[Objective] To explore the changes in the yield and composition of extracellular polymeric substances (EPS) ofAlternaria 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.

[Objective] To decipher the regulatory mechanism of a sensor histidine kinase (CusS) inEscherichia 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 ofcusCFBA andcusR aftercusS 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 genecusS was highly conserved inEscherichia andShigella. The colony PCR and first-generation sequencing confirmed the successful construction of ΔcusS. The deletion ofcusS had no influence on the growth or metabolism of the strain. However,cusS was the key gene forE.coli in response to the silver ion stress. [Conclusion] The deletion ofcusS did not affect the growth but attenuated the protective response ofE.coli to silver ion stress. Furthermore, the deletion ofcusS significantly down-regulated the mRNA levels of the downstream genescusCFBA andcusR. The bioinformatics analysis and phenotype characterization of CusS lays a foundation for unveiling the regulatory mechanism of CusS inE.coli in response to silver ion stress.

[Objective] Microsporidia are obligate intracellular parasites capable of infecting a wide range of animal species, including both humans and animals of economic interests. We exploredNosemabombycis hexokinase (NbHK) in terms of the expression, subcellular localization, regulatory functions, and interacting proteins inBombyxmori 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 inN.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 ofN.bombycis.

[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 ofListeria monocytogenes. [Methods] We constructed the mutant proteins LLO_Q253A and LLO_I254A and the mutant strainshly_Q253A andhly_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 strainshly_Q253A andhly_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 andhly_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 ofL.monocytogenes and holds significance for the construction of point-mutated strains ofL.monocytogenes.

[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 wereProteobacteria,Actinobacteriota, andBacteroidota, accounting for more than 70%.Novosphingobium andSphingomonas 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.

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 ofSerratia andEscherichia_Shigella and lower relative abundance ofOscillospiraceae UCG-005,Alloprevotella,Blautia andPrevotellaceae_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, andPrevotellaceae UCG-003 and lower relative abundance ofOscillospiraceae 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.

[Objective] To investigate the role of the small RNA (sRNA) RybB and the chaperone protein Hfq in regulating the expression of porin OmpD inSalmonella. [Methods] In this study,Salmonella Typhimurium (STM) was used as the research object. The pCE40 plasmid carrying the reporter genelacZ encoding β-galactosidase was transferred into the single mutant lackingompD to obtain thelacZ reporter strain. On this basis, we employed P22 phage-mediated transduction to construct the double mutants lacking full-lengthrybB, full-lengthhfq, partial sequence ofhfq, or truncatedhfq sequence and the triple mutantlacking full-lengthrybB and full-lengthhfq. 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 thelacZ reporter strain with truncated sequence (87 residues) ofhfq, 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 ofompD (P < 0.05), with the most significant up-regulation of 1.83-folds in the triple mutant. [Conclusion] The transcription and translation ofompD are mainly regulated by the negative feedback ofhfq and RybB. The distal end of Hfq plays a key role in the transcriptional repression ofompD. By construction of several mutants, this article illustrated the interactions of RybB and Hfq with OmpD and explored the key regions of Hfq in regulatingompD, which enriched the theory of sRNA regulation.

[Objective] The isolation and comparative genomic analysis ofAcidithiobacillus capable of oxidizing sulfur will enrich our knowledge about not only sulfur-oxidizing bacterial strains but also the molecular evolution and ecological adaptation mechanisms ofAcidithiobacillus. [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 withIgnacidithiobacilluscopahuensis VAN18-1. [Results] AnAcidithiobacillus 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 ofAcidithiobacillus. 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 withIgnacidithiobacilluscopahuensis 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 ofAcidithiobacillus and has unique genes that are not present in strains of the same species. Therefore, we hypothesize that the intra-species differentiation ofAcidithiobacillus can be attributed to adaptation to specific niches.

[Objective] Anthracnose is a major disease attackingCamellia oleifera plants.Colletotrichum fructicola with a wide distribution scope and a high isolation rate is the major pathogen causing anthracnose inC.oleifera. This study explored the roles of autophagy-related proteins CfAtg6 and CfAtg14 and the molecular mechanism for the pathogenicity ofC.fructicola, aiming to provide a theoretical basis for the prevention and control of anthracnose inC.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 onC.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 genesCfATG6 andCfATG14 are involved in the regulation of the growth, autophagy, and pathogenicity ofC.fructicola.

[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 inF.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 inF.proliferatum and then carried out the phylogenetic analysis. real-time PCR and SWISS-MODEL were employed to analyze the expression profiles ofFpACO genes and the protein structures, respectively. The homologous recombination method was used to construct theFpACO-deleted mutants ofF.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 ofF.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 inF.proliferatum.