EV-COMM: A database of interspecies and intercellular interactions mediated by extracellular vesicles.

Intra- and inter-organismal interactions play a crucial role in the maintenance and function of individuals, as well as communities. Extracellular vesicles (EVs) have been identified as effective mediators for the communication both within and between species. They can carry and transport molecular cargoes to transmit biological messages. Several databases (ExoBCD, ExoCarta, EVpedia, EV-TRACK, Vesiclepedia) complied the cargoes information including DNA, RNA, protein, lipid and metabolite associated with EVs. Databases that refer to the complete records on both donor and recipient information are warranted to facilitate the understanding of the interaction across cells and species. In this study, we developed a database that compiled the records equipped with a structured process of EV-mediated interaction. The sources of donor and recipient were classified by cell type, tissues/organs and species, thus providing an extended knowledge of cell-cell, species-species interaction. The isolation and identification methods were presented for assessing the quality of EVs. Information on functional cargoes was included, where microRNA was linked to a prediction server to broaden its potential effects. Physiological and pathological context was marked to show the environment where EVs functioned. At present, a total of 1481 data records in our database, including 971 cell-cell interactions belonging to more than 40 different tissues/organs, and 510 cross-species records. The database provides a web interface to browse, search, visualize and download the interaction records. Users can search for interactions by selecting the context of interest or specific cells/species types, as well as functional cargoes. To the best of our knowledge, the database is the first comprehensive database focusing on interactions between donor and recipient cells or species mediated by EVs, serving as a convenient tool to explore and validate interactions. The Database, shorten as EV-COMM (EV mediated communication) is freely available at http://sdc.iue.ac.cn/evs/list/ and will be continuously updated.

A database on the abundance of environmental antibiotic resistance genes.

Antimicrobial resistance (AMR) poses a severe threat to global health. The wide distribution of environmental antibiotic resistance genes (ARGs), which can be transferred between microbiota, especially clinical pathogens and human commensals, contributed significantly to AMR. However, few databases on the spatiotemporal distribution, abundance, and health risk of ARGs from multiple environments have been developed, especially on the absolute level. In this study, we compiled the ARG occurrence data generated by a high-throughput quantitative PCR platform from 1,403 samples in 653 sampling sites across 18 provinces in China. The database possessed 291,870 records from five types of habitats on the abundance of 290 ARGs, as well as 8,057 records on the abundance of 30 mobile genetic elements (MGEs) from 2013 to 2020. These ARGs conferred resistance to major common types of antibiotics (a total of 15 types) and represented five major resistance mechanisms, as well as four risk ranks. The database can provide information for studies on the dynamics of ARGs and is useful for the health risk assessment of AMR.

Temporal dynamics of microbial composition and antibiotic resistome in fermentation bed culture pig farms across various ages.

The discharge from pig farms presents significant challenges to the environment and human health, specifically regarding the dissemination of antimicrobial resistance (AMR). Fermentation bed culture has emerged as an increasingly popular and environmentally friendly pig farming model in China, as it minimizes the release of harmful substances into the environment. However, there remains a limited understanding of the occurrence and dynamics of microbiome and antibiotic resistome in fermentation bed culture. Herein, we collected fermentation bed materials (FBM) from four fermentation bed culture pig farms with varying service ages and investigated their bacterial communities, antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), metal resistance genes (MRGs) and potential antibiotic-resistant bacterial hosts through metagenomics. Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota were identified as the dominant phyla present in the FBM. In total, we detected 258 unique ARGs in the FBM samples, with 79 core ARGs shared by all FBM samples, accounting for 95 % of the total ARG abundance. Our results revealed significant variations in microbial communities and ARG profiles across varying service ages of FBM. Compared to long-term FBW, short-term FBM exhibited higher numbers and abundances of ARGs, MRGs and MGEs, along with higher levels of potential bacterial pathogens and high-risk ARGs. Further analysis of metagenome-assembled genome (MAG) indicated that the putative hosts of ARGs primarily belonged to Pseudomonadota, Actinomycetota and Bacillota. Alarmingly, among the 80 recovered ARG-carrying MAGs, 23 MAGs encoded multi-resistance, including clinically significant species that require urgent attention. Overall, this study provided valuable insights into the temporal patterns of antibiotic resistome and bacterial communities within FBM, enhancing our understanding of FBM in pig farming. The findings could potentially contribute to the development of effective strategies for evaluating and regulating fermentation bed culture practices in pig farming.

Antibiotic resistome and associated bacterial communities in agricultural soil following the amendments of swine manure-derived fermentation bed waste.

The practice of utilizing animal manures on land is widespread in agriculture, but it has raised concerns about the possible spread of antibiotic resistance genes (ARGs) and the potential risk it poses to public health through food production. Fermentation bed culture is an effective circular agricultural practice commonly utilized in pig farming that minimizes the environmental impact of livestock farming. However, this method generates a significant amount of fermentation bed waste (FBW), which can be turned into organic fertilizer for land application. The objective of this research was to examine the impacts of amending agricultural soil samples with swine manure-derived FBW on microbial communities, mobile genetic elements (MGEs), and ARG profiles over different periods. The study findings indicated that the amendment of swine manure-derived FBW significantly increased the diversity and abundance of ARGs and MGEs during the early stages of amendment, but this effect diminished over time, and after 12 months of FBW amendments, the levels returned to those comparable to control samples. The shift in the bacterial communities played a significant role in shaping the patterns of ARGs. Actinobacteriota, Proteobacteria, and Bacteroidetes were identified as the primary potential hosts of ARGs through metagenomic binning analysis. Furthermore, the pH of soil samples was identified as the most important property in driving the composition of the bacterial community and soil resistome. These findings provided valuable insights into the temporal patterns and dissemination risks of ARGs in FBW-amended agriculture soil, which could contribute to the development of effective strategies to manage the dissemination risks of FBW-derived ARGs.

Gut microbiota related response of Oryzias melastigma to combined exposure of polystyrene microplastics and tetracycline.

The co-existence of microplastics (MPs) and antibiotics in the coastal environment poses a combined ecological risk. Single toxic effects of MPs or antibiotics on aquatic organisms have been verified, however, the exploration of their combined toxic effects remains limited. Here, foodborne polystyrene microplastics (PS-MPs, 10 µm, 0.1 % w/w in food) and waterborne tetracyclines (TC, 50 µg/L) were used to expose an estuarine fish Oryzias melastigma for four weeks. We found that the aqueous availability of TC was not significantly altered coexisting with MPs. The fish body weight gain was significantly slower in TC alone or combined groups than the control group, consistent with the lower lipid content in livers. The body length gain was significantly inhibited by the combined presence compared to the single exposure. Both exposures led to a shift of gut microbiota composition and diversity. TC and the combined group possessed similar gut microbiota which is distinct from PS-MPs and the control group. The Firmicutes/Bacteroidetes (F/B) ratio in the TC and combined groups were significantly lower compared to the control, while the PS-MPs group showed no significant impact. Metabolomic analysis of the fish liver confirmed the shift of metabolites in specific pathways after different exposures. More, a number of gut microbiota-related metabolites on lipid metabolism was perturbed, which were annotated in arachidonic acid metabolism and linoleic acid metabolism. In all, TC modulates bacterial composition in the fish gut and disturbs their liver metabolites via the gut-liver axis, which led to the slower growth of O. melastigma. More, the adverse impact was aggravated by the co-exposure to foodborne PS-MPs.

Subsurface Manure Injection Reduces Surface Transport of Antibiotic Resistance Genes but May Create Antibiotic Resistance Hotspots in Soils.

Compared to surface application, manure subsurface injection reduces surface runoff of nutrients, antibiotic resistant microorganisms, and emerging contaminants. Less is known regarding the impact of both manure application methods on surface transport of antibiotic resistance genes (ARGs) in manure-amended fields. We applied liquid dairy manure to field plots by surface application and subsurface injection and simulated rainfall on the first or seventh day following application. The ARG richness, relative abundance (normalized to 16s rRNA), and ARG profiles in soil and surface runoff were monitored using shotgun metagenomic sequencing. Within 1 day of manure application, compared to unamended soils, soils treated with manure had 32.5-70.5% greater ARG richness and higher relative abundances of sulfonamide (6.5-129%) and tetracycline (752-3766%) resistance genes (p ≤ 0.05). On day 7, soil ARG profiles in the surface-applied plots were similar to, whereas subsurface injection profiles were different from, that of the unamended soils. Forty-six days after manure application, the soil ARG profiles in manure injection slits were 37% more diverse than that of the unamended plots. The abundance of manure-associated ARGs were lower in surface runoff from manure subsurface injected plots and carried a lower resistome risk score in comparison to surface-applied plots. This study demonstrated, for the first time, that although manure subsurface injection reduces ARGs in the runoff, it can create potential long-term hotspots for elevated ARGs within injection slits.

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity.

As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4-α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4-α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

Synthesis and Characterization of Cry2Ab-AVM Bioconjugate: Enhanced Affinity to Binding Proteins and Insecticidal Activity.

Bacillus thuringiensis insecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4"-O-succinyl avermectin (AVM) to synthesize Cry2Ab-AVM bioconjugate. It was found that Cry2Ab-AVM showed higher insecticidal activity against Plutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab-AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in the P. xylostella cadherin (PxCR10-11), with a much higher affinity (dissociation equilibrium constant KD = 3.44 nM) than Cry2Ab30 (KD = 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab-AVM ligand docking into the PxCR10-11 is a potential mechanism to enhance the binding affinity of Cry2Ab-AVM to PxCR10-11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.

Exposure of helices α4 and α5 is required for insecticidal activity of Cry2Ab by promoting assembly of a prepore oligomeric structure.

Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis, is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella, although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella. Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.

Proteolytic activation of Bacillus thuringiensis Vip3Aa protein by Spodoptera exigua midgut protease.

Proteolysis of Vip3Aa by insect midgut proteases is essential for their toxicity against target insects. In the present study, proteolysis of Vip3Aa was evaluated by Spodoptera exigua midgut proteases (MJ). Trypsin was verified involved in the activation of Vip3Aa and three potential cleavage sites (Lys195, Lys197 and Lys198) were identified. Four Vip3Aa mutants (KKK195197198AAA, KK197198AA, KK195198AA and KK195197AA) were designed and constructed by replacing residues Lys195,197,198, Lys197,198, Lys195,198 and Lys195,197 with Ala, respectively. Proteolytic processing assays revealed that mutants KK197198AA, KK195198AA and KK195197AA could be processed into 66kDa activated toxins by trypsin or MJ while mutant KKK195197198AAA was not cleaved by trypsin and less susceptible to MJ. Bioassays demonstrated that mutants KK197198AA, KK195198AA and KK195197AA were toxic against S. exigua resembled that of wild-type Vip3Aa, however, the LC50 of mutant KKK195197198AAA against S. exigua was higher than wild-type. Those results suggested that proteolysis by MJ was associated with the insecticidal toxicity of Vip3Aa against S. exigua. It also revealed that trypsin played an important role in the formation of Vip3Aa activated toxin. Our studies characterized the proteolytic processing of Vip3Aa and provided new insight into the activation of this novel Bt toxin.

PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis.

Lepidopteran midgut aminopeptidases N (APNs) are widely studied for their potential roles as one of the receptors for Bacillus thuringiensis (Bt) crystal toxins. In the present study, a loss of function analyses by RNAi (RNA interference) silencing of the Plutella xylostella APN5 (PxAPN5), a binding protein of Bt crystal toxin Cry2Ab, were performed. The knocking down of PxAPN5 in P. xylostella larvae greatly reduced their susceptibility to Cry2Ab and led to a decrease of Cry2Ab binding to P. xylostella midgut. Four truncated fragments of PxAPN5 were further constructed and expressed in Escherichia coli (E.coli) to find the binding region of PxAPN5 to Cry2Ab. The ligand blot result indicated that D1 domain (residues 1-262) and D3 domain (residues 510-620) of PxAPN5 could specially bind to Cry2Ab.

Proteolytic Activation of Bacillus thuringiensis Cry2Ab through a Belt-and-Braces Approach.

Proteolytic processing of Bacillus thuringiensis (Bt) crystal toxins by insect midgut proteases plays an essential role in their insecticidal toxicities against target insects. In the present study, proteolysis of Bt crystal toxin Cry2Ab by Plutella xylostella L. midgut proteases (PxMJ) was evaluated. Both trypsin and chymotrypsin were identified involving the proteolytic activation of Cry2Ab and cleaving Cry2Ab at Arg(139) and Leu(144), respectively. Three Cry2Ab mutants (R139A, L144A, and R139A-L144A) were constructed by replacing residues Arg(139), Leu(144), and Arg(139)-Leu(144) with alanine. Proteolysis assays revealed that mutants R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated toxins by PxMJ. Bioassays showed that mutants R139A and L144A were highly toxic against P. xylostella larvae, while mutant R139A-L144A was almost non-insecticidal. Those results demonstrated that proteolysis by PxMJ was associated with the toxicity of Cry2Ab against P. xylostella. It also revealed that either trypsin or chymotrypsin was enough to activate Cry2Ab protoxin. This characteristic was regarded as a belt-and-braces approach and might contribute to the control of resistance development in target insects. Our studies characterized the proteolytic processing of Cry2Ab and provided new insight into the activation of this Bt toxin.

Draft Genome Sequence of Bacillus muralis LMG 20238T (DSM 16288), a Spore-Forming Bacterium Isolated from Deteriorated Mural Paintings.

Bacillus muralis LMG 20238(T) is a Gram-positive, aerobic, and spore-forming bacterium. Here, we report the 5.18-Mb draft genome sequence of B. muralis LMG 20238(T), which is the first genome sequence of this species and will promote its fundamental research.

Draft Genome Sequence of Bacillus humi LMG 22167T (DSM 16318), an Endospore-Forming Bacterium Isolated from Soil.

Bacillus humi LMG 22167(T) is a Gram-positive, aerobic, and spore-forming bacterium Here, we report the 4.80-Mb draft genome sequence of B. humi LMG 22167(T), which is the first genome sequence of this species and will promote its fundamental research.

Bacillus gobiensis sp. nov., isolated from a soil sample.

A Gram-stain-positive, rod-shaped, endospore-forming, aerobic bacterium designated FJAT-4402T, was isolated from the weed rhizosphere soil of the Gobi desert in the Xinjiang Autonomous Region in the north-west of China. Isolate FJAT-4402T grew at 15-40 °C (optimum 30 °C), pH 5-10 (optimum pH 7) and in 0-3 % (w/v) NaCl (optimum 0 %). Phylogenetic analyses, based on 16S rRNA gene sequences, showed that isolate FJAT-4402T was a member of the genus Bacillus and was most closely related to Bacillus licheniformis DSM 13T (96.2 %). The isolate showed 33.3 % DNA-DNA relatedness to the closest reference isolate, B. licheniformis DSM 13T. The diagnostic diamino acid of the peptidoglycan of isolate FJAT-4402T was meso-diaminopimelic acid and the predominant isoprenoid quinone was MK-7. The major cellular fatty acids were anteiso-C15 : 0 (28.5 %), iso-C15 : 0 (20.1 %), anteiso-C17 : 0 (14.3 %), iso-C16 : 0 (9.6 %), C16 : 0 (8.4 %), iso-C17 : 0 (6.2 %) and iso-C14 : 0 (4.7 %) and the DNA G+C content was 42.0 mol%. The phenotypic, chemotaxonomic and genotypic properties indicated that strain FJAT-4402T represents a novel species within the genus Bacillus, for which the name Bacillus gobiensis sp. nov. is proposed. The type strain is FJAT-4402T ( = DSM 29500T = CGMCC 1.12902T).

Characterization of a new cry2Ab gene of Bacillus thuringiensis with high insecticidal activity against Plutella xylostella L.

Bacillus thuringiensis (Bt) strain FJAT-12 was a novel Bt strain isolated by Agricultural Bio-Resources Institute, Fujian Academy of Agricultural Science. In this study, a new cry2Ab gene was cloned from Bt strain FJAT-12 and named as cry2Ab30 by Bt delta-endotoxin Nomenclature Committee. The sequencing results showed there were two mutations in conservative sites which led to two amino acids modification. Homology modeling indicated that the two changes were located in ß-sheet of Domain II. A prokaryotic expression vector pET30a-cry2Ab30 was constructed and the expressed protein was analyzed by western blot using Cry2Ab antibody. The expression conditions including IPTG concentration, revolution and temperature were optimized to get the highest expression level by SDS-PAGE and BandScan. The bioassay results also showed that the Cry2Ab30 toxin had high insecticidal activity against Plutella xylostella and the LC50 value was 0.0103 µg.mL(-1). The two mutations in ß-sheet of Domain II might contribute to insecticidal activity of Cry2Ab30 toxin against Plutella xylostella.

A protein engineering of Bacillus thuringiensis δ-endotoxin by conjugating with 4"-O-succinoyl abamectin.

Conjugation of Bacillus thuringiensis δ-endotoxin (Bt toxin) with other toxins for insect pest control has been proposed as a new efficient strategy with increasing insecticidal toxicity and target range and delay the onset of insect resistance. A modified method was investigated by conjugating Bt toxin with 4"-O-succinoyl abamectin to form a new biocide which was named as BtA. 'Zero-length' cross-linker EDC in combination with NHS activated 4"-O-succinoyl abamectin and extended half-life period of active intermediate for binding to Bt toxin. The dissociation constant for 4"-O-succinoyl abamectin binding to Bt toxin was 6.44 µM by fluorescence quenching analysis. BtA showed a higher insecticidal toxicity against Plutella xylostella, while the relative-toxicity multiple of BtA to Bt toxin was calculated as 5.6. The interaction between Bt toxins with their receptors played a key role in toxicity of Bt toxins. The binding analysis showed the dissociation rate for the binding of BtA to its receptors (7.495 × 10(-3) S(-1)) was twice slower than that of Bt toxin (1.695 × 10(-2) S(-1)). The relative dissociation constant of BtA to Bt toxin was only 29% for the binding to the receptors. These results demonstrated that BtA bound to the receptor in BBMV with significantly higher affinity compared with Bt toxin.

Synthesis of 4'-thiosemicarbazonegriseofulvin and its effects on the control of enzymatic browning and postharvest disease of fruits.

4'-Thiosemicarbazonegriseofulvin, a new thiosemicarbazide derivative of griseofulvin, was synthesized and evaluated for its potential in the control of enzymatic browning and postharvest disease of fruits. Browning on fruits is mainly due to the enzymatic oxidation of phenolic compounds catalyzed by tyrosinase. 4'-Thiosemicarbazonegriseofulvin could effectively inhibit the activity of tyrosinase, and its 50% inhibitory concentration (IC(50)) against tyrosinase was determined to be 37.8 µM. It was a reversible and noncompetitive inhibitor of tyrosinase, and its inhibition constant (K(I)) was determined to be 38.42 µM. The antifungal activity of 4'-thiosemicarbazonegriseofulvin was studied against four fungi (Fusarium oxysporum, Fusarium moniliforme, Fusarium solani, and Colletotrichum truncatum) that often cause postharvest diseases of fruits. The results showed that 4'-thiosemicarbazonegriseofulvin could also strongly inhibit the mycelial growth of the four target fungi; the 50% lethal concentration (LC(50)) values were 5.4, 7.0, 15.3, and 1.5 mM, respectively.

Synthesis and antityrosinase activities of alkyl 3,4-dihydroxybenzoates.

In insects, tyrosinase plays important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. Thus, tyrosinase may be regarded as a potential candidate for novel bioinsecticide development. A family of alkyl 3,4-dihydroxybenzoates (C6-C9), new tyrosinsase inhibitors, were synthesized. Their inhibitory effects on the activity of tyrosinase have been investigated. The results showed all of them could inhibit the activity of tyrosianse effectively. The order of potency was nonyl 3,4-dihydroxybenzoate (C9DB) > octyl 3,4-dihydroxybenzoate(C8DB) > heptyl 3,4-dihydroxybenzoate(C7DB) > hexyl 3,4-dihydroxybenzoate (C6DB). The kinetic analysis of these four compounds on tyrosinase was taken to expound their inhibitory mechanism. The research of the control of insects in agriculture was taken as C6DB for example. C6DB could inhibit the development and molting of Plutella xylostella effectively. To clarify its insecticidal mechanism, we researched the expression of tyrosinase in the P. xylostella treated with C6DB by real-time quantitative PCR. The results showed C6DB could inhibit the expression of tyrosinase in the P. xylostella as expected.

Inhibitory effects of fatty acids on the activity of mushroom tyrosinase.

The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.