in the biosynthesis of various antibiotics, including glycopeptides (GPAs), aminoglycosides, aminocoumarins, and ramoplanin-like lipodepsipeptides (LDPs). In particular, the roles of StrR-like PSRs have been previously investigated in the biosynthesis of streptomycin, novobiocin, GPAs like balhimycin, teicoplanin, and A40926, as well as LDP enduracidin. In the current study, we focused on StrR-like PSRs from the ramoplanin biosynthetic gene cluster (BGC) in Actinoplanes ramoplaninifer ATCC 33076 (Ramo5) and the chersinamycin BGC in Micromonospora chersina DSM 44151 (Chers28). Through the analysis of the amino acid sequences of Ramo5 and Chers28, we discovered that these proteins are phylogenetically distant from other experimentally investigated StrR PSRs, although all StrR-like PSRs found in BGCs for different antibiotics share a conserved secondary structure. To investigate whether Ramo5 and Chers28, given their phylogenetic positions, might influence the biosynthesis of other antibiotic pathways governed by StrR-like PSRs, the corresponding genes (ramo5 and chers28) were heterologously expressed in Actinoplanes teichomyceticus NRRL B-16726 and Nonomuraea gerenzanensis ATCC 39727, which produce the clinically-relevant GPAs teicoplanin and A40926, respectively. Recombinant strains of NRRL B-16726 and ATCC 39727 expressing chers28 exhibited improved antibiotic production, although the expression of ramo5 did not yield the same effect. These results demonstrate that some StrR-like PSRs can "cross-talk" between distant biosynthetic pathways and might be utilized as tools for the activation of silent BGCs regulated by StrR-like PSRs.