Brevibacillus laterosporus (B. laterosporus) is a significant probiotic bacterium known for producing multiple secondary metabolites with notable antibacterial activity. In this study, bioinformatic tools such as FastANI and Roary were employed to analyze genomic similarity and construct the B. laterosporus pan-genome. The software platforms antiSMASH and BAGEL4 were used to predict biosynthetic gene clusters (BGCs) and identify bacteriocins. Core candidate bacteriocins were subsequently subjected to heterologous expression in E. coli, followed by nickel column purification and concentration. Their antibacterial activity was systematically evaluated using agar diffusion assays, minimum inhibitory concentration (MIC) determinations, time-kill curves, scanning electron microscopy (SEM), and live/dead bacterial staining in Listeria monocytogenes (L. monocytogenes). Pan-genome analysis revealed an open genome for B. laterosporus, comprising 2,840 core genes. KEGG enrichment analyses indicated that these core genes were significantly associated with pathways responsible for the biosynthesis of antimicrobial compounds, including secondary metabolite, pan-quinone, and terpenoid quinone biosynthesis. AntiSMASH predicted 1,654 BGCs, predominantly non-ribosomal peptide synthase (NRPS), polyketide synthase (PKS), and heterocyclic amine-associated antimicrobial protein synthesis clusters. Moreover, BAGEL4 predicted three putative core candidate bacteriocin variants (BreL1, BreL2, and BreL3), all of which exhibited pronounced antibacterial activity against L. monocytogenes. Among these, BreL1 demonstrated the most robust activity, causing morphological changes consistent with membrane-associated damage. Collectively, this study elucidates the open pan-genome architecture and the metabolically rich secondary metabolome of B. laterosporus, identifies three putative bacteriocin variants with active antibacterial efficacy against L. monocytogenes, which provides a theoretical foundation for developing natural antimicrobial agents.

Salmonella Typhimurium (S. Typhimurium) is a major foodborne Gram-negative pathogen, presenting significant health risks to both humans and animals. LuxR family regulatory factors are critically involved in bacterial adaption to environmental changes and virulence regulation. Nevertheless, the biological function of YjjQ, a member of this family, remains uncharacterized in S. Typhimurium. To clarify the regulatory function of YjjQ in S. Typhimurium, λ-Red homologous recombination was employed to construct the yjjQ gene deletion strain (SL1344-ΔyjjQ) and its complementation strain (SL1344-ΔyjjQ/comp). Then, the growth characteristics, biofilm formation, motility, cell adhesion and invasion, intracellular survival and proliferation, as well as virulence, were systematically analyzed, respectively. Furthermore, the expression profiles of those genes associated with biofilm, flagella, and virulence were evaluated, and the interaction between YjjQ and predicted target promoter was experimentally validated. Compared to SL1344 and SL1344-ΔyjjQ/comp, SL1344-ΔyjjQ exhibited notably enhanced biofilm formation, motility, cell invasion, and intracellular proliferation under hyperosmotic conditions, in addition to heightened virulence in mice. RT-qPCR analysis revealed these genes related to flagellar (flhD, fliA), biofilm (csgD, fliC), and SPI-1 (invF) were significantly upregulated. The EMSA confirmed that YjjQ protein directly binds the flhDC promoter region, suppressing transcription of the flhDC operon, thereby negatively regulating flagellar, biofilm, and virulence. These findings provide new insights into the molecular mechanisms underlying the coordinately modulated strategy of biofilm formation, flagellar biosynthesis and virulence in S. Typhimurium.

Mycoplasma bovis is a major etiological agent of bovine respiratory disease, causing substantial economic losses to the cattle industry. To address this challenge, we performed a pan-genome analysis of 80 globally sourced M. bovis genomes and identified 1058 core genes. Reverse vaccinology and immunoinformatics approaches were subsequently integrated to screen three potential vaccine candidates: namely lipoate protein ligase 2 (LplA), elongation factor 4 (LepA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Codon-optimized gene sequences were cloned into the pET-28a vector and successfully expressed and purified using E. coli expression system. Thirty female BALB/c mice were randomized into five groups (n = 6 per group). For primary immunization, each mouse received 100 μg of recombinant protein emulsified in Freund's complete adjuvant via subcutaneous injection, followed by booster immunizations with 50 μg of protein formulated with Freund's incomplete adjuvant at weeks 2 and 4. Fourteen days after primary immunization, all vaccinated groups exhibited significantly elevated antigen-specific antibody levels, which remained high through week 6. Notably, rLplA and rGAPDH induced higher antibody titers than rLepA. Analysis of the IgG2a/IgG1 subtype ratio indicated that rLplA, rGAPDH, and rLepA predominantly elicited Th2-type immune responses. In addition, all three recombinant proteins significantly increased cytokine production. Moreover, no adverse physiological reactions or histopathological abnormalities were observed throughout the immunization period, confirming the favorable safety profile of the candidates. Collectively, these findings identify promising targets for the development of M. bovis subunit vaccines to address pathogen variability, antibiotic resistance, and the limited efficacy of current vaccines.

This study aimed to identify and characterize a high-efficiency chitinase gene from Brevibacillus laterosporus (B. laterosporus), characterize its enzymatic traits, and assess its degrading activity against Caenorhabditis elegans (C. elegans) and Parascaris equorum (P. equorum) eggs. A nematicidal B. laterosporus isolate was subjected to whole-genome sequencing for chitinase gene screening, cloning, and molecular identification. The target gene was heterologously expressed in Escherichia coli BL21 (DE3), and biochemical properties including temperature, pH, metal ions, and substrate conditions were investigated, its kinetic parameters were determined, and its biological effects on C. elegans and P. equorum eggs were analyzed. The protein BLChi79 (79.3 kDa) belongs to GH18 chitinases with a typical carbohydrate-binding module. Its optimum activity occurred at 60 °C and pH 6.0, with colloidal chitin as the optimal substrate. Mg2+, Fe2+, and Mn2+ boosted its activity, while K+, Cu2+, Zn2+, and Ca2+ suppressed catalysis. Its Km was 6.14 mg·mL-1, Vmax 7.78 μmol·min-1·mg-1, and kcat 10.16 min-1. The purified recombinant enzyme could degradethe C. elegans chitin layer and the P. equorum egg vitelline envelope. In summary, the BLChi79 derived from B. laterosporus targets chitin-enriched eggshells, acting as a green biocatalyst for controlling livestock gastrointestinal nematodes.

Listeria monocytogenes (L. monocytogenes) is a zoonotic foodborne pathogen that triggers life-threatening invasive illnesses. MarR-type transcriptional regulators widely control bacterial stress adaptation, antibiotic tolerance and virulence, playing crucial roles in the pathogenesis of diverse bacterial pathogens. Lmo0840 is a conserved member of this family; however, its physiological functions and regulatory networks in L. monocytogenes remain completely unexplored. To this end, we constructed an lmo0840 deletion mutant and its complemented strain in the L. monocytogenes EGD-e background and systematically characterized the biological functions of Lmo0840 using phenotypic assays, transcriptomic profiling, and electrophoretic mobility shift assays (EMSA). Phenotypic characterization revealed that loss of lmo0840 impaired growth rate under hyperosmotic, oxidative, and iron-limitation stresses, while unexpectedly promoting growth rate at 30 °C and under tetracycline stress. Consistently, Disk diffusion assays further showed that the Δlmo0840 mutant exhibited significantly reduced inhibition zone diameters against tetracycline antibiotics compared to the EGD-e strain. Moreover, the Δlmo0840 mutant exhibited reduced early biofilm formation and attenuated macrophage adhesion, yet showed enhanced cellular invasion and decreased virulence in a murine model. EMSA further demonstrated that Lmo0840 directly binds to the promoter region of lmo0839 and represses its transcription, thereby contributing to bacterial adaptation to tetracycline stress. Collectively, these findings establish Lmo0840 as a pleiotropic regulator involved in environmental stress responses, tetracycline adaptation, and virulence in L. monocytogenes. Notably, this study identifies for the first time an Lmo0840–Lmo0839 regulatory cascade underlying tetracycline adaptation. These insights not only deepen our understanding of the molecular mechanisms governing Lm environmental adaptation and pathogenicity but also provide a potential target for the prevention and control of listeriosis.

The antibacterial activities of numerous medium and long-chain unsaturated fatty acids are well documented. However, the natural short-chain unsaturated fatty acid, 4-pentenoic acid (C5:1 Δ4) has not been systematically evaluated for its antibacterial activity, mechanism of action, or application potential. This study investigated these to provide a basis for novel preservatives. 4-Pentenoic acid showed broad-spectrum activity against 21 strains of pathogenic bacteria, with MICs 1.5-3 mg/mL, MBCs 3-6 mg/mL, and low resistance risk. It disrupted bacterial membrane integrity, inhibited biofilm formation, and regulated the pdu, nar and RND efflux pump genes, thereby affecting bacterial metabolism and stress responses. Checkerboard broth microdilution susceptibility assays confirmed that the combination of 4-pentenoic acid with antibiotics (such as meropenem) exerted additive antibacterial effects. At effective antibacterial concentrations, oral administration of 4-pentenoic acid caused no observable toxicity. It can markedly inhibit the proliferation of pathogenic bacteria in milk, beef, and peanut butter, while dosages close to the minimum inhibitory concentration exerted minimal adverse effects on the sensory properties and pH of these foods. In summary, this study provides theoretical and experimental basis for the development of new food preservatives and antibacterial agents.

Salmonella Typhimurium (S. typhimurium) is a significant zoonotic pathogen responsible for gastroenteritis and severe systemic infections in various hosts. The AraC family transcription factors are key gene expression regulators in prokaryotes, essential for bacterial adaptation to the environment and virulence. Despite their importance, the role of yeaM, a member of this family in S. typhimurium, remains unexplored. To elucidate yeaM regulatory function in virulence and biofilm formation, we engineered mutant and complementary strains of the yeaM gene using homologous recombination. We assessed their capabilities in biofilm formation under different conditions, macrophage adherence and invasion, and virulence in mice. Additionally, we identified potential target genes regulated by yeaM through transcriptome sequencing and confirmed these findings using an electrophoretic mobility shift assay (EMSA) and a dual-luciferase reporter assay. Our results demonstrate that, compared to the parental strain SL1344 and the complemented strain C Delta yeaM, the Delta yeaM strain exhibited significantly enhanced biofilm formation, increased invasion of mouse intestinal epithelial cells, enhanced intracellular proliferation within macrophages, and elevated induction of macrophage apoptosis. Furthermore, the Delta yeaM deletion strain displayed significantly increased virulence in mice and enhanced proliferation in milk. Transcriptome analysis revealed that S. typhimurium pathogenicity island 4 (SPI4) genes (siiA, siiB, siiC, siiD, siiF, and siiE) were significantly upregulated following the deletion of the yeaM gene. EMSA and dual-luciferase reporter assays further showed that the yeaM protein can bind to the promoter of the siiA gene and suppress its expression, thereby modulating the biofilm formation and virulence of S. typhimurium.

Introduction:Nematode-trapping fungi (NTFs) can produce various chitinases to degrade nematode body wall and eggshell chitin during predation. However, the regulatory mechanisms of their expression of chitinases still remain unclear. The primary objective of this study was to elucidate the differential protein profile of A. oligospora, an NTF, in response to chitin. Material and Methods:Colloidal chitin was added to induce the culture of A. oligospora, and the phenotypic differences before and after induction were observed under inverted microscope. The differential proteins before and after mycelium induction were screened by liquid chromatography-tandem mass spectrometry. The differentially expressed chitinase was expressed in Pichia yeast, and the recombinant enzyme was incubated with Caenorhabditis elegans and its egg suspension to explore its biological activity. Results:It was found that there was a significant acceleration in the mycelial growth post chitin interaction in A. oligospora. A total of 1,124 differentially expressed proteins (DEPs) were identified between the control group (AO-c) and the experimental group (AO-e), with 183 upregulated and 941 downregulated. Gene Ontology analysis revealed that the DEPs acted in various metabolic processes with catalysis and binding functions. Kyoto Encyclopedia of Genes and Genomes analysis associated these proteins primarily with signalling pathways related to glucose metabolism. Three chitinases were significantly modulated among DEPs. Moreover, enzymatic activity assays demonstrated that one of them effectively degraded C. elegans and its eggs. Conclusion:These findings suggest that A. oligospora can significantly alter its protein expression profile in response to chitin, thereby facilitating its sugar metabolism and mycelial development. Our study provided new insights into the regulatory mechanisms of nematode predation in A. oligospora.

The nematode-trapping fungus (NTF) Arthrobotrys oligospora (A. oligospora) is a promising biocontrol agent, but the transcriptional regulators governing its predation remain poorly understood. Here, we demonstrated that the APSES transcription factor AoMbp1 is a master regulator of its development and stress adaptation. Deletion of AoMbp1 severely impaired mycelial growth, conidiation, trap formation, and tolerance to oxidative and osmotic stresses. Transcriptome analysis revealed that these defects were associated with the widespread downregulation of genes, including those within the MAPK signaling pathway. Crucially, we showed that AoMbp1 directly binds to the promoter of AoSho1, a key upstream sensor of the MAPK cascade, and activates its expression. This finding establishes a direct AoMbp1-AoSho1 regulatory axis controlling trap morphogenesis and environmental adaptation. Our study provides novel mechanistic insights into the regulation of nematode trapping and identifies a potential target for enhancing the efficacy of A. oligospora as a biocontrol agent.

Exosomes are nanoscale vesicles secreted by cells that play vital regulatory roles in intercellular communication and immune responses. Listeria monocytogenes (L. Monocytogenes, LM) is a notable Gram-positive intracellular parasitic bacterium that infects humans and diverse animal species. However, the specific biological function of exosomes secreted by macrophages during L. Monocytogenes infection (hereafter EXO-LM) remains elusive. Here, we discovered that EXO-LM stimulated the secretion of inflammation-associated cytokines by macrophages, facilitating the intracellular survival of L. monocytogenes within macrophages. Transcriptomic analysis shows that EXO-LM significantly upregulates immune recognition and inflammation-related signaling pathways in macrophages. Furthermore, a ceRNA regulatory network comprising exosomal ncRNAs and macrophage RNAs was constructed through EXO-LM transcriptome sequencing. Utilizing bioinformatics and dual-luciferase reporter assays, we identified two potential binding sites between lncRNA Rpl13a-213 and miR-132-3p. Cell transfection experiments demonstrated that Rpl13a-213 overexpression augmented pro-inflammatory cytokine expression in macrophages, in contrast to the suppression by miR-132-3p overexpression. The decrease in Rpl13a-213 upon EXO-LM stimulation enhances miR-132-3p expression, dampening the inflammatory response in macrophages and aiding L. monocytogenes intracellular survival. This study unveils the immunomodulatory function of exosomal ncRNAs originating from macrophages, which provides fresh perspectives into the mechanisms underlying macrophage inflammatory response regulation by L. monocytogenes-infected cell-derived exosomes.

The genomes of 40 Brucella strains were retrieved from the NCBI database to investigate Brucellosis at the genomic level, focusing on secondary metabolites, resistance genes, and virulence factors. Genome analysis software, secondary metabolite mining tools, and relevant gene databases were employed for detailed analysis. The genome sizes of these strains range from 4.88 to 6.00 MB, with G+C content between 53.5 and 60.5%. Phylogenetic analysis classified the strains into three distinct clades: Brucella anthropi CCUG 34461, Brucella sp. NBRC 13694, and Brucella anthropi MAG47. Pan-genome analysis revealed 21,800 gene families, 198 core genes, and 10,371 unique genes, indicating an open pan-genome. The secondary metabolite mining software identified 18 categories and 350 gene clusters, predicting a total of 298 secondary metabolites, primarily arylpolyene, acyl-amino acids, betalactone, terpene, hydrogen cyanide, and NAGGN. Genome sequences were uploaded in FASTA format to the CARD resistance gene database, identifying seven resistance genes: rpsE, rpsL, rosA, golS, fabG, fabI, and uL3. B. anthropi SBA01 and B. media Q1108 were found to harbor the highest number of drug resistance genes. Likewise, the sequences were compared to the VFDB virulence gene database, revealing eight virulence genes: lpxC, acpXL, fliY, bspJ, lpxA, fliI, fliQ, and bvrR. The B. cytisi IPA7.2 strain exhibited the highest number of virulence genes, with lpxC and acpXL potentially being unique to Brucella compared to other species. This study provides comprehensive genomic data, elucidating the relationship between the pan-genome, core genome, and genome size, while predicting the types of secondary metabolites, resistance, and virulence genes. These findings provide a basis for comprehensively understanding Brucella and lay a solid foundation for its prevention and treatment.

Introduction. Salmonella Typhimurium (STM) is a food-borne Gram-negative bacterium, which can infect humans and a wide range of livestock and poultry, causing a variety of diseases such as septicaemia, enteritis and abortion.Hypothesis/Gap Statement. We will decipher the impacts of sRNA STnc1280 on STM virulence and provide a theoretical basis to reveal the regulatory role and molecular mechanism of STnc1280.Aim. The main objective of this study was to clarify whether sRNA STnc1280 exerts regulatory roles on STM pathogenicity.Methodology. The STnc1280 gene was amplified and its molecular characteristics were analysed in this study. Then, STnc1280 gene deletion strain (STM-ΔSTnc1280) and the complementary strain (ΔSTnc1280/STnc1280) were constructed by λ-Red homologous recombination method, respectively, to analyse of adhesion and invasive ability and pathogenicity of different strains. Subsequently, the potential target gene regulated by STnc1280 was predicted using target RNA2 software, followed by the verification of the interaction between STnc1280 and target mRNA using the dual plasmid reporter system (DPRS). Furthermore, the mRNA and protein level of target gene was determined using qRT-PCR and Western blot, respectively.Results. The results revealed that the cell adhesion and invasive ability and pathogenicity of STM-ΔSTnc1280 were significantly reduced compared to STM-SL1344 strain, indicating that the deficiency of STnc1280 gene significantly influenced STM pathogenicity. The DPRS results showed that STnc1280 can interact with the mRNA of target gene gldA, thus suppressing the expression of lacZ gene. Furthermore, the level of gldA mRNA was not influenced in STM-ΔSTnc1280, but the expression of GldA protein decreased significantly.Conclusion. Combining the bioinformatic analysis, these findings suggested that STnc1280 may bind to the SD sequence of gldA mRNA, hindering the binding of ribosomes to gldA mRNA, thereby inhibiting the expression of GldA protein to modulate the virulence of STM.

The effect of functionalized graphene on the growth and development of Vicia faba L. was investigated by analyzing its impact on the composition and diversity of the microbial community in rhizosphere peat soil. Seedlings of V. faba planted in this peat soil were treated with either distilled water (CK) or 25 mgL-1 (G25) of functionalized graphene solution. Results showed that the height and root length of V. faba seedlings in the G25 group were significantly larger than those in CK group. The microbial community was analyzed by amplifying and sequencing the 16S rRNA gene V3-V4 region of bacteria and internal transcribed spacer region of fungi in rhizosphere soil using Illumina MiSeq technology. Alpha and beta diversity analysis indicated that functionalized graphene increased the richness and diversity of bacteria and fungi in the V. faba rhizosphere peat soil. The abundances of three nitrogen cycling-related bacteria, Hydrogenophaga, Sphingomonas and Nitrosomonadaceae, were also altered after treatment with the functionalized graphene. The relative abundance of Basilicum, related to soil phosphorus solubilization, decreased in the fungal community, while the relative abundance of Clonostachys and Dimorphospora, which exhibited strong biological control over numerous fungal plant pathogens, nematodes and insects, increased in the soil after functionalized graphene treatment. Redundancy analysis revealed that the potential of hydrogen (pH), organic matter, and total phosphorus contributed the most to the changes in bacterial and fungal community composition in the rhizosphere soil. Overall, our findings suggested that the addition of functionalized graphene altered the relative abundances of nitrogen and phosphorus cycling-related microorganisms in peat soil, promoting changes in the physicochemical properties of the soil and ultimately leading to the improved growth of V. faba plants.

Salmonella Typhimurium (STM) is an important zoonotic Gram-negative pathogen that can cause infection in a variety of livestock and poultry. Meanwhile, as an important foodborne pathogen, the bacterium can survive in various stressful environments and transmits through the fecal-oral route, posing a serious threat to global food safety. To investigate the roles of STM1863, a member of the DUFs protein family, involved in STM environmental adaptation, biofilm formation, and virulence. We analyzed the molecular characteristics of the protein encoded by STM1863 gene and examined intra- and extracellular expression levels of STM1863 gene in mouse macrophages. Furthermore, we constructed STM1863 gene deletion and complementation strains and determined its environmental adaptation under stressful conditions such as acid, alkali, high salt, bile salt, and oxidation. And the capacity of biofilm formation and pathogenicity of those strains were analyzed and compared. In addition, the interaction between the promoter of STM1863 gene and RcsB protein was analyzed using DNA gel electrophoresis migration assay (electrophoretic mobility shift assay [EMSA]). The experiments revealed that acid adaptability and biofilm formation ability of STM1863 gene deletion strain were significantly weakened compared with the parental and complementary strains. Moreover, the adhesion and invasion ability of STM1863 deletion strain to mouse macrophages was significantly decreased, while the median lethal dose (LD50) increased by 2.148-fold compared with the parental strain. In addition, EMSA confirmed that RcsB protein could bind to the promoter sequence of STM1863 gene, suggesting that the expression of STM1863 gene might be modulated by RcsB. The present study demonstrated for the first time that STM1863, a member of the DUFs protein family, is involved in the modulation of environmental adaptation, biofilm formation, and virulence.

Reducing nonpoint-source pollution and improving soil quality by applying best management practices (BMPs) on sloping farmland are essential for ensuring water environmental safety and sustainable agricultural development. However, little is known about the accumulative effect of the long-term application of BMPs on reducing pollution and improving soil quality in major land use types of sloping farmland in watersheds. We conducted a nine-year field experiment for citrus orchard and cropland of sloping farmland in the Three Gorges Reservoir area of China. The treatment settings for the citrus experimental plots were as follows: (1) citrus intercropped with white clover (WC), (2) citrus orchard soil mulched with straw (SM), (3) citrus intercropped with Hemerocallis flava contour hedgerows (HF) and (4) conventional citrus planting pattern (C-CK). The treatment settings for the crop experimental plots were as follows: (1) wheat-peanut rotation intercropped with Toona sinensis contour hedgerows (TS), (2) wheat-peanut rotation intercropped with alfalfa contour hedgerows (AF), (3) ryegrass-sesame rotation (RS) and (4) conventional wheat-peanut rotation (W-CK). We found that compared with C-CK and W-CK, (1) runoff, sediment, total nitrogen and total phosphorus losses were reduced by 18.0 %, 66.1 %, 35.6 % and 40.2 %, respectively, in WC; by 22.4 %, 61.2 %, 43.9 % and 52.4 %, respectively, in SM; by 25.4%, 69.5 %, 52.0 % and 58.1 %, respectively, in HF; by 45.1 %, 73.7 %, 64.6 % and 66.0 %, respectively, in TS; by 41.8 %, 67.4 %, 59.3 % and 60.8 %, respectively, in AF; and by 24.1 %, 42.9 %, 39.3 % and 40.3 %, respectively, in RS; (2) the soil quality index values of the BMP treatments increased; and (3) the application of these BMPs did not significantly affect yield or product value. We suggest the application of these BMPs on sloping farmland to (1) efficiently reduce total runoff, total sediment, and soil total nitrogen and total phosphorus losses; (2) improve soil quality; and (3) have no significant effect on output, of which the BMP HF applied to citrus orchard and the BMP TS applied to cropland are the most effective.

Bacteriophages are key reservoirs of antibiotic resistance genes (ARGs) and are pivotal for their acquisition, maintenance, and dissemination. As extensively distributed contaminants, nanomaterials can interfere with the horizontal transfer of phage-borne ARGs. However, the effects of environmental factors on this process remain unclear. Therefore, the effect of nanoscale titanium dioxide (nTiO2) on the dissemination of ARGs via bacteriophage transduction under humic acid (HA) and Ultraviolet A illumination was investigated in this study. In the dark, 20 mg/L nTiO2 did not promote the propagation of ARGs and attenuated the positive effect of HA on the transductive transfer of ARGs. Further evidence revealed that nTiO2 can absorb HA on its surface and decrease the free HA content, thereby mitigating the instability of the outer membrane and leading to diminished transductant formation. Conversely, coexposure to HA and nTiO2 synergistically promoted transduction under Ultraviolet A illumination. Massive superoxide radicals produced by photoexcitation of the HA-nTiO2 complex elevated the membrane permeability and boosted intracellular oxidative stress, resulting in the enhanced dissemination of ARGs through phage transduction. The results of this study provide insights into the roles of environmental factors in transductive ARGs dissemination mediated by nTiO2 and are beneficial for the risk assessment of nanomaterials.

Extracellular proteases, such as chitinases secreted by Arthrobotrys oligospora (A. oligospora), play a crucial role in the process of nematode infection. However, post-transcriptional regulation of gene expression involving microRNAs (miRNAs) in A. oligospora remains scarcely described. Hereto, transcriptome sequencing was carried out to analyze the expression profiles of chitin-responsive miRNAs in A. oligospora. Based on the RNA-seq data, the differential expression of miRNAs (DEmiRNAs) in response to chitin was screened, identified and characterized in A. oligospora. Meanwhile, the potential target genes were predicted by the online tools miRanda and Targetscan, respectively. Furthermore, the interaction of DEmiRNA with it’s target gene was validated by a dual-luciferase reporter assay system. Among 85 novel miRNAs identified, 25 miRNAs displayed significant differences in expression in A. oligospora in response to chitin. Gene Ontology (GO) analysis showed that the potential genes targeted by DEmiRNAs were enriched in the biological processes such as bio-degradation, extracellular components and cell cycle. KEGG analysis revealed that the target genes were mainly involved in Hippo, carbon and riboflavin metabolic pathway. Outstandingly, chitinase AOL_s00004g379, which is involved in the hydrolysis metabolic pathway of chitin, was confirmed to be a target gene of differential miR_70. These findings suggest that chitin-responsive miRNAs are involved in the regulation of cell proliferation, predator hyphae growth and chitinase expression through the mechanisms of post-transcriptional regulation, which provides a new perspective to the molecular mechanisms underlying miRNAs-mediated control of gene expression in A. oligospora.

As important post-transcriptional regulators of gene expression, sRNAs play important modulatory roles in the environmental adaptation and virulence of bacteria. To investigate the regulatory role of sRNA STnc3020 in the virulence of Salmonella typhimurium (S. typhimurium). This study analyzed the impacts of STnc3020 deletion on adherence, invasion, intracellular survival, macrophage apoptosis, and pathogenicity of S. typhimurium in mice. Furthermore, potential regulatory target genes of STnc3020 were identified and its regulatory mechanism was validated. The results showed that at the cellular level, the deletion of STnc3020 significantly reduced the adhesion ability of S. typhimurium to intestinal epithelial cells (P < 0.01), as well as its proliferation and apoptosis-inducing abilities within macrophages (P < 0.01). Meanwhile, animal experiment results indicated that the deletion of STnc3020 significantly reduced the colonization rate of S. typhimurium in the liver and cecum of mice (P < 0.01), and increased the median lethal dose (4.28 × 105) in mice. Regulatory mechanism research results showed that STnc3020 can interact with the target gene prgJ of the Type III secretion system (T3SS), and the protein level of PrgJ significantly decreased after the deletion of STnc3020 (P < 0.01). These findings offer new insights into sRNA-mediated virulence control and may aid in developing new vaccines and drugs for S. typhimurium.

Graphene has promising applications in agriculture and forestry. In the current study, six different concentrations of graphene (0mg/L, 0.01mg/L, 0.10mg/L, 1.00mg/L, 10.00mg/L, and 100.00mg/L) were used to investigate its effect on the growth and development of V. angularis plants in soil culture. The results showed that the group treated with 1.00mg/L graphene (G-1) had significantly increased plant height (19.86%), stem diameter (24.33%), and leaf area (13.69%), compared to the control group (CK). Moreover, all concentrations of graphene had positive effects on the total root length, total root surface area, and the number of root tips of V. angularis. Compared to the CK group, the G-1 group had significantly increased leaf water potential (37.89%), leaf conductivity (2.25%), and SOD, POD, and CAT activities (47.67%, 35.22%, and 199.3%, respectively). The G-1 group also showed improved leaf net photosynthetic rate, chlorophyll content, and soluble sugar content (51.28%, 24.25%, and 38.35%, respectively), compared to the CK group. Additionally, 1.00mg/L graphene led to a 23.88% increase in the podding rate and a 17.04% increase in the yield of V. angularis plants. The rhizosphere soil of V. angularis treated with 1.00mg/L graphene had a 25.14% increase in hydrolyzable nitrogen content and a 66.67% increase in available phosphorus content. RNA-seq data indicated that 1.00mg/L graphene induced the expression of photosynthesis and nitrogen transmembrane transport genes, including ATP synthase subunit b, photosystem I reaction center subunit XI, photosystem I reaction center subunit IV A, ferredoxin, and psbP-like protein 1, as well as genes for photosynthesis antenna proteins, glutamine synthetase, glutamate dehydrogenase 1, cyanate hydratase, protein fluG-like, and NRT1/PTR family, suggesting that graphene promoted the growth and development of V. angularis by enhancing the photosynthesis and nitrogen metabolism processes in V. angularis plants. Our results indicated that a suitable concentration of graphene could significantly promote the growth of V. angularis plants in soil.