Abstract:2-phenylethanol (2-PE) is a rose-scented aromatic alcohol commonly used in the food, cosmetic, and pharmaceutical industries. The physical and chemical production methods of 2-PE are not suitable for industrial application due to the low yields. As a single-celled eukaryotic microorganism, yeast has the potential to efficiently synthesize natural 2-PE. Therefore, the strategy of using yeast as a chassis microorganism to synthesize 2-PE is favored by researchers. However, during the fermentation for 2-PE production, the yeast is inevitably affected by the toxic effects of 2-PE. Therefore, there is an urgent need to investigate the mechanisms of yeast tolerance to 2-PE, which will provide a theoretical basis for production practice and help to select yeast strains with high tolerance to 2-PE. In this paper, we review the research advances in 2-PE tolerance of yeast from the synthetic pathways of 2-PE and yeast tolerance mechanisms and introduce the methods for improving the 2-PE tolerance of yeast. Deciphering the mechanism of yeast tolerance to 2-PE for improving the yield and conversion efficiency of 2-PE in yeast is a top priority for the future research.

Abstract:Pyrroloquinoline quinone (PQQ), the third oxidoreductase coenzyme discovered in the nature after nicotinamide and riboflavin, is ubiquitous in bacteria, fungi, plants, and animals. PQQ participates in a variety of life activities and has anti-inflammation, anti-oxidation, cell metabolism-enhancing, and cardioprotective activities, demonstrating broad application prospects in pharmaceuticals, agriculture, food and other fields. Therefore, the large-scale production of PQQ is the primary problem that needs to be solved at present. Microbial fermentation is a primary production method of PQQ. Deciphering the biosynthesis pathway and regulatory mechanism of PQQ is essential for the screening and breeding of strains with short production periods and high yields by metabolic engineering, which has been a hot topic in this field. This paper summarizes the synthesis pathways, strain screening and breeding, microbial production, and purification processes of PQQ, aiming to provide a reference for further research and application of PQQ.

Abstract:People are exposed to environments containing various pathogens, which have multiple interactions with human cells or tissues. Pathogens can survive in the host environment by regulating pathogenic conditions such as virulence and invasiveness. At the same time, host cells resist the invasion of pathogens by mobilizing their own immune system. However, researchers mainly focus on the physiological functions of sRNAs in pathogens and have gained limited knowledge about the interactions between pathogens and hosts. How to use highly sensitive and high-resolution methods to study the interactions between pathogens and hosts have become a major challenge in the current research. By reviewing relevant studies, we summarize the commonly used techniques and experimental processes for studying the interactions between pathogens and hosts, aiming to improve the understanding about the mechanisms and principles of these experimental techniques and provide technical references for the research on interactions between pathogen sRNAs and host targets.

Abstract:In bacterial cells, RNase HI usually degrades RNA in the RNA/DNA hybrids to prevent the accumulation of primers in replication and the formation of R-loops in transcription, thus maintaining genomic stability and normal life activities. The recognition of substrates by RNase HI mainly depends on DNA- and RNA-binding grooves, and the catalysis of substrates by RNase HI mainly depends on the DEDD motif and a histidine located in a flexible loop near the active site. Metal ions represented by Mg²⁺ play an important role in the catalytic process. The mode of action of RNase HI is determined by the type of ssDNA overhangs on RNA/DNA hybrids. In the presence of a 5' ssDNA overhang or in the absence of any overhang on RNA/DNA hybrids, RNase HI functions as a non-sequence-specific endonuclease to degrade RNA randomly. In the presence of a 3' ssDNA overhang on RNA/DNA hybrids, RNase HI relies on 5'-exonuclease activity for the successive degradation of RNA. RNase HI, Rep, DinG, and UvrD are recruited near the replication forks by interacting with the six residues of the C-terminal tail of single-stranded DNA-binding protein (SSB), and may resolve replication-transcription conflicts in a cooperative manner. The deletion of RNase HI or the decrease in RNase HI activity will cause a series of harmful events such as DNA structural instability, gene mutation, transcriptional machinery backtracking, and replication incoordination. RNase HI has shown great application prospects in antisense technology, R-loop detection, and targeted therapy combined with antibiotics. The cooperative mechanism of primer degradation by RNase HI and other enzymes is also worth studying in the future.

Abstract:[Background] Long-term continuous cropping of tobacco (Nicotiana tabacum) leads to the accumulation of autotoxins, which aggravates the incidence of tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum and causing great economic losses of tobacco production. [Objective] To develop a compound bacterial agent capable of controlling TBW in the field with long-term continuous cropping of tobacco. [Methods] We formulated compound bacterial agents with screened strains capable of degrading autotoxins. Orthogonal design and single factor experiments were employed to optimize the strain ratio and the dosages of additives. The inhibition performance of the compound bacterial agent on TBW was evaluated in a greenhouse and in a field with continuous cropping of tobacco for 15 years. [Results] Orthogonal experiments showed that the optimal ratio of Bacillus sp. NO1, Brucella sp. NO8, Bacillus sp. NO9, and Bacillus sp. NO10 in the compound bacterial agent was 1:3:4:2. Single factor experiments showed that the best vector was silica. The best wetting agent and dispersant were sodium hexametaphosphate (SHMP) and sodium butylnaphthalene sulfonate (SBNS), respectively, which were optimized to be added at the dosages of 2%. The optimal concentration of the stabilizer glycerin was 1.0%. Pot experiments showed that both the degradation rate of six autotoxins and the inhibition rate of TBW by the compound bacterial agent reached over 78%. The results of the field experiment showed that the compound bacterial agent diluted by 100 folds had significant degradation effects on six autotoxins in the tobacco field with continuous cropping for 15 years. Moreover, the agent significantly increased the height, stem circumference, and waist leaf length and width of tobacco plants, thus promoting the growth and development of tobacco. In addition, the agent regulated the rhizosphere microbiota of tobacco, as manifested by the decreased relative abundance of Clostridium_sensu_stricto_1, Ralstonia, and Cellulomonas and the increased relative abundance of Devosia, Flavobacterium, and Sphingomonas. The agent decreased the incidence rate and disease index of TBW from 92.22% and 48.19% to 18.15% and 9.52%, respectively, with a control effect of 80%. [Conclusion] The optimized compound bacterial agent significantly reduces the incidence rate and disease index of TBW in the field with long-term continuous cropping of tobacco, which provides a solution for the prevention and control of TBW.

Abstract:[Objective] To study the spatial and temporal distribution characteristics, community structure, and diversity of uncultured myxobacteria in the rhizosphere soil of Phragmites australis in the Ebinur Lake wetland, so as to enrich the knowledge of myxobacteria resources in saline-alkaline wetlands, lay a foundation for exploiting the myxobacteria resources in extreme environments, and provide data support for the restoration of saline desert ecosystems. [Methods] The rhizosphere soil samples of P. australis were collected from 10 sites in Ebinur Lake wetland in 3 months, and high-throughput sequencing was conducted for the V4–V5 region of the 16S rRNA gene to reveal the diversity and spatial and temporal distribution of myxobacteria. [Results] The abundance of myxobacteria, as indicated by the presence of 16S rRNA gene tags, ranged from 0.22% to 3.54% of the total bacteria in the Ebinur Lake wetland. The highest diversity was observed in July and at the sampling site 4, suggesting the correlations of genus diversity with both seasons and sample sites. A total of 14 genera of myxobacteria belonging to 8 families of 3 suborders were identified. Among them, Haliangium was the dominant genus, with the relative abundance of 10.83%–71.01%. Network co-occurrence diagrams showed that most of the bacteria interacted with myxobacteria. Spearman correlation analyses showed that the Shannon, Chao1, and ACE indices of bacteria influenced the diversity and richness of myxobacteria. The redundancy analysis (RDA) showed that soil inorganic nitrogen (IN), organic matter (OM), and water-soluble magnesium ions (Mg²⁺) were the main abiotic factors influencing the diversity and community structure of myxobacteria. [Conclusion] The Ebinur Lake wetland is rich in myxobacteria, the diversity and community structure of which present spatial and temporal variations. Biotic factors (bacteria) and abiotic factors (soil physico-chemical properties) jointly affect the diversity of myxobacteria.

Abstract:[Objective] To analyze the influence of pH on sulfate-reducing properties of sulfate-reducing bacteria enriched from marine sediments, identify the bacterial community composition, predict the key genes of sulfate reduction, and explore the mechanism of sulfate reduction. [Methods] The sulfate reduction rates of sulfate-reducing bacteria at different pH conditions were determined. On this basis, high-throughput sequencing and PICRUSt were employed to analyze the dominant sulfate-reducing bacteria and the relative abundance of sulfate-reducing genes. [Results] The biomass (OD₆₀₀) and sulfate reduction rate of sulfate-reducing bacteria varied significantly under different pH conditions (P<0.01) and reached their peak values (0.34±0.01 and 96.52%±0.44%, respectively) at pH 5.0. According to the high-throughput sequencing data, the abundance and diversity of bacteria were the highest at pH 5.0. The dominant bacteria were Pseudomonas and Bacillus, and the assimilatory sulfate reduction-related genes had higher abundance. [Conclusion] Sulfate-reducing bacteria are suitable to be enriched and cultured at pH 5.0. Under this condition, the high sulfate reduction rate is attributed to the assimilatory sulfate reduction pathway. The findings provide experimental support for revealing sulfate reduction mechanism and broaden the germplasm resources of sulfate-reducing bacteria for application.

Abstract:[Objective] To investigate the effects and mechanism of Lactobacillus plantarum postbiotics at different doses on ameliorating Salmonella enterica Typhimurium (ST) infection in mice. [Methods] Sixty 5-week C57BL/6 mice were randomized into five groups: Control, ST, CFS-L+ST, CFS-M+ST, and CFS-H+ST. Lactobacillus plantarum postbiotics (cell-free supernatant, CFS) was administrated at low (L), medium (M), and high (H) doses (50, 100, and 200 μL, respectively) for 21 days. On day 22, mice were orally challenged with ST at 3×10⁸ CFU, and the samples were collected three days later. [Results] Compared with the control group, CFS-L+ST and CFS-M+ST groups showed no significant changes in body weight gain, while the CFS-H+ST group showed a significant decrease (P<0.05). The CFS-M+ST and CFS-H+ST groups alleviated ST-induced body weight loss (P<0.05). CFS pretreatment reduced ST-induced bacterial translocation in the colon, liver, spleen, and brain (P<0.05) and alleviated the pathological damages in the colon and spleen. ST reduced the levels of acetic acid and butyric acid in the cecum, which, however, increased in the CFS-M+ST group (P>0.05). Compared with the ST group, CFS-M+ST alleviated the inflammatory response by lowering the levels of pro-inflammatory cytokines including interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P<0.05) and elevating the levels of anti-inflammatory cytokines including interleukin-4 (IL-4) and interleukin-10 (IL-10) (P<0.05). Moreover, CFS-M+ST suppressed ST-induced inflammation by modulating the nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome, as indicated by the down-regulated mRNA levels of NLRP3, apoptosis-associated speck-like protein (ASC), cysteine-dependent aspartate-specific protease 1 (caspase-1), and gasdermin D (GSDMD) (P<0.05). Furthermore, CFS inhibited NLRP3 inflammasome by blocking the upstream key nuclear factor kappa beta (NF-κB) pathway, as indicated by the down-regulated expression levels of myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor beta-activated kinase 1 (TAK1), and NF-κB (P<0.05). [Conclusion] L. plantarum postbiotics CFS alleviated the S. enterica Typhimurium infection and inflammatory responses in mice by inhibiting the NF-κB-mediated NLRP3 inflammasome, and the pretreatment with medium-dose CFS showed the best effects.

Abstract:[Objective] Over a prolonged period of growth, the old tea plant ecosystem of Camellia sinensis ‘Fujian Shuixian’ in Gujing has acquired a distinctive fir flavor, with the mountainous environment playing a key role in shaping the tea quality. Microbial communities play a vital role in biogeochemical cycling within mountainous ecosystems. Nevertheless, the characteristics of the microbial community within the distinct old tea plant ecosystem remain incompletely understood. [Methods] Samples were collected from both the aboveground and belowground parts of the old C. sinensis ‘Fujian Shuixian’ tea garden in Gujing, situated in the central region of the Wuyishan National Park. The samples encompassed the phyllosphere and leaf endosphere, along with soils from the rhizosphere, non-rhizosphere, and surrounding regions. High-throughput sequencing of the 16S rRNA gene and internal transcribed spacer (ITS) region were carried out to assess the bacterial and fungal diversity, respectively. [Results] The richness and diversity of bacterial and fungal communities in the aboveground part were markedly lower than those in the belowground part. Within co-occurrence networks, the modularity index for taxa networks in each niche exceeded 0.4, and the positive correlations in interactions among microbial taxa are greater than the competition. The prevailing phyla, with the relative abundance greater than 1%, were Proteobacteria, Acidobacteriota, Actinobacteriota, Ascomycota, and Basidiomycota, which were present in both above and belowground habitats. Notably, the relative abundance of Proteobacteria, Actinobacteria, and Ascomycetes aboveground surpassed that belowground, whereas that of Acidobacteria and Basidiomycetes displayed an opposite pattern (P<0.05). The prominent genera (with the abundance exceeding 1%) identified were Methylobacterium-Methylorubrum, Sphingomonas, Pseudomonas, Amnibacterium, Bacillus, Cladosporium, and Fusarium. These genera potentially served as crucial biomarkers in the ecological niches of C. sinensis ‘Fujian Shuixian’ within the old tea plant ecosystem in Gujing. [Conclusion] The present study unveiled the unique attributes of bacterial and fungal communities within the old tea plant ecosystem of C. sinensis ‘Fujian Shuixian’ in Gujing, delivering valuable scientific insights for disease prevention and biocontrol in tea plant cultivation, tea quality improvement, and the exploration of functional microbial resources.

Abstract:[Objective] To compare the biotite weathering activities and mechanisms between Pseudomonas azotoformans F77 and Pseudomonas paracarnis P1. [Methods] During the mineral weathering process, the dissolved Fe and Al concentrations, cell number, pH, gluconic acid concentration, and residual glucose concentration in the culture medium were determined to reveal the biotite weathering effects and mechanisms of strains F77 and P1. Furthermore, RNA-seq was employed to explore the molecular mechanism for the difference in the biotite weathering effect between the two strains. [Results] During the 5 days of mineral weathering, strain F77 increased Fe and Al concentrations by 3.3-23.3 folds and gluconic acid concentration by 27.3-53.9 folds the compared with strain P1. Meanwhile, strain F77 showed decreased cell number and medium pH compared with strain P1. The data of comparative transcriptomics showed that strain F77 had more specific genes (2 872) and differentially expressed genes (1 832) than strain P1 (1 903 and 1 258 genes, respectively). Additionally, strain F77 carried more genes involved in the membrane transport, carbohydrate metabolism, cell motility, chemotaxis, and signal transduction than strain P1. Furthermore, strain F77 had higher fold changes in the expression levels of superoxide dismutase and catalase genes as well as higher number and fold changes of the genes involved in gluconic acid synthesis than strain P1. [Conclusion] Strain F77 surpassed strain P1 in weathering the biotite and producing gluconic acid. Strain F77 promoted the biotite weathering by producing gluconic acid. The addition of biotite significantly up-regulated the expression of genes involved in the transmembrane transport, cell movement and chemotaxis, signal induction, and carbon and energy metabolisms in mineral weathering. Furthermore, the genes involved in gluconic acid synthesis and encoding superoxide dismutase and catalase may play a role in the mineral weathering by strain F77.

Abstract:[Objective] To investigate the diversity and community structure of soil bacteria in the farmlands in Qinghai Province. [Methods] High-throughput sequencing was employed to analyze the bacterial community structure and diversity in the soil samples of farmlands growing wheat, oilseed rape, and highland barley in Dulan, Huzhu, Gonghe, and Datong counties. Furthermore, the relationship between bacterial community structure and soil physicochemical properties was analyzed. [Results] The pH, moisture, and organic matter of soil, as well as the Chao1 and Shannon indexes and linear discriminant analysis effect size (LEfSe) of soil bacteria, showed significant differences in some of these indexes (P<0.01) but no significant differences among the three crops (P>0.05). The results of principal component analysis (PCA) showed that the bacterial community structure was different among different regions but highly similar in the farmlands of the three crops. A total of 3 127 operational taxonomic units (OTUs) and 3 694 OTUs were common in the four regions and in the farmlands of the three crops, respectively. The OTUs of soil bacteria were identified as 423 species, 450 genera, 276 families, 192 orders, 93 classes of 36 phyla. The four regions or three crops had similar dominant phyla, genera, and species, while these taxa differed in relative abundance. Soil moisture, pH, and organic matter were significantly correlated with Chao1 and Shannon indexes. Soil pH and organic matter had significantly positive or negative correlations with unclassified species, unclassified RB41, and unclassified Sphingomonas. Chao1 and Shannon indexes had significantly negative correlations with unclassified Sphingomonas but positive correlations with unclassified RB41 and unclassified Vicinamibacteraceae. [Conclusion] Regional differences had significant effects on soil physicochemical properties, bacterial community structure and diversity, which were more obvious than those of crop differences.

Abstract:[Objective] To obtain the proteins of acetyl-CoA synthase (ACS_MU) and PHA synthase (PhaC_MU) from Massilia sp. UMI-21 by structuring an in vitro recombinant expression system, and to elucidate their roles in the biosynthesis of polyhydroxybutyrate (PHB) using the one-phase reaction system (OPRS). [Methods] Seamless cloning was employed to ligate the acetyl-CoA synthase gene acs_MU and the PHA synthase gene phaC_MU amplified from Massilia sp. UMI-21 to the pQE-80L plasmid to construct the recombinant plasmids. The recombinant plasmids were transformed into Escherichia coli BL21(DE3), and the recombinant strains were obtained. ACS_MU and PhaC_MU were purified using a 6×His tag, and their activities were determined by the 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) method. With 3HB as a substrate, the one-phase reaction system (OPRS) was employed to validate the functions of ACS_MU and PhaC_MU in the biosynthesis of PHB. [Results] The recombinant strains BL21-pQE-80L-acs_MU and BL21-pQE-80L-phaC_MU were successfully engineered, with the ACS_MU and PhaC_MU yields of 24.8 mg/L and 25.6 mg/L, respectively. The specific activity of ACS_MU was (0.148±0.011) U/mg, and that of PhaC_MU for (R)-3HBCoA was (0.102±0.011) U/mg. Nuclear magnetic resonance hydrogen spectroscopy (¹H-NMR) results showed that products from the all three PHB synthesis pathways, ACS_Pt-PCT_CP-PhaC_Re, ACS_MU-PCT_CP-PhaC_Re, and ACS_MU-PCT_CP-PhaC_MU, in OPRS were PHB. The yields of PHB via the three pathways were 0.62, 0.76, and 0.64 g/L, respectively. [Conclusion] The genes acs_MU and phaC_MU can be overexpressed in the E. coli expression system to yield active soluble proteins. Compared with the ACS_Pt-PCT_CP-PhaC_Re pathway, substitution of ACS_Pt with ACS_MU increased the PHB yield by 22.58%. The yield of PHB was contingent upon the stability of acetyl-CoA synthase (ACS), which provided acetyl-CoA for reaction under identical PhaC. Replacing PhaC_Re with PhaC_MU decreased the PHB yield by 15.79% compared with ACS_MU-PCT_CP-PhaC_Re. The polymerase PhaC plays a crucial role in PHB synthesis under identical precursor concentrations.

Abstract:[Objective] To explore the changes in the yield and composition of extracellular polymeric substances (EPS) of Alternaria sp. CGMCC 17463, a strain of dark septate endophyte (DSE), cultured for different time periods. [Methods] We conducted the shake flask experiment to compare the yield, structure, composition, and activity of EPS synthesized by a DSE strain cultured for different time periods. [Results] From day 4 to 12, the growth of the DSE strain entered the logarithmic and stationary phases. During this period, the EPS synthesis rate was high, with the yield reaching 1.41 g/L on day 12. Afterwards, the EPS synthesis rate gradually decreased. The component analysis revealed that the extracellular polysaccharide content was the highest on day 12 in the EPS samples of equal mass. As the growth of DSE continued and entered the decline phase, mycelial lysis occurred, significantly increasing the protein content in the EPS. Functional group analysis showed that as the incubation time was extended, the functional groups in the EPS presented changes only in the content but not species. The results of scanning electron microscopy and particle size analysis showed that the EPS composition gradually changed with the increase in incubation time. Specifically, the EPS components with the particle size smaller than 5 μm presented increased volume percentage, while those with the particle size larger than 100 μm showed gradually decreased volume percentage. Furthermore, the EPS possessed the ability to scavenge oxygen free radicals and retain water, which were significantly influenced by the changes in EPS composition. [Conclusion] The day 12 marks the optimal time point for the production of EPS with high polysaccharide content, while the day 24 marks the optimal time point for the production of EPS with high protein content. This result establishes a foundation for the application of EPS in the complex eco-environment of mines.

Abstract:[Objective] To decipher the regulatory mechanism of a sensor histidine kinase (CusS) in Escherichia coli K-12 in response to silver ion stress and provide scientific evidence for the prevention and treatment of this bacterium. [Methods] ProtParam, ProtScale, Protein-Sol, TMHMM, SignalP, LocTree3, NetNGlyc-1.0, NetPhosBac-3.0, SOPMA, I-TASSERF, STRING, and MEGA were employed to predict the physicochemical properties, hydrophilicity, solubility, transmembrane domain, signal peptides, subcellular localization, glycosylation sites, phosphorylation sites, secondary structure, tertiary structure, protein-protein interaction network of CusS, and the homology of CusS in Gram-negative bacilli, respectively. After that, ΔcusS was constructed by the Red homologous recombination system, and the growth of ΔcusS in different media was monitored. In addition, we evaluated the sensitivity of ΔcusS to silver and copper ions and common antibiotics based on the minimum inhibitory concentration (MIC). RT-qPCR was employed to determine the transcription levels of cusCFBA and cusR after cusS deletion. [Results] CusS was composed of 480 amino acid residues, with the relative molecular weight of 53 738.05, the atom number of 7 624, and the isoelectric point of 6.02. It was a hydrophilic and insoluble protein containing transmembrane domain, and no signal peptide, located in the intracellular membrane. CusS had 2 glycosylation sites, 24 serine phosphorylation sites, 14 threonine phosphorylation sites, and 3 tyrosine phosphorylation sites. In the secondary structure, α-helixes, β-sheets, β-turns, and random coils accounted for 55.42%, 11.67%, 3.75%, and 29.17%, respectively. The gene cusS was highly conserved in Escherichia and Shigella. The colony PCR and first-generation sequencing confirmed the successful construction of ΔcusS. The deletion of cusS had no influence on the growth or metabolism of the strain. However, cusS was the key gene for E. coli in response to the silver ion stress. [Conclusion] The deletion of cusS did not affect the growth but attenuated the protective response of E. coli to silver ion stress. Furthermore, the deletion of cusS significantly down-regulated the mRNA levels of the downstream genes cusCFBA and cusR. The bioinformatics analysis and phenotype characterization of CusS lays a foundation for unveiling the regulatory mechanism of CusS in E. coli in response to silver ion stress.

Abstract:[Objective] Microsporidia are obligate intracellular parasites capable of infecting a wide range of animal species, including both humans and animals of economic interests. We explored Nosema bombycis hexokinase (NbHK) in terms of the expression, subcellular localization, regulatory functions, and interacting proteins in Bombyx mori embryo cells, aiming to provide insights into the function and mechanism of this protein during infection. [Methods] We prepared a polyclonal antibody against NbHK to analyze the expression and localization of NbHK in N. bombycis-infected BmE cells by using Western blotting and the indirect immunofluorescent assay. Overexpression and RNA interference experiments were performed to assess the impact of NbHK on pathogen proliferation. RNA-seq was employed to analyze the transcriptional responses of the NbHK-transgenic BmE cells. A biotin-streptavidin system and mass spectrometry were employed to identify the interacting proteins of NbHK from NbHK::APEX2-transgenic BmE cells. [Results] NbHK was predominantly localized in the nucleus of infected cells, with consistently upregulated expression during infection. The overexpression of NbHK significantly increased the pathogen load, while the knock-down of NbHK suppressed pathogen proliferation, which indicated the crucial roles of NbHK during infection. RNA-seq analysis identified 94 differentially expressed genes (DEGs) responsive to infection, comprising 58 up-regulated genes and 36 down-regulated genes. The enrichment analysis of DEGs revealed significant activation of pathways related to cell lifespan regulation and protein processing in the endoplasmic reticulum while significant inhibition of the mitophagy pathway. Additionally, we identified host proteins including nucleoprotein translocated promoter region (NTPR) in the nucleus that potentially interacted with NbHK. [Conclusion] NbHK is secreted into silkworm nucleus to modulate the expression of genes involved in multiple pathways for promoting pathogen proliferation. Our study offers novel insights into the roles of NbHK in the infection of N. bombycis.

Abstract:[Objective] To investigate the impacts of the amino acid residues at position 253 (glutamine, Q) and 254 (isoleucine, I) in the β8 sheet of the D3 domain of listeriolysin O (LLO) on the biological functions of Listeria monocytogenes. [Methods] We constructed the mutant proteins LLO_Q253A and LLO_I254A and the mutant strains hly_Q253A and hly_I254A by homologous recombination. After the expression and purification, the mutant proteins examined for the hemolytic activity. Furthermore, the growth, adhesion, invasion, intracellular migration, and proliferation were compared between the mutant strains hly_Q253A and hly_I254A. [Results] After the mutation of the corresponding sites, LLO proteins could be expressed normally. However, the mutant proteins and strains lost hemolytic activity at pH 6.5, and the hemolytic activities of LLO_I254A and hly_I254A were restored at pH 5.5. The mutant strains showed no significant differences in extracellular growth, adhesion, and intracellular proliferation compared with the wild-type strain. However, the invasion and intercellular migration of the mutant strains were significantly lower than that of the wild-type strain. [Conclusion] The mutations of Q253A and I254A in LLO cause the loss of hemolytic activity at pH 6.5 and a reduction in the bacterial infection, the specific mechanisms of which remain to be explored. This study establishes a foundation for deeply understanding the impact of LLO structure on the biological function of L. monocytogenes and holds significance for the construction of point-mutated strains of L. monocytogenes.

Abstract:[Objective] To apply multifunctional plant growth-promoting rhizobacteria to enhance peanut growth and mitigate the inhibitory effects caused by continuous cropping. [Methods] Plant growth-promoting rhizobacteria were screened from the rhizosphere soil of peanut plants in a system with continuous cropping for ten years, and their growth-promoting and antagonistic abilities were determined. The strains were identified by 16S rRNA gene sequencing. Three plant growth-promoting rhizobacterial strains with complementary functions and no growth inhibition between each other were selected to prepare a compound microbial inoculant, the plant growth-promoting effect of which was examined by seed germination and pot experiments. High-throughput sequencing was carried out for the V3–V4 region of bacterial 16S rRNA gene. [Results] A total of 37 plant growth-promoting rhizobacterial strains capable of promoting plant growth and inhibiting pathogen growth were screened from the rhizosphere of peanut plants in a continuous cropping system. Three strains were selected to prepare the compound inoculant. Compared with the blank control, the compound inoculant increased the germination rate of peanut by 13.22%. Compared with the treatments with the three strains alone, the compound inoculant increased the germination rate by 6.99%, 7.51%, and 8.87%, respectively. The application of the compound inoculant had significant promoting effects on the root morphology, number of nodules, chlorophyll relative content (SPAD), photosynthetic parameters, and antioxidant enzyme activity of peanut plants. Specifically, it increased the total root length, number of root tips, taproot diameter, root volume, and root activity by 43.50%, 49.31%, 15.11%, 16.92%, and 112.16%, respectively. The application of the compound inoculant significantly increased the leaf SPAD value and promoted the photosynthesis of peanut plants at seedling stage and flowering stage. Furthermore, it increased the number of root nodules by 34 nodules per plant. However, the application of the compound inoculant had no significant effect on the bacterial diversity in peanut rhizosphere. The dominant phyla were Proteobacteria, Actinobacteriota, and Bacteroidota, accounting for more than 70%. Novosphingobium and Sphingomonas were the dominant genera. [Conclusion] The compound inoculant of plant growth-promoting rhizobacteria improved the seed germination, root growth, leaf SPAD value, and photosynthesis of peanut plants, providing technical support for alleviating continuous cropping obstacles and promoting the healthy growth of peanut plants.

Abstract:The intestinal mucosal microbiota plays an important role in regulating the physiological functions of the host, and its structure and composition are modulated by multiple factors. The host sex is regarded as a key factor shaping the gut microbiota. However, the effects of different sexes on intestinal mucosal microbiota remain unclear. [Objective] To investigate the differences of the composition and functions of the intestinal mucosal microbiota between male and female Jiangshan Black pigs. [Methods] This study analyzed the ileal and colonic mucosal microbiota of eight sexually mature female and eight male Jiangshan Black pigs by 16S rRNA gene high-throughput sequencing. [Results] The Chao1 index and Shannon index of the ileal mucosal microbiota in male pigs were higher than that in female pigs (P<0.05). However, the two indexes of the colonic mucosal microbiota had no significant differences between the male and female pigs (P>0.05). The ileal mucosa of female pigs had higher relative abundance of Serratia and Escherichia_Shigella and lower relative abundance of Oscillospiraceae UCG-005, Alloprevotella, Blautia and Prevotellaceae_NK3B31_group than that of male pigs (P<0.05). The colonic mucosa of female pigs had higher relative abundance of unclassified_Muribaculaceae, Rikenellaceae_RC9_gut_group, and Prevotellaceae UCG-003 and lower relative abundance of Oscillospiraceae UCG-005, Lachnospiraceae_NK4A136_group, and unclassified_Lachnospiraceae than that of male pigs (P<0.05). Functional prediction results showed that the intestinal mucosal microbiota of male Jiangshan Black pigs was mainly enriched with functional pathways such as amino acid metabolism, carbohydrate metabolism, and energy metabolism (P<0.05), while the colonic mucosal microbiota was mainly enriched with functional pathways such as ABC transporters and two-component signal transduction systems (P<0.05). [Conclusion] The structure and function of intestinal mucosal microbiota were different between male and female Jiangshan Black pigs. The results provide references for understanding and excavating the intestinal microbial resources of local breeds of domesticated animals in China.

Abstract:[Objective] To investigate the role of the small RNA (sRNA) RybB and the chaperone protein Hfq in regulating the expression of porin OmpD in Salmonella. [Methods] In this study, Salmonella Typhimurium (STM) was used as the research object. The pCE40 plasmid carrying the reporter gene lacZ encoding β-galactosidase was transferred into the single mutant lacking ompD to obtain the lacZ reporter strain. On this basis, we employed P22 phage-mediated transduction to construct the double mutants lacking full-length rybB, full-length hfq, partial sequence of hfq, or truncated hfq sequence and the triple mutantlacking full-length rybB and full-length hfq. The regulatory effects of RybB and Hfq on the expression of OmpD were probed by β-galactosidase activity assay and RT-qPCR. [Results] We successfully constructed the double and the triple mutant. Compared with that in the wild type (WT), the OmpD activity was down-regulated by 2.16% in the lacZ reporter strain with truncated sequence (87 residues) of hfq, and the β-galactosidase activity of OmpD increased in the rest strains. Compared with WT, except for STM LT2∆ompD::lacZ∆hfq6, all the mutants showed up-regulated transcript level of ompD (P<0.05), with the most significant up-regulation of 1.83-folds in the triple mutant. [Conclusion] The transcription and translation of ompD are mainly regulated by the negative feedback of hfq and RybB. The distal end of Hfq plays a key role in the transcriptional repression of ompD. By construction of several mutants, this article illustrated the interactions of RybB and Hfq with OmpD and explored the key regions of Hfq in regulating ompD, which enriched the theory of sRNA regulation.

Abstract:[Objective] The isolation and comparative genomic analysis of Acidithiobacillus capable of oxidizing sulfur will enrich our knowledge about not only sulfur-oxidizing bacterial strains but also the molecular evolution and ecological adaptation mechanisms of Acidithiobacillus.[Methods] The medium with sodium thiosulfate as the sole energy source was used to isolate the strain capable of oxidizing sulfur, which was followed by Illumina HiSeq X and Oxford Nanopore sequencing of strain M4-422-6. Bioinformatics tools were used for sequence assembly and gene annotation, and the comparative genomic analysis was performed with Ignacidithiobacillus copahuensis VAN18-1. [Results] An Acidithiobacillus strain M4-422-6 capable of oxidizing sulfur was isolated. The genome annotation results showed that the genome of strain M4-422-6 consisted of one chromosome and two plasmids, with a length of 2 917 823 bp and G+C content of 58.54%, encoding a total of 2 925 proteins. The 16S rRNA gene sequence and the phylogenetic tree built by the type (strain) genome server (TYGS) revealed that strain M4-422-6 represented a novel species of Acidithiobacillus. Functional gene annotation showed that strain M4-422-6 carried numerous genes involved in sulfur oxidation, CO₂ fixation, and acid resistance. The comparative genomic analysis revealed that although strain M4-422-6 had the closest genetic relationship with Ignacidithiobacillus copahuensis VAN18-1, and the two strains possessed numerous different genes, which were mainly involved in phage resistance and mobile element encoding. [Conclusion] Strain M4-422-6 represents a novel species of Acidithiobacillus and has unique genes that are not present in strains of the same species. Therefore, we hypothesize that the intra-species differentiation of Acidithiobacillus can be attributed to adaptation to specific niches.

Abstract:[Objective] Anthracnose is a major disease attacking Camellia oleifera plants. Colletotrichum fructicola with a wide distribution scope and a high isolation rate is the major pathogen causing anthracnose in C. oleifera. This study explored the roles of autophagy-related proteins CfAtg6 and CfAtg14 and the molecular mechanism for the pathogenicity of C. fructicola, aiming to provide a theoretical basis for the prevention and control of anthracnose in C. oleifera. [Methods] The homologous recombination principle and polyethylene glycol (PEG)-mediated transformation method were employed to construct the gene-deleted strains ΔCfATG6 and ΔCfATG14 and the complemented strains ΔCfatg6-C and ΔCfatg14-C. [Results] The yeast two-hybrid assay results showed that ΔCfatg6 and ΔCfatg14 might interact with each other. Compared with the wild type and complemented strains, ΔCfatg6 and ΔCfatg14 demonstrated significantly slow vegetative growth, and their appressorium formation rates were only 5% and 18% that of the wide type. In addition, ΔCfatg6 and ΔCfatg14 showed significantly weakened pathogenicity, causing the lesion areas only 1/3 of the wild type and complemented strains on C. oleifera leaves. In addition, ΔCfatg6 and ΔCfatg14 lost the ability of transporting and degrading CfAtg8 protein and became more sensitive to the cell wall stress. The conidium production of ΔCfatg6 decreased significantly, being only 20% that of wild type. The inhibition rate of hydrogen peroxide on the growth of the deleted strains was 10% higher than those on the wild type and complemented strains. ΔCfatg14 showed increased sensitivity to dithiothreitol stress. [Conclusion] The autophagy-related genes CfATG6 and CfATG14 are involved in the regulation of the growth, autophagy, and pathogenicity of C. fructicola.

Abstract:[Objective] Fusarium proliferatum is one of the major pathogens causing root rot of alfalfa. This study aims to investigate the function of the aconitase family in F. proliferatum and give insights into the molecular mechanisms underlying the physiological metabolism of this pathogen. [Methods] We employed the hmmsearch tool to identify the proteins containing the aconitase domain in F. proliferatum and then carried out the phylogenetic analysis. real-time PCR and SWISS-MODEL were employed to analyze the expression profiles of FpACO genes and the protein structures, respectively. The homologous recombination method was used to construct the FpACO-deleted mutants of F. proliferatum. Furthermore, we explored the growth, sporulation, spore morphology, stress responses, and pathogenicity of ΔFpACO3, ΔFpACO4-1, and ΔFpACO4-2 and measured the mitochondrial metabolism indicators of the mutants. [Results] FpACO4-1 and FpACO4-2 were involved in sporulation and spore morphogenesis. FpACO3, FpACO4-1, and FpACO4-2 were responsible for regulating the sensitivity of F. proliferatum to cell wall stress and metal ion stress. Moreover, FpACO3, FpACO4-1, and FpACO4-2 affected the mitochondrial metabolism indicators, including the total aconitase activity, the ATP level, the hydrogen peroxide level, and the expression of key genes in the tricarboxylic acid cycle. [Conclusion] The aconitase family members are involved in the regulation of the processes such as sporulation, spore morphogenesis, response to cell wall stress and metal ion stress, and mitochondrial metabolism in F. proliferatum.