Objective To investigate the transcriptional regulation of quorum sensing (QS) regulators AphA, ToxR, and QsvR on the expression of the phosphodiesterase (GepA) gene gepA in Vibrio parahaemolyticus. Methods Total RNAs were extracted from the wild type (WT) and the mutant strains of aphA, toxR, and qsvR. Quantitative real-time PCR (qPCR) was carried out to calculate the transcriptional variation of gepA between WT and mutant strains. The regulatory DNA region of gepA was cloned into the upstream region of promoterless luxCDABEreporter gene in the pBBRlux plasmid. The recombinant plasmid was respectively transferred into the WT and mutant strains. Luminescence assay was used to test the regulatory effect of QS regulators on the expression of gepA. The primer extension assay was employed to detect the transcription start site and the promoter activity of gepA. The effects of QS regulators on gepA were evaluated based on the abundance of primer extension products. The regulatory DNA region of gepA was cloned into the upstream region of lacZ in the pHRP309 plasmid. The LacZ recombinant plasmid was transformed into EC100 λpir harboring pBAD33 or PBAD33-qsvR. Two-plasmid LacZ reporter assay was conducted to investigate the regulatory effects of QS regulators on the transcription of gepA in EC100 λpir. The regulatory DNA region of gepA was amplified by PCR, and the His recombinant proteins of QS regulators were purified. The electrophoretic mobility shift assay (EMSA) was performed to investigate whether QS regulators directly regulated the expression of gepA. Results At low cell density, the qPCR results showed that expression of gepA in ΔaphA and ΔtoxR were significantly lower than that in WT, indicating that AphA and ToxR activated the transcription of gepA. The luminescence assay showed that the transcriptional activity of the promoter region of gepA in ΔaphA and ΔtoxR was significantly lower than that in WT, further indicating that AphA and ToxR promoted the transcription of gepA. The primer extension assay detected that the transcription start site of gepA was located at the A nucleotide 30 bp upstream of the start codon ATG, and its transcriptional activity was activated by AphA. The EMSA result indicated that His-AphA and His-ToxR were unable to bind the promoter DNA region of gepA. At high cell density, both the qPCR and primer extension assay indicated that QsvR inhibited the transcription of gepA. The EMSA result demonstrated that His-QsvR directly bound to the promoter DNA region of gepA. Two-plasmid lacZ reporter assay demonstrated that QsvR inhibited the transcriptional activity of the promoter region of gepA in EC100 λpir. Conclusion AphA and ToxR indirectly activate while QsvR directly inhibits the transcription of gepA. Therefore, the transcription level of gepA is higher at low cell density and significantly decreases at high cell density.

Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). It can adhere to and colonize uroepithelial cells, disseminate systemically, and induce severe sepsis and subsequent renal failure, posing a substantial threat to global public health. Emerging evidence indicates that lactylation, a key post-translational modification (PTM) in macrophages, plays a crucial role in the host defense against UPEC infection. Notably, the UPEC CFT073 strain harbors ldhA, which encodes lactate dehydrogenase (LDH), an enzyme critical for lactate biosynthesis. However, the mechanism by which LdhA (the ldhA-encoded LDH) regulates macrophage lactylation during UPEC infection remains elusive. Objective To elucidate how LdhA modulates macrophage lactylation and thereby impacts UPEC pathogenicity. Methods Online bioinformatics tools were used to predict the functional domains and transmembrane regions of LdhA. The recombinant protein rLdhA was generated via molecular cloning, and its LDH activity was measured by a commercial LDH activity assay kit. Western blotting was performed to assess the cellular entry of rLdhA into macrophages and its regulatory effect on macrophage lactylation. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the secretion of inflammatory cytokines in macrophages treated with rLdhA. The drug resistance profile of UPEC CFT073 (wild-type and ldhA-knockout strains) was analyzed via an automated microbial identification system. To evaluate the role of LdhA in UPEC pathogenicity, we treated mice with the wild-type UPEC CFT073 (CFT073^wt), ldhA-deficient mutant (CFT073^ΔldhA ), or rLdhA (with or without pretreatment with an LDH inhibitor) through intraperitoneal injection or tail vein injection, and then observed and quantified pathogenic phenotypes. Results LdhA harbored a LDH domain and was secreted extracellularly. We successfully established an expression system for ldhA and achieved efficient expression and purification of rLdhA. Functional assays confirmed that rLdhA exhibited LDH activity and can enter macrophages via clathrin-mediated endocytosis, subsequently enhancing macrophage lactylation in a dose-dependent manner. Additionally, rLdhA significantly inhibited lipopolysaccharide (LPS)-induced inflammatory cytokine production in macrophages. Furthermore, LdhA was found to substantially modulate the drug resistance profile of UPEC CFT073. In vivo studies demonstrated that LdhA promoted the pathogenicity of UPEC in a mouse infection model. Conclusion Collectively, our findings demonstrate that LdhA enhances UPEC pathogenicity by upregulating macrophage lactylation and suppressing the production of proinflammatory cytokines. However, the underlying molecular mechanisms mediating this regulatory cascade remain to be fully elucidated and warrant further exploration. This study offers a new theoretical basis for deciphering the pathogenic mechanisms of UPEC infections.

The enzootic nasal tumor of sheep and goats is a progressive and contagious disease caused by enzootic nasal tumor virus. It is mainly characterized by tumor growth in the mucosal epithelial tissue of the ethmoid bone and nasal turbinates within the nasal cavity of sheep and goats. In the later stage of the disease, the significant enlargement of tumor volume can lead to upper respiratory tract obstruction, which subsequently causes the affected animals to die of asphyxiation. This disease markedly reduces the production performance of infected animals, causing economic losses to the livestock industry. Moreover, it poses a threat to precious local breeds and core breeding flocks, resulting in the loss of high-quality breeding sheep. Consequently, it has become one of the major diseases threatening the sheep and goat industry. Currently, there are no vaccines or specific treatments for this disease. This article reviews enzootic nasal tumor virus in terms of the etiology, epidemiology, clinical symptoms, main pathological changes, diagnosis, and prevention and control, providing references and ideas for the prevention and control of this disease.

Objective Mixotrophy that combines phototrophic autotrophy and phagotrophic heterotrophy is widespread among unicellular eukaryotic microalgae and plays a key ecological role in energy flow within food webs and in elemental biogeochemical cycles. However, identifying and characterizing mixotrophic microalgae in natural waters remains technically challenging. Improving current approaches to accurately reveal the diversity of mixotrophic microalgae is an urgent task in this field. Methods Fluorescently labeled prey surrogates and feeding experiments were employed to trace phagotrophic microalgae within plankton communities. Target organisms were captured at the single-cell level through fluorescence-activated cell sorting (FACS), followed by multiple-displacement amplification (MDA) and 18S rRNA gene sequencing for taxonomic identification. On the basis of this FACS-MDA workflow, we established a methodological framework for studying the functional groups of microalgae. Results Applying this approach to multiple freshwater and seawater samples from China, we identified twenty phagotrophic microalgal species belonging to six classes and twelve genera, as well as heterotrophic consumers representing one class and three genera, demonstrating the robustness and broad applicability of this method. Conclusion This study applies the combined FACS-MDA technology to the identification of functional groups of microalgae in natural water bodies. The established technology has broad application prospects in microbial ecology. It enables deeper insights into the functional diversity and in situ feeding activities of environmental microalgae.

Objective Senecavirus A (SVA) keeps posing a serious threat to the swine industry in China. This study aimed to characterize the biological properties and genetic evolutionary features of the latest circulating SVA strains. Methods In November 2024, vesicular lesion tissue samples were collected from pigs suspected of foot-and-mouth disease at a farm in eastern China. RT-PCR was performed, and positive samples were inoculated onto BHK-21 cells following standard virus isolation procedures. Indirect immunofluorescence assay was employed to preliminarily confirm viral isolation. The complete viral genome was amplified and sequenced, followed by genetic evolution analysis. Amino acid variations in the VP1 protein of the strain were identified by comparison with representative strains from key epidemic nodes. Viral replication characteristics were evaluated through plaque formation assay and one-step growth curve analysis. Viral particle morphology was observed via transmission electron microscopy (TEM). Results RT-PCR and virus isolation confirmed SVA as the causative agent of the disease, and the isolated strain was designated SDWF/11/2024. The viral genome of this strain was 7 292 bp in length, and its overall organization was highly consistent with that of previously reported SVA strains. Phylogenetic analysis revealed that SDWF/11/2024 belonged to the USA-like evolutionary clade, showing genetic divergence from Chinese strains circulating between 2015 and 2018, while exhibiting the closest relationship to a Chilean strain isolated in 2022. The isolate replicated efficiently in BHK-21 cells and induced typical cytopathic effects. Its replication kinetics was comparable to that of the early Chinese isolate HN/11/2017, although differences in plaque morphology were observed. TEM examination identified spherical viral particles with diameters of 25-35 nm, consistent with typical SVA virions. Conclusion This study successfully isolated and characterized the SVA strain SDWF/11/2024 circulating in China, 2024, and elucidated its molecular evolutionary features. The isolated SDWF/11/2024 provides a new reference strain for SVA surveillance in China and suggests that the virus may still persist at low levels in pig populations. These findings enhance our understanding about the genetic diversity and epidemic dynamics of SVA and support the improvement of molecular epidemiological monitoring and the development of prevention and control strategies.

[Objective] To establish a method of semi-thin section preparation and scanning electron microscopy (SEM) observation with a simple operation, a short cycle, and a low technical threshold, which can be used as a rapid pre-screening strategy for the complex process of transmission electron microscopy (TEM) ultra-thin sections, in response to the requirements of rapid identification and dynamic observation of typical subcellular structures (such as cell wall, cytoplasm, spore, and vacuole) of microbial samples in the process of fermentation production. [Methods] Bacillus subtilis, Escherichia coli, and Pichia pastoris, taken as the research objects, were embedded in Embed 812 epoxy resin to prepare semi-thin sections with different thicknesses (200, 500, and 1 000 nm) and resin embedding blocks (> 1 000 nm) with samples. After platinum coating by ion sputtering, the subcellular structure was observed by SEM. Ultra-thin sections (70 nm) of the above microbial samples were also prepared, stained with lead and uranium, and imaged by TEM. The imaging effect and operation efficiency of the above two methods were compared. [Results] The 200 nm semi-thin section can clearly and completely display various kinds of microbial subcellular structures under a scanning electron microscope. The image quality was significantly better than that of 500 and 1 000 nm sections and resin block samples. Its resolution was close to the TEM observation level, and it can save about 6.5 h. [Conclusion] The method of SEM combined with 200 nm semi-thin sections was successfully applied to the high-resolution imaging of microbial subcellular structure for the first time, which can clearly identify the typical ultrastructural morphology, and has the advantages of a simple operation, a short cycle, a low cost, and high safety. It has strong versatility and promotion value, and provides a new and effective solution for the biological electron microscopy technology system.

As a widely conserved interspecies quorum sensing signaling molecule, autoinducer-2 (AI-2) is involved in regulating various crucial physiological processes such as bioluminescence, chemotaxis, and biofilm formation. However, the effects of AI-2 on Halomonas elongata and its underlying mechanisms remain unreported. [Objective] To reveal the receptor that regulates the chemotaxis and biofilm formation of H. elongata in response to AI-2. [Methods] The quantitative capillary assay was employed to examine the chemotactic response of H. elongata to AI-2. We conducted protein domain identification, sequence alignment, and molecular docking of methyl-accepting chemotaxis proteins to identify the key amino acid sites in Tar1, the potential AI-2 receptor. The ligand-binding domain (LBD) of Tar1 and single-point mutants were expressed and purified, and the binding between Tar1-LBD and AI-2 was measured by the Vibrio harveyi MM32 bioluminescence assay. tar1 was deleted by homologous recombination, and the effects of AI-2 on the chemotaxis and biofilm formation of H. elongata were evaluated by quantitative capillary and biofilm formation assays. [Results] The quantitative capillary assay revealed that H. elongata exhibited chemotaxis to AI-2. Four methyl-accepting chemotaxis proteins were identified in H. elongata. Protein domain identification, sequence alignment, molecular docking, and V. harveyi MM32 bioluminescence assay demonstrated that Tar1-LBD bound to AI-2. The tar1-deleted mutant of H. elongata was successfully constructed by homologous recombination. The deletion of tar1 impaired the chemotaxis of H. elongata to AI-2, whereas the complementation of this gene restored the chemotaxis to level comparable to that in the wild-type. Furthermore, biofilm formation assay revealed that AI-2 enhanced the biofilm formation in H. elongatavia Tar1. [Conclusion] H. elongata exhibits chemotaxis to AI-2, and this signal molecule binds to the LBD of Tar1, thereby modulating chemotaxis and biofilm formation.

[Objective] Citric acid is the main metabolite of Aspergillus niger at pH≤5.0, while l-malic acid becomes the main metabolite at pH 6.0. In this study, we employed transcriptomics to analyze the differences in the expression of key genes in metabolic pathways, aiming to explore the biosynthesis mechanisms of the two organic acids. [Methods] The cells at 48 h and 72 h of the fermentation processes for citric acid and l-malic acid production were selected for transcriptomics analysis. [Results] The transcriptome data of 72 h and 48 h were compared. GO enrichment analysis showed that the upregulated genes related to the synthesis of citric acid were concentrated in carbohydrate metabolism, while those related to the synthesis of l-malic acid were concentrated in ion transport process. The acid protease genes ANI_1_62014 (aspergillin II) and ANI_1_654124 (aspartic protease pepA) showed extremely high transcription levels during citric acid synthesis, while the key genes ANI_1_2494074 [3-oxoacyl-(acyl carrier protein) synthase] and ANI_1_2488074 (biosynthetic fatty acid synthase subunit β) essential for fatty acid chain synthesis showed extremely high transcription levels in the l-malic acid synthesis pathway. The transcription level of zinc cluster transcription factor [Zn(II)₂Cys₆ transcription factor] was higher in the synthesis process of l-malic acid. HacA, AP-1, and AtfA in the bZIP family showed higher transcriptional levels in response to environmental low pH stress during citric acid synthesis. Compared with l-malic acid synthesis, citric acid synthesis was accompanied by upregulated transcription levels of ANI_1_66114 (hexokinase), ANI_1_2950014 (citrate synthase), and ANI_1_478154 (citrate transporter) and a downregulated transcription level of ANI_1_3136024 (isocitrate dehydrogenase). Efficient glycolysis, citric acid synthesis, and citric acid transport capacity and low isocitrate dehydrogenase level were the key factors for citric acid production. In the process of l-malic acid synthesis, cytoplasmic ANI_1_440184 (pyruvate carboxylase), cytoplasmic ANI_1_12134 (malate dehydrogenase), ANI_1_914104 (isocitrate lyase), and ANI_1_2040144 (malate transporter) showed upregulated transcriptional levels. The cytoplasmic rTCA pathway and glyoxylic acid carboxylation pathway were thereby determined to be the main pathways for l-malic acid synthesis. [Conclusion] This study inferred the key differential metabolic pathways for the production of citric acid and l-malic acid by analyzing integrated transcriptomic data, and screened significant differentially expressed core genes, transcription factors, and potential transporters. These results provide important clues and a theoretical basis for elucidating the regulatory mechanisms of citric acid and l-malic acid synthesis.

[Objective] To investigate the biostimulatory effects of Bacillus velezensis XZT106 on the tuber crop sweet potato (Ipomoea batatas) and elucidate the potential mechanisms underlying its yield increase. [Methods] Sweet potato plants were treated by foliar spraying with B. velezensis fermentation broth, with the plants treated with inactivated B. velezensis fermentation broth as the control. We analyzed the chloroplast content, chloroplast ultrastructure, and antioxidant enzyme activity as well as the structure and metabolite composition of endophyte communities in different ecological niches of sweet potato plants to delve into the mechanisms by which B. velezensis fermentation broth increases the sweet potato yield. [Results] Foliar application of B. velezensis increased the sweet potato yield, enhanced the antioxidant enzyme activity in the roots, induced changes of chloroplast ultrastructure, and led to a more compact matrix structure with enlarged intracellular starch granules. In addition, foliar application of B. velezensis caused significant changes of endophyte community structures in various parts of sweet potato plants, significantly reducing the relative abundance of Fusarium and increasing the relative abundance of Pantoea. Moreover, the foliar application significantly altered the metabolome profiles of leaves and soil. Riboflavin metabolism, zeatin biosynthesis, and isoflavone biosynthesis, which regulate growth and enhance stress resistance, were significantly upregulated in leaves. The axon regeneration pathway promoting lateral root development and the glycerophospholipid metabolism pathway promoting cell proliferation were significantly upregulated in soil. [Conclusion] B. velezensis fermentation broth exerts a plant growth-promoting effect by enhancing antioxidant capacity, improving leaf cell ultrastructure, reshaping the endophyte community structure, and activating key growth-promoting and stress response metabolic pathways in sweet potato plants. These findings provide a new theoretical foundation for the application of B. velezensis-based microbial inoculants in enhancing the sweet potato yield.

In recent years, microbially mediated mineralization, a widespread form of biomineralization in nature, has emerged as a research hotspot. This process not only exerts profound influences on mineral formation and global biogeochemical cycling but also contributes to mineral deposition within living organisms, thereby holding significant ecological and biological importance. Among microorganisms, bacteria—characterized by high metabolic activity and remarkable environmental adaptability—represent the most prominent agents in microbial mineralization. This review summarizes the mechanisms of bacteria-mediated mineralization and their applications in the biomedical field, with a particular emphasis on three principal mechanisms: bacteria-controlled mineralization, bacteria-induced mineralization, and bacteria-influenced mineralization. Furthermore, the potential applications of these processes in medical imaging, targeted therapy, and tissue engineering are discussed. The overarching aim is to provide valuable references and scientific insights to inform future research and facilitate their translation into practical applications.