[Objective] To identify the diguanylate cyclase activity of the intracellular domain and its mutants of PA0847 fromPseudomonas aeruginosa and preliminarily probe into its catalytic mechanism. [Methods] Congo red plate staining was employed to verify the diguanylate cyclase activity of the intracellular domain of PA0847. PCR was employed to construct the PA0847 PAS-GGDEF domain and its single-point mutants, and the corresponding proteins were expressed and purified. Gel filtration chromatography was utilized to analyze the aggregation states of proteins in solution. The diguanylate cyclase activity of the proteins was identified byin-vitro enzymatic reactions. Based on thiazole orange fluorescence staining, the production of cyclic diguanylate monophosphate (c-di-GMP) was determined after the enzymatic reactions, and the amino acid residues closely related to the activity of diguanylate cyclase were screened. The structural models of PA0847 PAS-GGDEF and its complex with guanosine triphosphate (GTP) were obtained by structure prediction combined with molecular docking. [Results] PA0847 PAS-GGDEF primarily exerted its catalytic activity as a dimer, with the PAS domain facilitating the dimer formation and increasing the activity of the diguanylate cyclase. Mutant screening revealed a significant increase in the activity of the non-catalytic site mutant Y700A compared with the wild type at a low protein concentration (< 0.6 mg/mL). Gel filtration chromatography indicated that the heightened activity may be attributed to the enhanced GGDEF (Gly-Gly-Asp-Glu-Phe) dimerization driven by Y700A. Structural modeling revealed that PA0847 PAS-GGDEF had a conserved GTP binding site, in which the amino side chain of K722 played an important role in binding to the phosphoryl group of GTP. The aromatic ring of Y700 engaged in a hydrophobic interaction with the alpha-helix containing K722. Therefore, the mutation Y700A may alter the spatial orientation of the K722-containing helix, which promoted the binding of K722 to the substrate GTP and the dimerization of GGDEF. [Conclusion] The non-catalytic site Y700 of PA0847 fromPseudomonas aeruginosa can indirectly regulate the diguanylate cyclase activity.

[Objective] Riemerella anatipestifer is a Gram-negative bacterium infecting ducks, causing serious economic losses to the duck industry. After infection,R.anatipestifer regulates gene expression to adapt to the 42 ℃ body temperature of ducks. To identify the adaptation mechanism ofR.anatipestifer CH-1 in ducks, we sequenced and compared the transcriptomes ofR.anatipestifer CH-1 at 37 ℃ and 42 ℃. [Methods] R.anatipestifer CH-1 was cultured to the exponential growth phase at 37 ℃ and then subjected to heat stress at 37 ℃ and 42 ℃, respectively, for 1 h. The cells were then collected for the extraction of total RNA. The raw transcriptome data of the bacteria cultured at 37 ℃ and 42 ℃ were obtained by transcriptome sequencing, and differentially expressed genes (DEGs) were screened. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses were carried out for the DEGs. The genednaK involved in the response to heat stress was selected for preliminary functional identification. [Results] A total of 234 DEGs were screened out, including 169 genes with up-regulated expression and 65 genes with down-regulated expression. The GO enrichment analysis showed that the DEGs were mainly enriched in the nucleotide metabolic process, glycosyl compound metabolic process, and core RNA polymerase binding transcription factor activity. The KEGG enrichment analysis indicated that the DEGs were mainly involved in oxidative phosphorylation, ribosomes, and bacterial secretion systems. The deletion ofdnaK impaired the growth ofR.anatipestifer CH-1 at 42 ℃, compared with that at 37 ℃. [Conclusion] Compared with that at 37 ℃, the growth ofR.anatipestifer CH-1 was not affected at 42 ℃. The strain up-regulated or down-regulated the expression of heat shock response proteins and other factors to cope with heat stress.

Arbuscular mycorrhizal fungi play important roles in ecosystems and plant growth. The physiological and ecological roles of arbuscular mycorrhizal fungi, especially in promoting plant growth, have attracted much attention. The colonization characteristics of arbuscular mycorrhizal fungi, as vital indicators of the symbiotic relationship between arbuscular mycorrhizal fungi and plants, are key parameters to evaluate the ecological adaptability of plants. However, systematic and comprehensive studies remain to be carried out regarding the colonization characteristics of arbuscular mycorrhizal fungi in the plants growing in China. [Objective] This study explored the colonization parameters of arbuscular mycorrhizal fungi in the plants growing in China, as well as their distribution in different ecosystems and climate regions, aiming to provide fundamental data to advance the research on mycorrhizae. [Methods] A database of arbuscular mycorrhizal fungi colonizing plants growing in China was established with the data from FungalRoot and 605 Chinese papers. Furthermore, the database was supplemented with 47 700 data sets including mycorrhizal colonization characteristics, ecosystem types, climate regions, plant types, and plant growth years. The analysis in this study was then performed based on this database. [Results] In China, 69.7% and 30.3% plants exhibited the arbuscular mycorrhizal fungal colonization rates ranging from 0 to 55% and from 55% to 100%, respectively. The majority of plants showed the arbuscular mycorrhizal fungal colonization intensity, hyphal abundance, vesicular abundance, and arbuscular abundance below 40.0%. Among the symbiotic forms of arbuscular mycorrhizal fungi, type A was the most prevalent, existing in 56.3% plants. Arbuscular mycorrhizal fungi demonstrated similar colonization rates in farmland, desert, and grassland ecosystems (51.8%, 51.6%, and 51.8%, respectively) and a low colonization rate (40.4%) in forest ecosystems. The plants in desert ecosystems showcased the highest arbuscular mycorrhizal fungal colonization intensity, hyphal abundance, vesicular abundance, and arbuscular abundance, which reached 46.0%, 47.1%, 37.2%, and 31.2%, respectively. In different climate regions, the colonization level followed the trend of warm temperate zone (53.3%) > tropical zone (50.0%) > temperate zone (45.2%) > subtropical zone (42.2%). The colonization level was higher in herbaceous plants than in woody plants, and higher in perennial plants than in annual plants. Among woody plants, shrubs had the highest colonization rate at 46.3%, followed by trees and vines, both at 43.9%. Herbaceous plants had higher colonization intensity (30.2%) and hyphal abundance (32.5%) than woody plants, while woody plants had higher vesicular abundance (19.5%) and arbuscular abundance (23.4%). Among the plants colonized by arbuscular mycorrhizal fungi, angiosperms accounted for the majority (90.2%, belonging to 110 families), while ferns, gymnosperms, and lycophytes were less common. [Conclusion] In China, arbuscular mycorrhizal fungi showed the colonization rate below 55% and colonization indicators below 40.0% in most plants. Different ecosystems, climate regions, plant types, and plant growth years affected the colonization status to different degrees.

The proposal of ban on antibiotics in feed is a turning point for the animal husbandry, marking that the feed industry and animal husbandry have entered a new stage of transformation and upgrading. [Objective] To investigate the effects of addingBacillus licheniformis HDTN to feed on the growth performance and intestinal flora of '817' broilers. [Methods] B.licheniformis HDTN powder (7.0×10¹⁰ CFU/g) was added to the feed for '817' broilers. The effects of high-, medium-, and low-dose (1 000, 500, and 250 g/t) bacterial addition on the growth performance, serum biochemical indexes, intestinal morphology, and intestinal flora structure of the '817' broilers during 1–56 days were studied, with the group without bacterial addition as the control. [Results] Compared with the control group, the addition of low-doseB.licheniformis HDTN increased the average body weight by 105.47 g (P < 0.01) and reduced the feed-to-weight ratio by 0.25 (P < 0.05) during days 1–35. The antioxidant indexes in the broiler serum were positively correlated with the dose of HDTN, which reduced oxidative stress in broilers. The content of malondialdehyde was negatively correlated with the dose of HDTN, which suggested that HDTN reduced cellular damage in broilers. The ratio of villus height to crypt depth in the duodenum of the low-dose group was higher than that of the control group (P < 0.01). In addition, compared with the control group, medium-dose (P < 0.01) and low-dose (P < 0.05) groups showed increased relative abundance ofFirmicutes, and the medium-dose group showed increased relative abundance ofBacteroides (P < 0.05). [Conclusion] Addition of low-doseB.licheniformis HDTN for '817' broilers can improve the growth performance, reduce the feed-to-weight ratio, improve the intestinal morphology, and accelerate the assembly of intestinal flora during days 1–35.

Phenolic acids autotoxic substances secreted by plant roots, such as p-hydroxybenzoic acid (PHBA), are the main factors causing continuous cropping obstacles in plants. [Objective] In order to obtain PHBA degrading bacteria and improve its degradation efficiency. [Methods] A PHBA-degrading strain was isolated by screening medium and inorganic salt medium, and it was identified asMicrobacterium aurantiacum. The initial content of PHBA, culture temperature, pH and nitrogen source were optimized by single factor experiment and response surface methodology. [Results] The highest degradation rate was obtained when the PHBA content was 0.4 g/L, the temperature was 30 ℃, the pH was 8.0, and the nitrogen source was ammonium sulfate. The optimal degradation conditions were temperature 30.2 ℃, pH 8.3, and PHBA concentration 0.18 g/L, and the degradation rate reached 100%. Combined with pot experiments and high performance liquid chromatography, the results showed that the strain could effectively mitigate the PHBA stress effect in cucumber rhizosphere. [Conclusion] The strain ofMicrobacterium aurantiacum screened in this study has a high ability to degrade PHBA, and has the potential value in continuous cropping obstacles.

[Objective] The biosafety of silver nanoparticles (AgNPs) has been a subject of concern due to the narrow therapeutic window. Expanding the therapeutic window could facilitate the application of AgNPs in the treatment of multi-drug resistant bacterial infections in humans and animals. This study aimed to enhance the biosafety of AgNPs by modifying their surface with alpha-ketoglutaric acid (AKG), a crucial component of the tricarboxylic acid cycle. [Methods] Silver ion was reduced to AgNPs by rutin at room temperature, and then AgNPs were stabilized with 1 mmol/L polyvinylpyrrolidone (PVP) solution to generate PVP-AgNPs. AKG (10 mmol/L) was added to generate PVP-AgNPs@AKG. The prepared AgNPs were characterized by a full-wavelength spectrophotometer, a particle size analyzer, and a transmission electron microscope. The antibacterial activities of PVP-AgNPs and PVP-AgNPs@AKG were evaluated based on minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill curve, and post-antibiotic effect. The cytotoxicity of the prepared AgNPs to human cervical epithelial cells (HCerEpic) was examined by the MTT assay and flow cytometry. Furthermore, the effects of the prepared AgNPs on the energy metabolism, oxidative stress, and expression of genes involved in anaerobic respiration ofEscherichia coli BW25113 were studied. [Results] The MIC and MBC of PVP-AgNPs@AKG against Gram-positive and Gram-negative bacteria were 50% or above 50% lower than those of PVP-AgNPs. PVP-AgNPs@AKG and PVP-AgNPs showed no significant difference in the cytotoxicity to HCerEpic cells. Compared with PVP-AgNPs, PVP-AgNPs@AKG at the MIC showed significantly enhanced inhibitory effect on the α-ketoglutarate dehydrogenase inEscherichia coli, increased accumulation of AKG, lowered ATP level, and elevated reactive oxygen species level. Moreover, PVP-AgNPs@AKG significantly up-regulated the expression ofsoxS and down-regulated the expression of genes involved in anaerobic respiration, such asarcA,fnr, andfdnH. [Conclusion] The findings suggested that PVP-AgNPs@AKG disrupted the energy metabolism by targeting α-ketoglutarate dehydrogenase, rending bacteria more vulnerable to oxidative damage. Modifying with AKG would be a potential method to expand the therapeutic window of AgNPs.

[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) ofCorynebacteriumpseudotuberculosis and evaluate the role of this gene in the pathogenicity ofC.pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flankingcp40 ofC.pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred intoC.pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion ofcp40. The roles ofcp40 in the pathogenicity ofC.pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between thecp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WTinvitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WTinvivo. [Results] We successfully constructed thecp40-deleted strain XH02Δcp40 by using the established dual-plasmid CRISPR/Cas9 editing system. Compared with WT (XH02), XH02Δcp40 showed no obvious difference in the colony morphology or growth curve. However, the J774A.1 cells infected with XH02Δcp40 showed decreased lactate dehydrogenase (LDH) release (P=0.06) and propidium iodide (PI) staining ratio (P < 0.01) compared with those infected with XH02. The mortality of XH02Δcp40-infected mice reduced by 50% and the bacterial loads in the liver and kidney of XH02Δcp40-infected mice significantly reduced compared with those of XH02-infected mice (P < 0.001). [Conclusion] The CRISPR/Cas9 gene editing system established in this study can effectively deletecp40 ofC.pseudotuberculosis. The results confirm thatcp40 is a virulence-related gene, providing a foundation for subsequent research on the infection ofC.pseudotuberculosis based on this gene.

[Objective] To study the population diversity, spatial distribution characteristics, and correlations with physicochemical factors of yeasts in the Napahai plateau wetland. [Methods] Yeast strains were isolated from the water samples of the Napahai plateau wetland by membrane filtration and plate culture. The strains were identified based on the D1/D2 sequences of the 26S rRNA gene and morphological characteristics. R 4.2.2 and Canoco 5 were used to analyze the yeast diversity and its correlations with physicochemical factors. [Results] A total of 565 yeast strains were isolated from the water bodies in the Napahai plateau wetland and identified as 36 species of 21 genera. The dominant species wereRhodotorula mucilaginosa,Meyerozyma guilliermondii, andRhodotorula graminis. Total nitrogen was the main physicochemical factor affecting the yeast count, which presented a negative correlation. [Conclusion] The water bodies of Napahai plateau wetland harbor abundant yeast resources which present significant spatial heterogeneity. Human activities and total nitrogen have significant impacts on the yeast count. The yeast community composition in the water bodies of the Napahai plateau wetland is significantly different from that in other plateau freshwater lakes and has unique yeast resources that require further research.

Soil viruses are the most abundant biological entities on Earth. They play important roles in the regulation of host dynamics and soil ecosystem, including microbial diversity, community composition, and element cycling. However, due to the tremendous complexity of the soil ecosystem, the incredible functions of soil viruses and the underlying functioning mechanisms remain largely unexplored and represent a hot topic and frontier in soil biology, microbial ecology, and environmental science. The recent advances in molecular biotechnology and the widespread application of ecological theories have significantly boosted the research on soil viruses. To provide a comprehensive overview of the state-of-the-art knowledge of soil virology, we conducted a literature review using bibliometric methods. This paper offers an overview of the current status and hotspots in soil virus research, shedding light on the development in this field. Taking into account the characteristics of soil environments, the study presents the future prospects for soil virus research, including the diversity, biological characteristics, environmental factors, mechanisms, and applications of soil viruses. With the interdisciplinary integration and advancements in research technologies, soil virus research is experiencing rapid growth. The noteworthy achievements in nutrient cycling, ecosystem services, and biological regulation through soil virus research can contribute to soil health and carbon neutrality initiatives.