Elucidation of the ferrichrome siderophore biosynthetic pathway in albomycin-producing Streptomyces sp. ATCC 700974.

Sideromycins are a unique subset of siderophores comprising of a siderophore conjugated to an antimicrobial agent. The "Trojan horse" antibiotic albomycins are unique sideromycins consisting of a ferrichrome-type siderophore conjugated to a peptidyl nucleoside antibiotic. They exhibit potent antibacterial activities against many model bacteria and a number of clinical pathogens. Earlier studies have provided significant insight into the biosynthetic pathway of the peptidyl nucleoside moiety. We herein decipher the biosynthetic pathway of the ferrichrome-type siderophore in Streptomyces sp. ATCC 700974. Our genetic studies suggested that abmA, abmB, and abmQ are involved in the formation of the ferrichrome-type siderophore. Additionally, we performed biochemical studies to demonstrate that a flavin-dependent monooxygenase AbmB and an N-acyltransferase AbmA catalyze sequential modifications of L-ornithine to generate N5-acetyl-N5-hydroxyornithine. Three molecules of N5-acetyl-N5-hydroxyornithine are then assembled to generate the tripeptide ferrichrome through the action of a nonribosomal peptide synthetase AbmQ. Of special note, we found out that orf05026 and orf03299, two genes scattered elsewhere in the chromosome of Streptomyces sp. ATCC 700974, have functional redundancy for abmA and abmB, respectively. Interestingly, both orf05026 and orf03299 are situated within gene clusters encoding putative siderophores. In summary, this study provided new insight into the siderophore moiety of albomycin biosynthesis and shed light on the contingency of multiple siderophores in albomycin-producing Streptomyces sp. ATCC 700974.

Next-Generation Drug Discovery to Combat Antimicrobial Resistance.

The widespread emergence of antibiotic-resistant pathogens poses a severe threat to public health. This problem becomes even worse with a coincident decline in the supply of new antibiotics. Conventional bioactivity-guided natural product discovery has failed to meet the urgent need for new antibiotics, largely due to limited resources and high rediscovery rates. Recent advances in cultivation techniques, analytical technologies, and genomics-based approaches have greatly expanded our access to previously underexploited microbial sources. These strategies will enable us to access new reservoirs of microorganisms and unleash their chemical potentials, thus opening new opportunities for the discovery of next-generation drugs to address the growing concerns of antimicrobial resistance.

The Streptococcus mutans irvA gene encodes a trans-acting riboregulatory mRNA.

In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation.

Biochemical Insights into a Novel Family 2 Glycoside Hydrolase with Both ß-1,3-Galactosidase and ß-1,4-Galactosidase Activity from the Arctic.

A novel GH2 (glycoside hydrolase family 2) ß-galactosidase from Marinomonas sp. BSi20584 was successfully expressed in E. coli with a stable soluble form. The recombinant enzyme (rMaBGA) was purified to electrophoretic homogeneity and characterized extensively. The specific activity of purified rMaBGA was determined as 96.827 U mg-1 at 30 °C using ONPG (o-nitrophenyl-ß-D-galactopyranoside) as a substrate. The optimum pH and temperature of rMaBGA was measured as 7.0 and 50 °C, respectively. The activity of rMaBGA was significantly enhanced by some divalent cations including Zn2+, Mg2+ and Ni2+, but inhibited by EDTA, suggesting that some divalent cations might play important roles in the catalytic process of rMaBGA. Although the enzyme was derived from a cold-adapted strain, it still showed considerable stability against various physical and chemical elements. Moreover, rMaBGA exhibited activity both toward Galß-(1,3)-GlcNAc and Galß-(1,4)-GlcNAc, which is a relatively rare occurrence in GH2 ß-galactosidase. The results showed that two domains in the C-terminal region might be contributed to the ß-1,3-galactosidase activity of rMaBGA. On account of its fine features, this enzyme is a promising candidate for the industrial application of ß-galactosidase.

Stepwise increase of thaxtomins production in Streptomyces albidoflavus J1074 through combinatorial metabolic engineering.

Herbicide-resistance in weeds has become a serious threat to agriculture across the world. Thus, there is an urgent need for the discovery and development of herbicides with new modes of action. Thaxtomin phytotoxins are a group of nitrated diketopiperazines produced by potato common scab-causing phytopathogen Streptomyces scabies and other actinobacterial pathogens. They are generally considered to function as inhibitors of cellulose synthesis in plants, and thus have great potential to be used as natural herbicides. Generation of an overproducing strain is crucial for the scale-up production of thaxtomins and their wide use in agriculture. In the present study, we employed a stepwise strategy by combining heterologous expression, repressor deletion, activator overexpression, and optimization of fermentation media for high-level production of thaxtomins. The maximum yield of 728 mg/L thaxtomins was achieved with engineered Streptomyces albidoflavus J1074 strains in shake-flask cultures, and it was approximately 36-fold higher than S. albidoflavus J1074 carrying the unmodified cluster. Moreover, the yield of thaxtomins could reach 1973 mg/L when the engineered strain was cultivated in a small-scale stirred-tank bioreactor. This is the highest titer reported to date, representing a significant leap forward for the scale-up production of thaxtomins. Our study presents a robust, easy-to-use system that will be broadly useful for improving titers of bioactive compounds in many Streptomyces species.

Engineered biosynthesis of thaxtomin phytotoxins.

Herbicide-resistant weeds are a growing problem worldwide. Thaxtomin phytotoxins are a group of nitrated diketopiperazines produced by the potato common scab-causing pathogen Streptomyces scabies and other actinobacterial plant pathogens. They represent a unique class of microbial natural products with distinctive structural features and promising herbicidal activity. The biosynthesis of thaxtomins proceeds through multiple steps of unusual enzymatic reactions. Advances in understanding of thaxtomins biosynthetic machinery have provided the basis for precursor-directed biosynthesis, pathway refactoring, and one-pot biocombinatorial synthesis to generate thaxtomin analogues. We herein summarize recent findings on the biosynthesis of thaxtomins and highlight recent advances in the rational generation of novel thaxtomins for the development of potent herbicidal agents.

[Functional characterization of gouC and gouD in gougerotin biosynthesis].

OBJECTIVE: To determine the functions of gouC and gouD in gougerotin biosynthesis, disruption of these two genes was performed. As gougerotin producing strain Streptomyces graminearus lacks efficient genetic manipulation system, the gene cluster for gougerotin biosynthesis was heterologously expressed in Streptomyces coelicolor M1146 to facilitate genetic manipulations of gouC and gouD. METHODS: By using fosmid D6-4H containing the complete gougerotin biosynthetic gene cluster, gouC and gouD were disrupted by PCR-targeting method to generate pGOUe-ΔC and pGOUe-ΔD. Both pGOUe-ΔC and pGOUe-ΔD were introduced into Streptomyces coelicolor M1146 by intergeneric conjugation, thus gouC and gouD disrpution mutants (Ml146-GOUe-AC and M1146-GOUe-ΔD) were obtained. The gougerotin production of M1146-GOUe-ΔC and M1146-GOUe-ΔD were assayed by HPLC analysis. The intermediates accumulated in these mutants were purified and subjected to MS and NMR analyses for structure determinations. Bioassay of these intermediates against tumor cell line were also carried out. RESULTS: Disruption mutants of gouC and gouD failed to produce gougerotin and the mutants accumulated different gougerotin intermediates, which lost their ability to inhibit cancer cell proliferation. CONCLUSION: gouC and gouD are key structual genes in the biosynthesis of gougerotin peptidyl moieties. This study will pave the way for the elucidation of gougerotin biosynthetic pathway.

A γ-butyrolactone-sensing activator/repressor, JadR3, controls a regulatory mini-network for jadomycin biosynthesis.

Two regulatory genes, jadR2 and jadR3, in the jadomycin (jad) biosynthetic gene cluster of Streptomyces venezuelae encode homologues of γ-butyrolactone receptor. JadR2 was previously shown to be a pseudo γ-butyrolactone receptor. jadR3 is situated at the upstream of jadW123 encoding putative enzymes for γ-butyrolactone biosynthesis. Disruption of jadR3 resulted in markedly decreased production of jadomycin. Transcriptional analysis revealed that JadR3 represses jadW1, jadR2 and jadR3 but activates jadR1, the key activator gene for jadomycin biosynthesis. DNase I footprinting showed that JadR3 has four binding sites in the intergenic regions of jadR2-jadR1 and jadR3-jadW1. A JadR3 interactive molecule, SVB1, was purified from a large-scale fermentation and its structure found to be the same as SCB3, a γ-butyrolactone from Streptomyces coelicolor, and was absent from a jadW123 mutant lacking jadomycin production. Addition of SVB1 or extract from S. coelicolor to the mutant restored jadomycin production. Overall, our results revealed that the association of JadR3 and SVB1 plays an important role in controlling a regulatory mini-network governing jadomycin biosynthesis, providing new insights into the ways in which γ-butyrolactone/receptor systems modulate antibiotic biosynthesis in Streptomyces.

[Engineering and heterologous expression of a nikkomycin biosynthetic gene cluster].

OBJECTIVE: We expressed a nikkomycin biosynthetic gene cluster in the well-characterized surrogate Streptomyces coelicolor M1146. METHODS: By using PCR-targeting method, we replaced the promoters of sanG and sanF in pNIK, which contains nikkomycin biosynthetic gene cluster, with the hrdB promoter to generate pNIKm. We transferred pNIK and pNIKm into S. coelicolor M1146 by intergeneric conjugation and obtained M1146-NIK and M1146-NIKm, respectively. We then evaluated expression of the gene cluster in the heterologous host by RT-PCR. Furthermore, we also compared the antifugal activity and nikkomycin production of M1146-NIK and M1146-NIKm by bioassay against Alternaria longipes and HPLC analysis. RESULTS: M1146-NIK and M1146-NIKm exhibited antifungal activity, and they can produce a trace amount of nikkomycin X, nikkomycin Z and pseudo-Z. There was a substantial accumulation of uridine in M1146-NIK, whereas substantial accumulations of uridine, ribofuranosyl-4-formyl-4-imidazolone and pyridylhomothreonine were observed in M1146-NIKm. CONCLUSION: We successfully expressed the nikkomycin biosynthetic gene cluster in the heterologous host and identified nikkomycins and some of its key biosynthetic intermediates. This study will provide the basis for enzymatic reaction of the condensation between the two nikkomycin moieties and for the generation of hybrid antibiotics by combinatorial biosynthesis.

GouR, a TetR family transcriptional regulator, coordinates the biosynthesis and export of gougerotin in Streptomyces graminearus.

Gougerotin is a peptidyl nucleoside antibiotic. It functions as a specific inhibitor of protein synthesis by binding ribosomal peptidyl transferase and exhibits a broad spectrum of biological activities. gouR, situated in the gougerotin biosynthetic gene cluster, encodes a TetR family transcriptional regulatory protein. Gene disruption and genetic complementation revealed that gouR plays an important role in the biosynthesis of gougerotin. Transcriptional analysis suggested that GouR represses the transcription of the gouL-to-gouB operon consisting of 11 structural genes and activates the transcription of the major facilitator superfamily (MFS) transporter gene (gouM). Electrophoresis mobility shift assays (EMSAs) and DNase I footprinting experiments showed that GouR has specific DNA-binding activity for the promoter regions of gouL, gouM, and gouR. Our data suggested that GouR modulates gougerotin production by coordinating its biosynthesis and export in Streptomyces graminearus.

Novel nikkomycin analogues generated by mutasynthesis in Streptomyces ansochromogenes.

BACKGROUND: Nikkomycins are competitive inhibitors of chitin synthase and inhibit the growth of filamentous fungi, insects, acarids and yeasts. The gene cluster responsible for biosynthesis of nikkomycins has been cloned and the biosynthetic pathway was elucidated at the genetic, enzymatic and regulatory levels. RESULTS: Streptomyces ansochromogenes ΔsanL was constructed by homologous recombination and the mutant strain was fed with benzoic acid, 4-hydroxybenzoic acid, nicotinic acid and isonicotinic acid. Two novel nikkomycin analogues were produced when cultures were supplemented with nicotinic acid. These two compounds were identified as nikkomycin Px and Pz by electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR). Bioassays against Candida albicans and Alternaria longipes showed that nikkomycin Px and Pz exhibited comparatively strong inhibitory activity as nikkomycin X and Z produced by Streptomyces ansochromogenes 7100 (wild-type strain). Moreover, nikkomycin Px and Pz were found to be more stable than nikkomycin X and Z at different pH and temperature conditions. CONCLUSIONS: Two novel nikkomycin analogues (nikkomycin Px and Pz) were generated by mutasynthesis with the sanL inactivated mutant of Streptomyces ansochromogenes 7100. Although antifungal activities of these two compounds are similar to those of nikkomycin X and Z, their stabilities are much better than nikkomycin X and Z under different pHs and temperatures.

Characterizing and Engineering Rhamnose-Inducible Regulatory Systems for Dynamic Control of Metabolic Pathways in Streptomyces.

Fine-tuning gene expression is of great interest for synthetic biotechnological applications. This is particularly true for the genus Streptomyces, which is well-known as a prolific producer of diverse natural products. Currently, there is an increasing demand to develop effective gene induction systems. In this study, bioinformatic analysis revealed a putative rhamnose catabolic pathway in multiple Streptomyces species, and the removal of the pathway in the model organism Streptomyces coelicolor impaired its growth on minimal media with rhamnose as the sole carbon source. To unravel the regulatory mechanism of RhaR, a LacI family transcriptional regulator of the catabolic pathway, electrophoretic mobility shift assays (EMSAs) were performed to identify potential target promoters. Multiple sequence alignments retrieved a consensus sequence of the RhaR operator (rhaO). A synthetic biology-based strategy was then deployed to build rhamnose-inducible regulatory systems, referred to as rhaRS1 and rhaRS2, by assembling the repressor/operator pair RhaR/rhaO with the well-defined constitutive kasO* promoter. Both rhaRS1 and rhaRS2 exhibited a high level of induced reporter activity, with no leaky expression. rhaRS2 has been proven successful for the programmable production of actinorhodin and violacein in Streptomyces. Our study expanded the toolkit of inducible regulatory systems that will be broadly applicable to many other Streptomyces species.

Engineering of tnaC-Based Tryptophan Biosensors for Dynamic Control of Violacein Production.

Tryptophan not only serves as a fundamental building block for protein synthesis but also acts as a metabolic precursor for a diverse array of high-value chemicals. Although a few tryptophan-responsive biosensors are currently available, there is a growing interest in developing high-performance biosensors. In this study, we create a miniature toolkit of tryptophan biosensors based upon the leader regulatory region of the tnaCAB operon, which is responsible for tryptophan catabolism in Escherichia coli. Four variants are generated by engineering the tnaC leader sequence, which encodes a leader peptide composed of 24 amino acid residues. Subsequently, the performance of both the natural tnaC sequence and its engineered variants is assessed in a reporter strain based on the MazEF toxin-antitoxin system. The results demonstrate that two engineered variants exhibit increased sensitivity to low levels of tryptophan. Moreover, the engineered biosensors are further optimized by replacing the native promoter of tnaC with a phage-derived constitutive promoter. Intriguingly, the engineered biosensors can be reconstructed for extended application in Pseudomonas putida, a robust microbial chassis for metabolic engineering. In summary, our study expands the toolkit of tryptophan biosensors that can be broadly used for the bioproduction of many other high-value tryptophan-derived products.

Inhibition of pathogenic microorganisms in solid organic waste via black soldier fly larvae-mediated management.

Inadequately managed solid organic waste generation poses a threat to the environment and human health globally. Biotransformation with the black soldier fly larvae (BSFL) is emerging as talent technology for solid waste management. However, there is a lack of understanding of whether BSFL can effectively suppress potential pathogenic microorganisms during management and the underlying mechanisms. In this study, we investigated the temporal variations of microorganisms in two common types of solid waste, i.e., kitchen waste (KW) and pig manure (PM). Natural composting and composting with BSFL under three different pH levels (pH 5, 7, and 9) were established to explore their impact on microbial communities in compost and the gut of BSFL. The results showed that the compost of kitchen waste and pig manure led to an increase in relative abundance of various potentially pathogenic bacteria. Temporal gradient analyses revealed that the most substantial reduction in the relative abundance and diversity of potentially pathogenic microorganisms occurred when the initial pH of both two wastes were adjusted to 7 upon the introduction of BSFL. Through network and pls-pm analysis, it was discovered that the gut microbiota of BSFL occupied an ecological niche in the compost, inhibiting the proliferation of potentially pathogenic microorganisms. This study has revealed the potential of BSFL in reducing public health risks during the solid waste management process, providing robust support for sustainable waste management.

Combined gene cluster engineering and precursor feeding to improve gougerotin production in Streptomyces graminearus.

Gougerotin is a peptidyl nucleoside antibiotic produced by Streptomyces graminearus . It is a specific inhibitor of protein synthesis and exhibits a broad spectrum of biological activities. Generation of an overproducing strain is crucial for the scale-up production of gougerotin. In this study, the natural and engineered gougerotin gene clusters were reassembled into an integrative plasmid by λ-red-mediated recombination technology combined with classic cloning methods. The resulting plasmids pGOU and pGOUe were introduced into S. graminearus to obtain recombinant strains Sgr-GOU and Sgr-GOUe, respectively. Compared with the wild-type strain, Sgr-GOU led to a maximum 1.3-fold increase in gougerotin production, while Sgr-GOUe resulted in a maximum 2.1-fold increase in gougerotin production. To further increase the yield of gougerotin, the effect of different precursors on its production was investigated. All precursors, including cytosine, serine, and glycine, had stimulatory effect on gougerotin production. The maximum gougerotin yield was achieved with Sgr-GOUe in the presence of glycine, and it was approximately 2.5-fold higher than that of the wild-type strain. The strategies used in this study can be extended to other Streptomyces for improving production of industrial important antibiotics.

Improvement of gougerotin and nikkomycin production by engineering their biosynthetic gene clusters.

Nikkomycins and gougerotin are peptidyl nucleoside antibiotics with broad biological activities. The nikkomycin biosynthetic gene cluster comprises one pathway-specific regulatory gene (sanG) and 21 structural genes, whereas the gene cluster for gougerotin biosynthesis includes one putative regulatory gene, one major facilitator superfamily transporter gene, and 13 structural genes. In the present study, we introduced sanG driven by six different promoters into Streptomyces ansochromogenes TH322. Nikkomycin production was increased significantly with the highest increase in engineered strain harboring hrdB promoter-driven sanG. In the meantime, we replaced the native promoter of key structural genes in the gougerotin (gou) gene cluster with the hrdB promoters. The heterologous producer Streptomyces coelicolor M1146 harboring the modified gene cluster produced gougerotin up to 10-fold more than strains carrying the unmodified cluster. Therefore, genetic manipulations of genes involved in antibiotics biosynthesis with the constitutive hrdB promoter present a robust, easy-to-use system generally useful for the improvement of antibiotics production in Streptomyces.

PLGA/BGP/Nef porous composite restrains osteoclasts by inhibiting the NF-κB pathway, enhances IGF-1-mediated osteogenic differentiation and promotes bone regeneration.

BACKGROUND: Novel bone substitutes are urgently needed in experimental research and clinical orthopaedic applications. There are many traditional Chinese medicines that have effects on bone repair. However, application of natural medicines in traditional Chinese medicine to bone tissue engineering and its mechanism were rarely reported. RESULTS: In this study, the osteogenic ability of bioactive glass particles (BGPs) and the osteogenic and osteoclastic ability of neferine (Nef) were fused into PLGA-based bone tissue engineering materials for bone regeneration. BGPs were prepared by spray drying and calcination. Particles and Nef were then mixed with PLGA solution to prepare porous composites by the phase conversion method. Here we showed that Nef inhibited proliferation and enhanced ALP activity of MC3T3-E1 cells in a dose- and time-dependent manner. And the composites containing Nef could also inhibit RANKL-induced osteoclast formation (p < 0.05). Mechanistically, the PLGA/BGP/Nef composite downregulated the expression of NFATC1 by inhibiting the NF-κB pathway to restrain osteoclasts. In the other hands, PLGA/BGP/Nef composite was first demonstrated to effectively activate the IGF-1R/PI3K/AKT/mTOR pathway to enhance IGF-1-mediated osteogenic differentiation. The results of animal experiments show that the material can effectively promote the formation and maturation of new bone in the skull defect site. CONCLUSIONS: The PLGA/BGP/Nef porous composite can restrain osteoclasts by inhibiting the NF-κB pathway, enhance IGF-1-mediated osteogenic differentiation and promotes bone regeneration, and has the potential for clinical application.

Comparative and Functional Analyses Reveal Conserved and Variable Regulatory Systems That Control Lasalocid Biosynthesis in Different Streptomyces Species.

Lasalocid, a representative polyether ionophore, has been successfully applied in veterinary medicine and animal husbandry and also displays promising potential for cancer therapy. Nevertheless, the regulatory system governing lasalocid biosynthesis remains obscure. Here, we identified two conserved (lodR2 and lodR3) and one variable (lodR1, found only in Streptomyces sp. strain FXJ1.172) putative regulatory genes through a comparison of the lasalocid biosynthetic gene cluster (lod) from Streptomyces sp. FXJ1.172 with those (las and lsd) from Streptomyces lasalocidi. Gene disruption experiments demonstrated that both lodR1 and lodR3 positively regulate lasalocid biosynthesis in Streptomyces sp. FXJ1.172, while lodR2 plays a negative regulatory role. To unravel the regulatory mechanism, transcriptional analysis and electrophoretic mobility shift assays (EMSAs) along with footprinting experiments were performed. The results revealed that LodR1 and LodR2 could bind to the intergenic regions of lodR1-lodAB and lodR2-lodED, respectively, thereby repressing the transcription of the lodAB and lodED operons, respectively. The repression of lodAB-lodC by LodR1 likely boosts lasalocid biosynthesis. Furthermore, LodR2 and LodE constitute a repressor-activator system that senses changes in intracellular lasalocid concentrations and coordinates its biosynthesis. LodR3 could directly activate the transcription of key structural genes. Comparative and parallel functional analyses of the homologous genes in S. lasalocidi ATCC 31180T confirmed the conserved roles of lodR2, lodE, and lodR3 in controlling lasalocid biosynthesis. Intriguingly, the variable gene locus lodR1-lodC from Streptomyces sp. FXJ1.172 seems functionally conserved when introduced into S. lasalocidi ATCC 31180T. Overall, our findings demonstrate that lasalocid biosynthesis is tightly controlled by both conserved and variable regulators, providing valuable guidance for further improving lasalocid production. IMPORTANCE Compared to its elaborated biosynthetic pathway, the regulation of lasalocid biosynthesis remains obscure. Here, we characterize the roles of regulatory genes in lasalocid biosynthetic gene clusters of two distinct Streptomyces species and identify a conserved repressor-activator system, LodR2-LodE, which could sense changes in the concentration of lasalocid and coordinate its biosynthesis with self-resistance. Furthermore, in parallel, we verify that the regulatory system identified in a new Streptomyces isolate is valid in the industrial lasalocid producer and thus applicable for the construction of high-yield strains. These findings deepen our understanding of regulatory mechanisms involved in the production of polyether ionophores and provide novel clues for the rational design of industrial strains for scaled-up production.

The best practice for microbiome analysis using R.

With the gradual maturity of sequencing technology, many microbiome studies have published, driving the emergence and advance of related analysis tools. R language is the widely used platform for microbiome data analysis for powerful functions. However, tens of thousands of R packages and numerous similar analysis tools have brought major challenges for many researchers to explore microbiome data. How to choose suitable, efficient, convenient, and easy-to-learn tools from the numerous R packages has become a problem for many microbiome researchers. We have organized 324 common R packages for microbiome analysis and classified them according to application categories (diversity, difference, biomarker, correlation and network, functional prediction, and others), which could help researchers quickly find relevant R packages for microbiome analysis. Furthermore, we systematically sorted the integrated R packages (phyloseq, microbiome, MicrobiomeAnalystR, Animalcules, microeco, and amplicon) for microbiome analysis, and summarized the advantages and limitations, which will help researchers choose the appropriate tools. Finally, we thoroughly reviewed the R packages for microbiome analysis, summarized most of the common analysis content in the microbiome, and formed the most suitable pipeline for microbiome analysis. This paper is accompanied by hundreds of examples with 10,000 lines codes in GitHub, which can help beginners to learn, also help analysts compare and test different tools. This paper systematically sorts the application of R in microbiome, providing an important theoretical basis and practical reference for the development of better microbiome tools in the future. All the code is available at GitHub github.com/taowenmicro/EasyMicrobiomeR.

Growth substrates alter aboveground plant microbial and metabolic properties thereby influencing insect herbivore performance.

The gut microbiome of plant-eaters is affected by the food they eat, but it is currently unclear how the plant metabolome and microbiome are influenced by the substrate the plant grows in and how this subsequently impacts the feeding behavior and gut microbiomes of insect herbivores. Here, we use Plutella xylostella caterpillars and show that the larvae prefer leaves of cabbage plants growing in a vermiculite substrate to those from plants growing in conventional soil systems. From a plant metabolomics analysis, we identified 20 plant metabolites that were related to caterpillar feeding performance. In a bioassay, the effects of these plant metabolites on insects' feeding were tested. Nitrate and compounds enriched with leaves of soilless cultivation promoted the feeding of insects, while compounds enriched with leaves of plants growing in natural soil decreased feeding. Several microbial groups (e.g., Sporolactobacillus, Haliangium) detected inside the plant correlated with caterpillar feeding performance and other microbial groups, such as Ramlibacter and Methylophilus, correlated with the gut microbiome. Our results highlight the role of growth substrates on the food metabolome and microbiome and on the feeding performance and the gut microbiome of plant feeders. It illustrates how belowground factors can influence the aboveground properties of plant-animal systems, which has important implications for plant growth and pest control.