[Objective] To construct a strain XH02Δlpxtg270 with knockout of the LPXTG motif protein-coding gene from Corynebacterium pseudotuberculosis XH02 and explore the role of lpxtg270 in the growth, biofilm formation, and infection of XH02. [Methods] CRISPR/Cas9 was employed to construct XH02Δlpxtg270. The knockout strain and the wild strain XH02 were compared in terms of biological characteristics, invasion into J774A.1 macrophages, and pathogenicity in mice. [Results] Compared with XH02, XH02Δlpxtg270 did not change significantly in the colony morphology, growth curve, adhesion to J774A.1 macrophages, or intracellular proliferation, while it demonstrated reductions in the biofilm formation and invasion into J774 A.1 cells. Moreover, the release of lactate dehydrogenase and secretion of interleukin-1β from J774 A.1 cells infected with the knockout strain decreased compared with those infected with the wild strain. Compared with XH02, XH02Δlpxtg270 showed weakened pathogenicity in mice and decreased loads in the liver, spleen, kidney, lung, and brain, causing milder pathological changes of above organs in mice. [Conclusion] LPXTG270 of C. pseudotuberculosis is related to the biofilm formation and invasion into macrophages, playing a key role in the pathogenicity of this bacterium in mice.

Double-crossover homologous recombination using the SacB negative selection system is commonly employed for genome editing in Gram-negative bacteria. However, its negative selection efficiency varies significantly across strains, being frequently compromised by differences in metabolic characteristics or genomic composition. [Objective] To develop a novel counterselection system based on the type VI secretion system (T6SS) effector Txe1 to enhance the efficiency of seamless genome editing. [Methods] We first modified the conventional suicide plasmid pDM4 by introducing a kanamycin resistance gene, generating the derivative plasmid pDM4K. Subsequently, we replaced sacB with an l-arabinose-inducible expression cassette encoding the C-terminal domain of Txe1 (araC-P_BAD::txe1^CTD ), constructing a novel counterselection plasmid pTL1010. Using the virulence gene tssB of Edwardsiella piscicida FC2 as the target, we systematically evaluated and compared the counterselection efficiency of the Txe1 system with that of the conventional SacB system. [Results] Under induction with l-arabinose, the Txe1-based counterselection system achieved the efficiency of 91.1% (false-positive rate of 8.9%), outperforming the SacB system which had a false-positive rate of 100% (P<0.01). [Conclusion] The newly developed Txe1-based counterselection plasmid pTL1010 significantly enhances the efficiency of seamless genome editing in E. piscicida and provides a highly effective tool for precise genetic manipulation in Gram-negative bacteria.

[Objective] To explore the effects of plant diversity and soil improvement on the soil microbial community in abandoned lead-zinc mine wasteland. [Methods] A pot experiment was conducted with nine commonly used plants for ecological restoration in mining areas. Different plant diversity levels (S1 to S5) were set up, and for each level, a control group (Y: without soil amendment) and an improvement group (G: with organic fertilizer and polyacrylamide) were established. [Results] The plant height of the improvement group was generally higher than that of the control group. After soil improvement, the content of available nitrogen, available phosphorus, and available potassium all significantly increased, while that of heavy metals Cd and Pb decreased the most when the species richness was 9 (S5), dropping by 37.20% and 14.85% respectively. The diversity of soil microorganisms increased with the increase in plant diversity. The application of soil amendments reduced the richness and diversity of the fungal community, while enhancing the richness and diversity of the bacterial community. In the improvement group, the soil fungal abundance reached the highest level when GS4 configuration was adopted, with the Observed index and Chao1 index being 110.50 and 169.23, respectively. The soil bacterial abundance reached the highest level when GS2 was adopted, with the Observed index and Chao1 index being 1 081.59 and 1 116.79, respectively. In the fungal community, the abundance ofascomycetesat both the phylum and genus levels increased to varying degrees in the improvement group compared with the control group, and the abundance of Ascomycota increased with the increase in plant diversity. Soil improvement reduced the abundance of Mucoromycota and Rhizopus, but increased the abundance of Sordaria. Moreover, the abundance of Mucoromycota and Rhizopus decreased with the increase in plant diversity. In the bacterial community, soil improvement increased the abundance of Sphingomonas and Gemmatimonas. [Conclusion] Soil microbial diversity increases as the plant diversity increases. The application of soil amendments reduces the richness and diversity of soil fungi and improves the richness and diversity of soil bacteria. As the plant diversity increases, the abundance of Ascomycota increases, while that of Mucoromycota and Rhizopus decreases.

[Objective] To investigate the effects of fluoride exposure on the gut microecosystem of the model insect Bombyx mori and elucidate the toxic mechanisms of fluoride under different conditions on insect hosts. [Methods] We measured physiological enzyme activity changes in the gut tissue of B. mori following fluoride exposure and observed histopathological changes by using hematoxylin and eosin (HE) staining. We also analyzed the dynamic changes in gut microbiota by using 16S rRNA amplicon sequencing and examined the expression changes of immune-related genes in gut tissue by qPCR. [Results] Fluoride exposure significantly increased the malondialdehyde (MDA) level and decreased the reduced glutathione (GSH) level in the gut tissue. Enzyme activities of catalase (CAT), superoxide dismutase (SOD), acetylcholinesterase (AChE), carboxylesterase (CarE), alkaline phosphatase (AKP/ALP), and lactate dehydrogenase (LDH) were also markedly suppressed. Additionally, we observed epithelial cell rupture and separation from the basement membrane. The Toll immune pathway was inhibited, which resulted in significantly reduced expression of antimicrobial peptide genes, such as attacin, cecropin, lebocin, and lysozyme. Gut microbiota analysis revealed significant declines in the relative abundances of Glutamicibacter, Staphylococcus, Acinetobacter, and Methylobacterium. The gut microbiota exhibited notable structural heterogeneity with strengthened functions related to metabolic pathways and biosynthesis of secondary metabolites. [Conclusion] Fluoride exposure significantly weakened the host insect’s antioxidant capacity, basal metabolism, and immune function, damaged the integrity of gut tissue, and caused gut microbiota dysbiosis. This disruption of the gut microecosystem might be a major factor contributing to reduced insect species diversity and biomass.

[Objective] To develop a recombinant influenza A virus (IAV) expressing a reporter (enhanced green fluorescent protein, EGFP), study its biological characteristics in vitro, and explore its application in antiviral drug screening and neutralizing antibody (nAb) detection. [Methods] The EGFP gene was inserted into the C-terminus of NA (derived from H1N1-PR8) by reverse genetics of influenza virus, and the recombinant IAV was successfully constructed, rescued, and named PR8NAEGFP/WSN. The EGFP insertion position and genetic stability were analyzed by RT-PCR and sequencing. PR8NAEGFP/WSN and the control virus (PR8NA/WSN, without EGFP insertion) were identified based on EGFP reporter gene expression, replication kinetics, and plaque morphology. The in vitro antiviral efficacy of favipiravir, a positive drug for influenza virus, was evaluated by focus-forming assay (FFA), qPCR, and EGFP fluorescence detection. The focus reduction neutralization test (FRNT) and reporter gene activity detection were conducted for IAV nAb detection. [Results] PR8NAEGFP/WSN remained genetically stable after five passages in chicken embryos. Compared with PR8NA/WSN, PR8NAEGFP/WSN showed a slightly declined titer at the time point of 48 h and similar crystal violet plaque morphology. The EC₅₀ of favipiravir measured with the recombinant virus is consistent with that with the control virus, and the EC₅₀ values obtained through various detection methods, including FFA, qPCR, and EGFP fluorescence, show good correlations. The correlation coefficient r of the nAb titer determined by FRNT and EGFP fluorescence was 0.930 4, which indicated good consistency. [Conclusion] PR8NAEGFP/WSN, a recombinant IAV carrying the reporter gene, might be used as a real-time visualization tool for the basic research on IAV and the evaluation of antivirals and vaccines for IAV.

[Objective] We systematically analyzed the growth and compared the ectoine accumulation of Halomonas campaniensis XH26 cultured with nine different amino acids, aiming to clarify the optimal amino acid for ectoine accumulation of strain XH26. [Methods] Under the optimal salt concentration of 1.5 mol/L, nine amino acids (l-monosodium glutamate, l-glutamine, l-aspartic acid, l-asparagine, l-histidine, l-tryptophan, l-glycine, l-serine, and l-lysine) were selected as the single carbon/nitrogen source of the culture medium and added within the concentration range of 20-50 mmol/L (interval of 5 mmol/L), on the basis of which the optimal concentration and optimal amino acid for ectoine accumulation were screened. l-aspartic acid was selected to culture the cells at low (L, 20 mmol/L), medium (M, 35 mmol/L), and high (H, 50 mmol/L) concentrations for targeted metabolomics sequencing and analysis. [Results] The amount of ectoine synthesis first increased and then decreased as the amino acid concentration increased and reached the highest at optimum concentration (30/35 mmol/L). Metabolomics analysis screened out 28 (L vs. M), 27 (L vs. H), and 26 (H vs. M) significantly differential metabolites, such as glyceric acid, lactose, adenosine 5′-monophosphate, α-ketoglutaric acid, glucose-1-phosphate, fumaric acid, and citric acid. KEGG metabolic pathway enrichment analysis showed that l-alanine, l-aspartic acid, and l-glutamate metabolic pathways were the most significantly enriched pathways. [Conclusion] Targeted metabolomics of differential metabolites of bacteria discovers that the strain achieves a rebalance between nitrogen homeostasis and energy supply through the aspartate-alanine axis and the arginine-proline metabolic axis.

[Objective] To deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang. [Methods] Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined. [Results] Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588. [Conclusion] The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.

[Objective] To investigate the effects of calcium ion-free (Ca²⁺-free) conditions on the gene expression, biofilm formation, and virulence of Vibrio parahaemolyticus. [Methods] Ethylene glycol-tetraacetic acid was used to chelate Ca²⁺ in culture media to create Ca²⁺-free conditions. Crystal violet staining was employed to evaluate the biofilm formation of V. parahaemolyticus. Swimming and swarming assays were performed to assess the motility. Additionally, the Kanagawa phenomenon test, HeLa cell adhesion assay, and cytotoxicity experiment were conducted to analyze the virulence phenotypes of V. parahaemolyticus. By comparing the expression profiles, we analyzed the effect of Ca²⁺-free conditions on the gene expression in V. parahaemolyticus. [Results] Ca²⁺-free conditions inhibited the growth and significantly reduced the biofilm formation, intracellular c-di-GMP levels, and motility of V. parahaemolyticus. Furthermore, Ca²⁺-free conditions suppressed the hemolytic activity and reduced the bacterial adhesion to HeLa cells, while enhancing the cytotoxicity of V. parahaemolyticus. Transcriptomic analysis revealed 359 differentially expressed genes (DEGs) under Ca²⁺-free conditions. These DEGs were mainly associated with biofilm formation, virulence factors, and regulators. Notably, the genes involved in lateral flagella and polar flagellum were downregulated, while most virulence genes were upregulated. The majority of putative regulator genes were downregulated. [Conclusion] Ca²⁺-free conditions significantly affect the biofilm formation, motility, virulence, and gene expression of V. parahaemolyticus.

[Objective] To clarify the succession patterns of Lactobacillus and their regulatory effects on the ethyl acetate-to-ethyl lactate ratio during Xiaoqu Qingxiangxing Baijiu fermentation. [Methods] High-throughput sequencing was employed to analyze the species-level succession patterns of Lactobacillus in Xiaoqu and fermented grains (Jiupei). Modified culture media and enrichment methods were employed to isolate Lactobacillus from Jiupei. The dominant Lactobacillus strains were then evaluated for tolerance, subjected to single-carbon-source fermentation experiments, sorghum hydrolysate fermentation experiments, and lab-scale simulated solid-state fermentation with Lactobacillus. The physiological and biochemical characteristics of different Lactobacillus strains, their fermentation characteristics on single carbon sources, and their impact on the ethyl acetate-to-ethyl lactate ratio in the Baijiu were investigated. [Results] A total of 15 Lactobacillus strains were isolated from Jiupei, and high-throughput data analysis identified eight dominant species: Limosilactobacillus pontis, Lactobacillus helveticus, Lentilactobacillus buchneri, Lentilactobacillus hilgardii, Levilactobacillus brevis, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, and Lactobacillus acetotolerans. Further tolerance tests and single-carbon-source fermentation characterization revealed that all the eight dominant strains could withstand actual fermentation conditions (ethanol/acetic acid/lactic acid). However, L. plantarum exhibited impaired maltose utilization, while L. acetotolerans showed defective d-galactose metabolism. L. helveticus and L. pontis displayed a single-product fermentation profile in single-carbon-source cultures, whereas L. brevis, L. buchneri, and L. hilgardii exhibited multi-product fermentation patterns in both single-carbon-source and sorghum hydrolysate media. In lab-scale simulated solid-state fermentation with Lactobacillus, compared with the control, supplementation with L. hilgardii increased the ethyl lactate and ethyl acetate content by 175% and 44%, respectively, reducing the ethyl acetate-to-ethyl lactate ratio to 0.357. Supplementation with L. buchneri increased the ethyl acetate content by 50% while decreasing the ethyl lactate by 71%, raising the ethyl acetate-to-ethyl lactate ratio to 4.496. [Conclusion] The dominant Lactobacillus strains in Xiaoqu Qingxiangxing Baijiu fermentation exhibit strong overall environmental tolerance, and their metabolic profiles vary in single-carbon-source fermentation. The supplementation with different dominant Lactobacillus strains differentially modulates ester formation. These findings provide a theoretical foundation for dynamically regulating key flavor compounds during Xiaoqu Qingxiangxing Baijiu fermentation.

[Objective] To investigate the effects of fertilization regime alterations on microbial communities in paddy soils. [Methods] We conducted a long-term field experiment with three treatments: chemical fertilizer (H) and H converted to a conventional amount of organic fertilizer (HC), a conventional amount of organic fertilizer (C) and C converted to chemical fertilizer (CH), and a high amount of organic fertilizer (G) and G converted to chemical fertilizer (GH). Metagenomic sequencing was combined with bioinformatics analysis to assess the structures, diversity, and co-occurrence networks of microbial communities in paddy soils. [Results] Compared with the H treatment, the HC treatment increased the soil organic carbon, dissolved organic carbon, total nitrogen, and alkali-hydrolyzable nitrogen (P<0.05). The CH and GH treatments exhibited lower soil carbon and nitrogen levels than C and G treatments, respectively. The HC treatment markedly altered the relative abundance of Acidobacteriota, Nitrospirota, Candidatus Rokubacteria, Mucoromycota, and Thaumarchaeota. The CH treatment showed no significant changes in microbial composition at the phylum level, whereas the GH treatment significantly modified the relative abundance of Nitrospirota and the archaeal community structure. Although fertilization regime alterations showed no significant effect on microbial alpha diversity (P>0.05), the beta diversity differed across treatments (P<0.05). Co-occurrence network analysis demonstrated that the HC treatment enhanced the network complexity relative to the H treatment, with increased nodes (181), edges (2 935), average degree (16.215), modularity (0.757), and clustering coefficient (0.495). The CH treatment showed more edges (3 894) and higher average degree (21.514) but lower modularity (0.599) than the C treatment (0.751). Conversely, the GH treatment diminished all network topology parameters relative to G. Redundancy analysis identified dissolved organic carbon (22.1%), soil pH (16.8%), total nitrogen (15.6%), ammonium nitrogen (14.6%), available potassium (11.8%), and available phosphorus (10.6%) as the primary drivers of microbial community variations. [Conclusion] The findings indicate that fertilization regime alterations influence the microbial community structure and network characteristics in paddy soils by modifying soil physicochemical properties. Transitioning from chemical to organic fertilization enhances microbial community stability and soil ecological functions, while replacing organic with chemical fertilization reduces soil carbon and nitrogen availability, potentially compromising microbial network complexity and resilience.