[Objective] To construct a recombinant vaccinia virus strain WR that expresses dual reporters (luciferase Fluc and red fluorescent protein mCherry). [Methods] Firstly, the gRNA CRISPR/Cas9 plasmid targeting the J2R region of WR and the plasmid pJSE-Fluc/mCherry carrying the dual reporter genes were constructed. Then, the CRISPR/Cas9 gene editing tool was used to insert the dual reporter genes into the TK region, and thus the recombinant vaccinia virus strain rWR-Fluc/mCherry (rWR) was constructed. The location and sequence of insertion in rWR were analyzed by PCR and sequencing. The recombinant strain rWR was characterized by mCherry/Fluc activity and plaque assays. The recombinant strain rWR was subcultured for 12 passages, and the expression levels of the dual reporter genes and E3L were determined to reveal the genetic stability of the strain. To analyze the replication dynamics of the virus in Vero and HeLa cells, we determined the cytopathic effect (CPE), TCID₅₀, and dual reporter expression of rWR and the wild type (WR) in the infected cells. Furthermore, we evaluated the inhibitory effects of ST-246 as a positive drug on both rWR and WR in vitro by the plaque assay, qPCR, and dual reporter activity measurement. [Results] Fluc and mCherry were accurately inserted into the TK region of WR. The Vero cells infected with rWR showed the activities of dual reporters and the plaque morphology consistent with that of WR. After 12 passages, the dual reporter activities and E3L expression were stably detected in rWR, which indicated that rWR was successfully constructed and genetically stable. The CPE, TCID₅₀, and dual reporter activity in Vero and HeLa cells indicated that replication peaked 48–72 h post-infection with rWR, which was consistent with the replication dynamics of WR. The median effective concentration (EC₅₀) of ST-246 against rWR was in agreement with that against WR, and the EC₅₀ (2–7 nmol/L) obtained by the plaque assay, qPCR, and dual reporter activity measurement showed good consistency (r > 0.500 0 and P < 0.05). [Conclusion] A recombinant vaccinia virus strain rWR simultaneously expressing Fluc and mCherry was successfully constructed, and it was genetically stable. This strain might be used as an in vitro system for rapid screening and characterization of anti-orthopoxvirus drugs with simple operation.

Coastal wetlands are crucial blue carbon ecosystems and play an indispensable role in regulating global climate change and preserving biodiversity. Microorganisms are key drivers of biogeochemical cycles, maintaining ecosystem functions and stability by coupling carbon, nitrogen, and sulfur cycles. Synthetic microbial ecology aims to understand microbial interaction mechanisms and environmental applications. In view of the critical issues in coastal wetlands, this paper focuses on the theoretical studies of synthetic microbial ecology and explores the applications of microbiome engineering in reducing greenhouse gas emissions and enhancing carbon sinks, providing scientific and technological support for mitigating global climate change.

[Objective] To study the therapeutic effect of Bifidobacterium adolescentis strains with strong antioxidant capacity on a mouse model of ulcerative colitis (UC). [Methods] The B. adolescentis strains with strong antioxidant capacity were screened based on 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging rate, reducing capacity, and hydrogen peroxide tolerance. Subsequently, we established a mouse model of dextran sulfate sodium (DSS)-induced colitis to investigate the alleviating effects of the B. adolescentis strains with strong antioxidant capacity on UC. [Results] Among the 26 strains of B. adolescentis, TH02767, TH03658, and TH03664 demonstrated strong antioxidant capacity. Only TH02767 showed an alleviating effect on UC in terms of disease activity index and spleen index in the mouse model (P < 0.05). Moreover, the intervention with TH02767 lowered the levels of tumor necrosis factor-α, interleukin-6, interleukin-1β, and myeloperoxidase (P < 0.05), while enhancing the production of interleukin-10 (P < 0.05) in the colon. In addition, TH02767 modulated gut microbiota in the mice by reducing the relative abundance of Deferribacterota and increasing the relative abundance of Bacteroidetes. At the genus level, it increased the relative abundance of Muribaculum and Muribaculaceae (P < 0.05). [Conclusion] B. adolescentis TH02767 screened out in this study for its robust antioxidant capacity not only ameliorates the clinical symptoms associated with DSS-induced colitis in mice but also significantly reduces the levels of pro-inflammatory cytokines and modulates the gut microbiota.

[Objective] To explore the effects of rice-crab co-culture on physicochemical indicators and microbial community structure of soil in the Yellow River irrigation area of Ningxia and thus provide a theoretical basis for promoting rice-crab farming in the Yellow River irrigation area in northwest China. [Methods] The physicochemical indicators of soil were measured, and high-throughput sequencing of 16S rRNA gene amplicons was employed to reveal the microbial community structure in the fields with only rice planting (CK) and rice-crab co-culture (EG) by introduction of crabs into the ditches around rice plots. [Results] EG had higher soil pH, total nitrogen (TN), and soil organic matter (SOM) and lower total salt content than CK from April to August. The TN, available phosphorus (AP), and SOM in EG were significantly higher than those in CK in June and July. In terms of microbial community structure characteristics, EG significantly altered the bacterial community structure in soil, increasing the bacterial diversity and abundance in April, and August. EG had higher cumulative relative abundance of the top 20 bacterial phyla than CK from April to August. The dominant phylum of both CK and EG from April to August was Proteobacteria. At the genus level, EG increased the relative abundance of Pseudomonas and decreased the relative abundance of Thiobacillus and Methanosaeta. The comparison showed that EG had stronger effect on the bacterial community structure than the sampling month. Correlation analysis shows that TN is the main factor affecting the relative abundance of soil bacteria, and it is significant (P < 0.05). [Conclusion] Compared with traditional rice monoculture (CK), sampling rice crab intercropping (EG) enhances soil fertility, increases the relative abundance of Proteobacteria and Pseudomonas in the soil, and helps improve soil fertility and nutrient cycling efficiency, as well as clean up pollutants in soil and water bodies.

[Objective] To evaluate the biocontrol and growth-promoting effects and reveal the antifungal mechanisms of the pepper endophyte Bacillus velezensis XY40-1, thus providing elite bacterial strains and technical support for the development of biocontrol agents for pepper Phytophthora blight. [Methods] Plate confrontation, in vivo inoculation assay, and microscopic observation were employed to determine the biocontrol and growth-promoting effects of B. velezensis XY40-1 on pepper. Genomics and metabolomics methods were adopted to determine the antifungal metabolites of the strain and the synthesis pathways of substances involved in antifungal effects, on the basis of which the antifungal mechanisms were explored. [Results] Strain XY40-1 exhibited strong inhibitory effects on six pathogenic fungi: Phytophthora capsici, Sclerotium rolfsii, Colletotrichum scovillei, Alternaria alternate, Fusarium oxysporum, and F. solani, that infected pepper, and it demonstrated broad-spectrum resistance. The strain suspension at 10⁷ CFU/mL showcased the control effect of 66.13%. Moreover, it possessed the abilities to solubilize phosphorus and potassium and fix nitrogen, thus promoting the growth of pepper seedlings. XY40-1 carried a large number of genes involved in the synthesis of ketone and terpenoid antibiotics, which enabled it to produce extracellular metabolites such as nogalamycin, megalomicin, rifaximin, avermectin ala, avermectin, and ansamitocin P-3 to inhibit pathogenic fungi. The synthesis and degradation of ketone bodies, as well as the biosynthetic pathways of terpenoid skeletons and antibiotics, contributed to the antagonistic function of XY40-1. The core metabolic pathway was the biosynthesis of secondary metabolites. [Conclusion] B. velezensis XY40-1, an endophyte of pepper, possesses excellent biocontrol and growth-promoting effects on pepper, being a multifunctional and high-quality strain for the biocontrol of pepper diseases.

[Objective] To investigate the biosynthetic potential of the endophyte Streptomyces sp. SZC001 in Sinomenium acutum and explore the unknown active natural products. [Methods] SZC001 was isolated by surface sterilizing method and its full-length genome sequence was obtained by third-generation and second-generation sequencing. Then, the biosynthetic potential of SZC001 was evaluated by antiSMASH analysis. Fermentation was carried out with four media, and the compounds were separated and identified by silica gel column chromatography, high-performance liquid chromatography, high-resolution mass spectrometry and nuclear magnetic resonance. The CCK-8 assay was employed to examine the cytotoxicity of the target compounds. [Results] The strain was identified as Streptomyces sp. SZC001, with a genome length of 9 109 166 bp and the G+C content of 71.08%. The antiSMASH analysis showed that the genome of the strain contained a total of 31 biosynthetic gene clusters (BGCs), among which 17 BGCs had less than 40% similarity with the known BGCs. The strain produced several anthracyclines in four media, and the two most abundant compounds 1 and 2 were isolated and identified. Compound 1 is the known compound ε-rhodomycinone and compound 2 is a new compound η-rhodomycinoe, which is derived from α₂-rhodomycinone by derivatization of the hydroxyl group at the C-10 position and transfer of the hydroxyl group at the 6-position to the 11-position of the backbone. Both compounds 1 and 2 showed relatively strong inhibitory effects on two tumor cell lines, with IC₅₀ of 1.55–4.59 μmol/L. [Conclusion] SZC001 is a strain that holds the potential for the exploration of active natural products. The anthracyclines produced by the strain exhibit anti-tumor activities and warrant further development and utilization. This study enriches ε-rhodomycinone derivatives, furnishing a foundation for the subsequent exploitation of the endophytic resources of S. acutum.

A mutant (G₉-72) of Halomonas campaniensis exhibiting high ectoine production was obtained by ultraviolet (UV) mutagenesis. The mutation sites, molecular variations, and high ectoine production mechanism of this mutant remain unknown.[Objective] To investigate the mutation sites and genetic variations of G₉-72 compared with the wild type strain XH26 and identify the potential causes of ectoine accumulation outbreak. [Methods] PacBio Sequel Ⅱ was used for whole-genome sequencing, and the mutation sites in the genome of the mutant were identified based on the sequencing results. The amino acid metabolic pathways were analyzed to reveal the association between mutated genes and ectoine synthesis, and the results were verified by RT-PCR. [Results] The genome of strain XH26 was 4.11 Mb, encoding 3 927 genes. Compared with strain XH26, G₉-72 showed 35 mutation sites, including 18 single nucleotide polymorphism mutations, 14 insertion mutations, and 3 deletion mutations. The mutated genes argF, coaBC, and livH, which encoded ornithine transcarbamylase (100.00% similarity with ArgF proteins in NCBI database), phosphopantothenoylcysteine decarboxylase (99.28% similarity with CoaBC proteins in NCBI database), and branched-chain amino acid ABC transporter permease (96.27% similarity with LivH proteins in NCBI database), were implicated in the synthesis of fumaric acid, citric acid and the absorption and transport of branched-chain amino acids, respectively. The increased flow of upstream metabolites may be the key reason for the sharply increased accumulation of ectoine in the mutant. RT-PCR verified 20 genes related to the ectoine metabolic pathway, and the transcriptional expression levels were consistent with the expected analysis. [Conclusion] The overexpression of genes argF, coaBC, and livH enhanced the anabolic flow of ectoine, which contributed to a significant increase in ectoine accumulation in the mutant. This finding provides a reference point for subsequent studies on the reaction mechanisms of enzymes in the mutant and the fermentation production.

Dissolved organic matter (DOM) in the ocean encompasses complex and diverse organic compounds, and heterotrophic bacteria, the main DOM decomposers, also exhibit high biodiversity. The interactions between heterotrophic bacteria and DOM play an important role in biogeochemical cycles, which, however, are not fully understood.[Objective] To explore the dynamics of microbial communities with the addition of marine-derived concentrated DOM. [Methods] DOM with a molecular weight exceeding 1 kDa and enriched from coastal seawater was introduced into microcosm culture systems. Illumina amplicon sequencing, dissolved organic carbon (DOC) concentration measurement, and bacterial isolation were performed on different days of incubation. [Results] The addition of DOM significantly influenced bacterial community composition, inducing more pronounced changes in the high-DOM group. Specifically, the relative abundance of Campylobacterota, Nitrosococcales, and Nitrincolaceae increased in the high-DOM group on days 3, 10, and 30, respectively. The alpha diversity and evenness of the microbial community decreased during days 0-3 and increased during days 10-30, with a transition point occurring between days 3 and 10. The network analysis revealed that the high-DOM group exhibited a more tightly interconnected and complex network than the control group. In addition, bacterial isolates from the culture systems added with different concentrations of DOM were distinct. The specific genera of different DOM treatments were identified, which may be key groups in DOM degradation. [Conclusion] The addition of DOM triggers the succession of microbial community structures within microcosm culture systems, and the community composition may be associated with specific DOM components, which influence the direction of community succession. Furthermore, the varying DOM concentrations select for culturable bacteria with diverse survival strategies. This study provides a basis for enriching our understanding about the mechanisms underlying microbial responses to marine-derived DOM.

Cyclic adenosine monophosphate (cAMP) is a second messenger widely present in eukaryotes. It is synthesized by adenylate cyclase (AC) and regulates downstream protein activity by binding to protein kinase A, thereby regulating fungal growth and development, virulence, cell wall integrity, environmental stress responses, and sexual/asexual reproduction. This article introduces the research progress of the cAMP signaling pathway in phytopathogenic fungi and the cooperation of this pathway with other signaling pathways in regulating cellular processes. At the same time, it elucidates the role of the cAMP signaling pathway in the infection of plant phytopathogenic fungi. This review is expected to provide reference for screening the agents for inhibiting phytopathogenic fungi that target the genes or proteins in the cAMP pathway. Additionally, the cAMP signaling pathway could be targeted to prevent and control the growth, development, and pathogenicity of phytopathogenic fungi in the future.

[Objective] To reveal the dynamics of bacterial communities in surface water and groundwater in the densely populated area of the Jianghan Plain across seasons and explore the underlying factors causing the temporal variations. [Methods] Water samples were collected from both surface water and groundwater in the densely populated area of the Jianghan Plain, and metagenomic sequencing was employed to investigate the seasonal variations of bacterial communities. The variations in environmental factors, bacterial community structure, and bacterial community assembly processes in the surface water and groundwater were compared considering rainy and dry seasons. [Results] For the surface water, the salinity (P < 0.01) and conductivity (P < 0.01) during the rainy season were lower than those during the dry season, while no significant seasonal variation was observed in turbidity. For the groundwater, the turbidity was higher in the rainy season than in the dry season (P < 0.05), while neither salinity nor conductivity showcased seasonal variations. The Shannon index of bacteria in the surface water in the rainy season was higher than that in the dry season (P < 0.01), whereas that in the groundwater presented no significant seasonal difference. The principal coordinates analysis and permutational multivariate analysis of variance (P=0.001) revealed significant seasonal variations in bacterial communities between surface water and groundwater. The Mantel test showed no significant correlations between the bacterial community in the groundwater and environmental factors during the dry season, while other communities were significantly correlated with at least one environmental factor. The neutral community model, modified stochasticity ratio (MST), and β nearest taxon index all indicated that stochastic processes exerted stronger effects on the bacterial community in the surface water during the rainy season than during the dry season, and the trend was similar but not significant for the bacterial community in the groundwater. The migration rate of surface water was higher during the rainy season than during the dry season, whereas that of groundwater showed an opposite trend. (5) The seasonal variations in the diversity of antibiotic resistance genes exhibited a negative correlation with MST (ρ=−0.164, P=4.942E−2) in the surface water, whereas the correlation was positive in the groundwater (ρ=0.393, P=1.452E−6). [Conclusion] The temporal dynamics of bacterial communities in surface water and groundwater in the densely populated area of the Jianghan Plain showed notable differences. These differences can potentially be attributed to distinct basic properties between surface water and groundwater, frequent environmental disturbances during the rainy season, and water exchange processes between surface water and groundwater.