[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 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 unclassifiedSphingomonas. Chao1 and Shannon indexes had significantly negative correlations with unclassifiedSphingomonas but positive correlations with unclassified RB41 and unclassifiedVicinamibacteraceae. [Conclusion] Regional differences had significant effects on soil physicochemical properties, bacterial community structure and diversity, which were more obvious than those of crop differences.

[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 werePseudomonas andBacillus, 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.

[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.

[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 byRalstoniasolanacearum 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 ofBacillus sp. NO1,Brucella sp. NO8,Bacillus sp. NO9, andBacillus 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 ofClostridium_sensu_stricto_1,Ralstonia, andCellulomonas and the increased relative abundance ofDevosia,Flavobacterium, andSphingomonas. 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.

[Objective] To compare the biotite weathering activities and mechanisms betweenPseudomonas azotoformans F77 andPseudomonas 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.

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.

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.

[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] 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.