Abstract:[Background] As an essential part of freshwater ecosystem, virioplankton plays a key role in shaping the planktonic community structure and regulating the biogeochamical cycle of nutrients in water. The hydrodynamic process of reservoir is different from that of lake, and the disturbance may affect the virioplankton-host dynamics. [Objective] To reveal the effect of hydraulic disturbance on virioplankton-host dynamics and thus to lay a scientific basis for explaining the ecological function of virioplankton in reservoir. [Methods] The water sample from Xiangxi Bay was used and different flows were simulated. The parameters such as virioplankton abundance, host abundance, host lysis rate, and percentage of lysogenic host were monitored, and the relationship of the parameters with environmental factors was analyzed. [Results] The flow at 0.05 m/s and 0.10 m/s significantly promoted the growth of phytoplankton and bacteriaplankton but had no significant influence on the abundance of virioplankton. Disturbance enhanced the virus-mediated lysis of phytoplankton and bacteriaplankton, particularly the flow at 0.05 m/s. Meanwhile, the disturbance significantly reduced the phytoplankton lysogenic induction rate but caused the significant increase in the bacteriaplankton lysogenic induction rate (P <0.05). [Conclusion] The simulated disturbance had significant impact on the virioplankton-host dynamics, indicating that the survival strategy of reservoir virioplankton may be different from that of lake virioplankton.

Abstract:[Background] Research on the causes of the pollution in reservoir water often focuses on water eutrophication, and the changes of pH value, dissolved oxygen, ammonia nitrogen, and total bacterial count. However, a few studies on the seasonal variation pattern of heavy metal content and environmental factors and the correlation between the heavy metal content and different environmental factors are available. In addition, there is no report on the diversity of in-situ microbial populations in typical seasons. [Objective] To detect the seasonal variation of the concentration of Mn²⁺ and Fe²⁺ and different environmental factors in the bottom water of Changtan reservoir, Taizhou city, Zhejiang province, analyze their correlation, enrich functional microbial consortia at the bottom of the reservoir in normal (February) and wet (August) periods, and elucidate the difference in the species and diversity of the enriched microbial communities. [Methods] The bottom water of the reservoir was filtrated and the functional microbial consortia was enriched. The V3–V4 regions of 16S rRNA genes were sequenced and the community structure was analyzed. All tests were carried out according to the Standard Examination Methods for Drinking Water (GB5750-2006). [Results] The concentration of Mn²⁺ and Fe²⁺ in the water of Changtan reservoir changed seasonally. The concentration of Mn²⁺ and Fe²⁺ (initial 0 mg/L) in the water began to slowly increase in the spring-summer succession period every year, peaked in summer and autumn, and then decreased to below the detection limit in the late autumn and early winter. Among the detected environmental factors, the concentration of dissolved oxygen in water, ambient temperature of reservoir, and precipitation showed obvious seasonal changes. The concentration of Mn²⁺ and Fe²⁺ had a positive correlation with temperature, precipitation, and turbidity but a negative correlation with dissolved oxygen concentration, pH value, and total phosphorus concentration. In the correlation analysis, the concentration of two metal ions had the strongest correlation with dissolved oxygen concentration, followed by ambient temperature and precipitation. The species and diversity of functional microbes were significantly different between the normal and wet periods. Only Acinetobacter and Sediminibacterium were detected in the wet period, accounting for about 50% each, while the normal period was dominated by 9 genera, such as Bacillariophyta (47.62%) and Limnohabitans (9.52%). The culturable consortia from water in the normal period and wet period both can remove the Mn²⁺ in the reservoir water, and the removal rates were about 35.9% and 11.4%, separately. [Conclusion] The concentration of Mn²⁺ and Fe²⁺ in the bottom of Changtan reservoir changes seasonally with different environmental factors, and the concentration of Mn²⁺ and Fe²⁺ shows different correlation with different environmental factors. The structures of the two functional microbial communities enriched in wet and normal periods are different. This study provides microbial resources for the treatment of heavy metal-polluted water by microorganisms, and has a certain reference value for realizing the goal of “beautiful countryside” in China.

Abstract:[Background] Food waste tends to increase and thus it is an urgent task to rapidly digest it. The thermophilic aerobic biodegradation technology turns to be an effective solution. [Objective] To screen the thermophilic aerobic bacteria which can adapt to food waste and decompose the organic matter in the waste and thereby to improve the degradation efficiency and reduce the waste. [Methods] Experiments on the temperature tolerance in casamino acids, yeast extract, salts (CYS) medium and high-oil and high-salt tolerance in food waste leachate medium were conducted for the primary screening of strains. The enzyme production test was used for re-screening of strains, and food waste biodegradation test for functional verification. [Results] Strains N3-1, C7, N3-3 and G6-1 were finally screened out. The degradation rate of volatile solid (VS) was 36.95%, 33.23%, 32.83% and 31.91%, respectively, 3.02, 2.71, 2.68 and 2.61 times that of the control group. They were identified as Geobacillus thermoleovorans, Bacillus smithii, G. caldoxylosilyticus and G. lituanicus, separately. [Conclusion] The 4 strains have strong adaptability to food waste and high efficiency of degrading the waste. This study lays a foundation for the development of thermophilic aerobic microbial agent and provides technical support for the reduction, harmlessness treatment and resource utilization of food waste.

Abstract:[Background] In open-pit coal mining in arid areas, the dust is destined to aggravate soil environment deterioration and air quality decline. A few studies on the microbial community structure in soil and dust particles in coal districts are available. [Objective] To study the microbial community structure and diversity of soil, dust, and atmospheric PM_2.5 from different functional regions in the open-pit coal mine in Nanhu Township, Hami, Xinjiang. [Methods] Illumina NovaSeq was employed for high-throughput sequencing to characterize the community structure and functional diversity of bacteria and fungi in the three media in the open pit area and fly ash area. [Results] Ascomycota and Basidiomycota dominated the fungi, while Proteobacteria and Actinobacteria were the dominant bacterial phyla in the coal district. The abundance and diversity of fungal and bacterial communities showed no significant difference in the entire coal district, but the niche breadth of bacterial communities in atmospheric PM_2.5 was significantly larger than that in the open pit area and the fly ash area. Some functional groups with significant difference in abundance were found between soil and atmospheric PM_2.5 in the coal district, such as saprophytic trophic fungi, methanotrophic bacteria, and chitinase-producing bacteria. [Conclusion] The dust produced in open-pit coal mining has important impact on the microbial community structure in soil and atmospheric PM_2.5 in the coal district. The specific coal components-degrading microbes are among the main contributors to the soil ecological safety in mining area.

Abstract:[Background] Algae, bacteria and their interspecies interactions play important roles in aquatic ecosystem. In recent years, salinization of some rivers, lakes and other freshwater resources has become more serious, which greatly impacts the aquatic ecosystem. However, how salt stress influences the algae-bacteria interaction and whether certain bacteria can enhance algal salt tolerance remain unclear. [Objective] To isolate and identify the bacteria that can promote the salt tolerance of freshwater alga, Chlamydomonas reinhardtii, and analyze the relevant mechanism. [Methods] Effective bacteria were screened through enrichment culture, isolation and co-culture experiments. The algal growth ability under different conditions was assessed based on cell concentration, chlorophyll content and other parameters. In addition, 16S rRNA gene sequence analysis and genome sequence analysis were carried out to predict the possible mechanism of algae-bacteria interaction. [Results] A bacterial strain, MEZX29, was isolated, which could significantly enhance the salt tolerance of C. reinhardtii under the condition of 250-290 mmol/L NaCl. The 16S rRNA gene sequence analysis indicated that MEZX29 might belong to Rhodococcus qingshengii. The genome sequence analysis showed that MEZX29 contained the genes involved in glucose metabolism, ethylene metabolism, and biofilm formation, which might play roles in this salt tolerance enhancement. [Conclusion] R. qingshengii strain MEZX29 could enhance the salt tolerance of C. reinhardtii, which provided a new material for studying the beneficial interaction between algae and microorganisms.

Abstract:[Background] The second aerobic bioreactor O₂ of the oxic-hydrolytic-oxic (O/H/O) system contributes to the biomineralization and complete nitrification of residual pollutants, which is important for the standard discharge of wastewater. [Objective] To elucidate the structure and functions of the microbial community in bioreactor O₂. [Methods] The 16S rRNA gene was sequenced to investigate the microbial diversity and composition, predict the microbial functional pathways, and reveal the microbial co-occurrence and the environmental driving factors in bioreactor O₂. [Results] Proteobacteria, Bacteroidetes, and Chlorobi were the dominant phyla in the bioreactor. Among the dominant genera, Rhodoplanes, Lysobacter, and Thiobacillus were involved in the degradation of residual pollutants, such as chemical oxygen demand (COD), phenols, and thiocyanate (SCN^-), and Nitrosovibrio and Nitrospira were the ammonia-oxidizing bacteria (AOB) and the dominant nitrite-oxidizing bacteria (NOB), respectively. Functional profiling suggested that the benzoate degradation, aminobenzoate degradation, chloroalkane and chloroalkene degradation, eluorobenzoate degradation, and nitrotoluene degradation were the top five pathways in the xenobiotics biodegradation and metabolism. These major functional pathways were distributed widely in the dominant genera, implying their roles in biodegradation of residual pollutants. The pmoA / B / C-amoA / B / C, hao, and nxrA / B encoding related enzymes constituted the nitrification pathway. According to the result of microbial co-occurrence network, Lysobacter, Candidatus Solibacter, and Rhodoplanes occupied an important position in the O₂ ecosystem. Redundancy analysis (RDA) suggested that the microbial community in the bioreactor was mainly affected by COD and NH₃ [Conclusion] Rhodoplanes and Lysobacter were the key genera for biomineralization and ecological stability of the community. Nitrosovibrio and Nitrospira played an important part in nitrification. The metabolic pathways in O₂ were dominated by biomineralization and complete nitrification of residual pollutants. COD and ammonia (NH₃) were the main influencing environmental factors. This study illustrates the structure and functions of microorganisms in bioreactor O₂, which is expected to lay a microbial basis for improving the treatment of coking wastewater by O/H/O system.

Abstract:[Background] Wild Grammoplites scaber is nutritious, but the microorganisms in its gastrointestinal tract (GI) are rarely studied. [Objective] To study the microbial community in GI of G. scaber, reveal the potential probiotics and pathogens, and thus to provide a reference for the regulation of beneficial microbes. [Methods] We collected the GI samples of G. scaber from the Pearl River Estuary and investigated them with the culture-independent method and pure culture method. [Results] Through high-throughput sequencing of V3 regions of 16S rRNA genes, a total of 456 operational taxonomic units (OTUs) were identified. At the phylum level, Proteobacteria and Firmicutes dominated the microbes in the GI. At the genus level, Clostridium was prevalent in the samples, accounting for 57.11%. The 16S rRNA gene-based phenotypic and functional prediction suggested that the GI of G. scaber harbored both probiotics and pathogens, and they tended to restrict each other. A total of 99 strains were screened out with 12 different selective media, and they belong to 13 genera, 10 families, 10 orders, 4 classes, and 3 phyla (Proteobacteria, Firmicutes, and Actinobacteria). The dominant group was Proteobacteria (50.51%), and Psychrobacter was the predominant genus. [Conclusion] This study reveals the microbial composition and diversity in the GI of wild G. scaber, laying a basis for the research on the core intestinal microflora of teleost. In addition, the probiotics and pathogens identified in this study can serve as a reference for the safety of food made with G. scaber and for the development of marine fishery.

Abstract:[Background] The methylotrophic yeast Komagataella phaffii (K. phaffii) or Pichia pastoris has received extensive attention as a cell factory to produce recombinant proteins and build biosynthetic pathways. Real-time quantitative PCR (RT-qPCR) is a rapid and efficient technique for detecting gene expression levels in the study of the K. phaffii expression system. However, normalization is required to ensure the reliability of the results obtained. [Objective] To screen and verify the most stable reference genes in K. phaffii at different growth stages for accurate normalization of RT-qPCR. [Methods] Sixteen candidate reference genes (rps8b, rpl35a, rpl10, eif5a, rpl19a, por1, rpl23b, 0887, tif1, ole1, rpl14b, gs, sun, sdh2, trx1, and ccp1) were preliminarily screened by transcriptome data analysis. After the C _t values of the candidate reference genes were detected by RT-qPCR, their expression stability was analyzed by applying qBASE geNorm combined with NormFinder. [Results] The optimal number of reference genes by geNorm analysis was 2, and the most stable genes were rpl19a and tif1. The NormFinder analysis demonstrated that the most stable gene was tif1. In addition, we validated the candidate reference genes using fdh encoding formate dehydrogenase and afdh encoding the bifunctional alcohol/formaldehyde dehydrogenase. [Conclusion] Two reference genes, tif1 and rpl19a, were required for accurate normalization of RT-qPCR at different growth stages of K. phaffii. This study provided a reliable basis for the analysis of expression quantification of related functional genes and enriched the reference genes in RT-qPCR analysis, facilitating the study of gene expression regulation and application at different growth stages of K. phaffii.

Abstract:[Background] Conventional prokaryotic expression of heterologous protein usually requires ultrasonication or enzymolysis to lyse bacteria, the process of which is quite cumbersome. [Objective] We aimed to construct a type of plasmid-based opportunistic autolytic bacteria that exploited the lysis (lys) gene-mediated autolytic property of MS2 bacteriophage to facilitate the process of obtaining heterologous proteins. [Methods] The gene lys was cloned from the MS2 bacteriophage into a recombinant expression plasmid, which was heterologously-expressed in Escherichia coli BL21(DE3), and a novel type of plasmid-based opportunistic autolytic bacteria was generated. By L-arabinose induction, the lytic efficiency of the above-mentioned bacteria was assessed by growth curves and colony forming units (CFUs). Finally, the release levels of the heterologous protein were examined by SDS-PAGE. [Results] Three strains of plasmid-based opportunistic autolytic bacteria, pBAD- lys BL21(DE3), pBAD-Opti- lys BL21(DE3), and pCDF-BAD-Opti- lys BL21(DE3), were constructed. Following L-arabinose induction, all three strains of plasmid-based opportunistic autolytic bacteria achieved over 99.99% lytic efficiency. CFU data indicated that the host carrying pCDF-BAD-Opti- lys plasmid had better lytic effect than the other two. Upon L-arabinose-induced lysis of this host strain with heterologous expression of His-tagged enhanced green fluorescent protein (eGFP), more than 63% of eGFP were released to the outside of the cell and a single target protein of approximately 30 kDa was obtained directly from the culture medium by Ni-NTA. [Conclusion] In this study, we have successfully constructed a type of lys -mediated, plasmid-based opportunistic autolytic bacteria, which is capable of releasing most of the cytosolic heterologous proteins through L-arabinose-induced autolysis, simplifying the traditional process to acquire heterologous proteins.

Abstract:[Background] The establishment of cyclodipeptide synthase (CDPS)-associated tailoring enzymes offers particular promise for the generation of diketopiperazines with novel structures and good bioactivities. In the previous studies, coded diketopiperazines of the dmtA3B3C3 in CDPS gene cluster-terpenoid drimentines (DMTs) was identified in Streptomyces aidingensis CGMCC 4.5739. It was speculated that dmtD3-E3 in the downstream of CDPS participated in the biosynthesis of DMTs. However, the function of dmtD3 _ E3 has not been characterized. [Objective] To characterize the cyclodipeptide oxidase dmtD3_E3 in the gene cluster dmt3 of CDPS in S. aidingensis CGMCC 4.5739, and provide functional elements for structural diversity study of diketopiperazines. [Methods] dmtD3_E3 was cloned from the genome of S. aidingensis CGMCC 4.5739, and the recombinant plasmid pWLI209 was constructed and expressed soluble in Escherichia coli BL21(DE3). In vitro enzymatic reactions were performed, and high performance liquid chromatography-mass spectrometry (HPLC-MS) and nuclear magnetic resonance (NMR) were used to ensure the structure of catalysate. [Results] The cyclodipeptide oxidase dmtD3_E3 catalyzed the oxidative dehydrogenation of C14-C17 in cyclo- (L-Trp-L-Leu) (cWL) to generate cWΔL, and the dehydrogenation of C10-C11 in cyclo -(L-Trp-L-Ala) (cWA) to generate cΔWA. The results showed that dmtD3_E3 demonstrated broad substrate specificity. [Conclusion] This study explored and characterized the novel cyclodipeptide oxidase dmtD3_E3 in the CDPS biosynthesis pathway, laying a foundation for the further generation of “non-natural” diketopiperazines through strategies of combinatorial biosynthesis and synthetic biology.

Abstract:[Background] Multidrug efflux pump mostly presents in the form of membrane protein complex, which makes major contributions to bacterial drug resistance. The transport function and assembly of the efflux pump are among the important issues for bacterial drug resistance and drug development. [Objective] Taking AcrAB-TolC, an important member of resistance-nodulation cell division (RND) family, as the research example, this study aimed to investigate and analyze the transport activity and in vitro assembly of the multidrug efflux pump complex. [Methods] Basing on the gene sequence of AcrAB-TolC complex in Escherichia coli, the recombinant plasmids containing acrA, acrB and tolC genes were constructed. Each subunit of the complex was expressed and purified. This study then explored the transport functions of the complex and subunits, the interaction between subunits and substrates, as well as the interaction between subunits and their dynamic assembly by means of fluorescence spectroscopy and isothermal titration calorimetry (ITC). [Results] The expression and purification of the subunits of the AcrAB-TolC complex have been achieved with the homogeneity to over 98%. It was confirmed that living cells with expressed individual subunits had increased activity to transport ethidium bromide (EB). The activities of AcrB and TolC to transport EB were affected by N-hexanoyl-L-homoserine lactone (C6-HSL), a quorum-sensing signal molecule. ITC results further confirmed the interactions between C6-HSL and AcrB or TolC. In addition, there were obvious correlations in AcrA-AcrB and AcrA-TolC, while no significant interaction was found in AcrB-TolC. In the in vitro assembly experiments, the monomolecular fluorescence intensity of AcrAB-TolC subunit increased with time, which validated the dynamic assembly of the complex subunits on the membrane. [Conclusion] The AcrAB-TolC efflux pump and its subunits were expressed and purified. The activity of AcrAB-TolC to transport and its interaction with substrates were verified. The dynamic assembly of AcrAB-TolC was observed. These results facilitated the study of bacterial drug resistance and antimicrobial strategies in associating with multidrug efflux pumps.

Abstract:[Background] Keratinase is a kind of hydrolase that specifically degrades keratin, and has important application potential in animal feed, biological fertilizer, medicine, washing, tanning, and environmental treatment. [Objective] The keratinase gene of Pseudomonas aeruginosa Gxun-7 from marine environment was cloned and expressed, and the enzymatic properties of recombinant enzyme were investigated, which laid a foundation for the application of keratinase in industrial production. [Methods] Based on the putative keratinase gene of P. aeruginosa Gxun-7 genome, primers were designed to obtain keratinase gene kp2. The recombinant expression plasmid pET22b- kp2 was constructed, and transformed into E. coli RosettagamiB (DE3) for induced expression. Meanwhile, the expression conditions of the recombinant expression strain were optimized. The recombinant keratinase was isolated and purified by nickel column and its enzymatic properties were studied. [Results] The molecular weight of recombinant keratinase was about 33 kDa, and the optimal temperature was 40 ℃ at pH 8.0. The recombinant keratinase had good stability at temperature of 30-60 ℃ and pH 6.5-8.0. Metal ions including Co²⁺ and Cu²⁺, and chemical reagents including sodium dodecyl sulfonate (SDS), ethylenediaminetetraacetic acid (EDTA), and phenylmethylsulfonyl fluoride (PMSF) inhibited the enzyme activity, whereas Mg²⁺, K⁺, mercaptoethanol, and dithiothreitol (DTT) promoted the enzyme activity. The recombinant keratinase showed good salt tolerance, and the relative enzyme activity was 87.55% under the action of 12.5% NaCl. When casein was used as substrate, the K _m and V _max of the enzyme were 60.92 mg/mL and 9.70 U/mL. [Conclusion] The recombinant keratinase from the marine-derived P. aeruginosa Gxun-7 has good stability in temperature, alkali and salt, which can be applied to industrial production in the future.

Abstract:[Background] Vibrio parahaemolyticus is a major pathogen of Penaeus vannamei. Posing a threat to food safety, environment, and sustainable development, conventional antibiotic therapy is no longer effective. Biocontrol of this bacteria seems to inevitable for the sustainable development of P. vannamei aquaculture. Phage, a natural safe antibacterial, is highly specific and typically only infects or kills an individual species of bacteria with high efficiency. [Objective] To identify virulent phage that can efficiently lyse V. parahaemolyticus so that the set of methodology was established to explore the phage agents for the control of V. parahaemolyticus. [Methods] Double-layer agar technique was used to enrich phages from sewage samples of seafood market with 28 strains of V. parahaemolyticus derived from diseased shrimps. The lytic spectra of phages were determined through spot test. The one with broad spectrum was characterized by transmission electron microscopy, biological characterization, and whole-genome sequencing. [Results] Vpas_PP24, a virulent phage against V. parahaemolyticus was screened out. It had an icosahedral head with about 92 nm in length and around 46 nm in width and a long tail of about 147 nm in length, which was thus identified as a member of Siphoviridae in Caudovirales. The full-length genome of Vpas_PP24 was 83 482 bp, and it harbored 118 open reading frames (ORFs), with 13 known protein-coding genes, and no non-coding RNA, virulence genes, or resistance genes. Genome alignment suggested that Vpas_PP24 may be a new phage species against Vibrio. The phage could lyse 15 (54%) of the 28 V. parahaemolyticus strains and 18 (16%) of 116 other Vibrio species. The optimal multiplicity of infection (MOI) of Vpas_PP24 was 0.000 1 and titer was 3.0×10¹⁰ PFU/mL. One-step growth curve indicated that it had a latent period of 10 min, rise period of 150 min, and burst size of 30 PFU/cell. The optimal temperature and pH for the phage growth were<50 ℃ and pH 4.0–11.0, respectively. The phage was tolerant to chymotrypsin, papain, and shrimp hepatopancreas extract but sensitive to proteinase K. Vpas_PP24 was also sensitive to ultraviolet radiation. The emergence frequency of Vpas_PP24-insensitive mutants was 2×10^-5. [Conclusion] A virulent phage Vpas_PP24 with new genotype was screened out. It has the potential to be further developed into novel anti- V. parahaemolyticus agent, thanks to its broad lytic spectrum, biological properties, as well as its promising stability.

Abstract:[Background] Arbuscular mycorrhizal (AM) fungi have a wide range of host species, environmental adaptation and remarkable plant growth-promoting capability. However, high phosphate level in soils has seriously inhibited the growth of AM fungi and the AM formation. [Objective] This study aimed to isolate and identify indigenous AM fungal strains resistant to high phosphate in south China, and to provide novel materials for mycorrhiza research. [Methods] The AM fungi from high phosphate soils were identified by classical morphology and molecular systematics. [Results] Twenty-five AM fungi species belonging to 7 genera were identified from root-zone soils with the available phosphorus concentration of 53-131 (88.2±17.6 as average±SD) mg/kg, including 12 species in Acaulospora, 7 in Glomus, 2 in Septoglomus, 1 in Claroideoglomus, 1 in Rhizophagus, 1 in Sclerocystis and 1 in Paraglomus, among which Claroideoglomus etunicatum and Acaulospora mellea were dominant. At the high phosphate level of (87.7±8.0) mg/kg, AM fungi still formed arbuscules and vesicles. However, when the phosphate level reached (99.7±1.2) mg/kg, total colonization frequency and arbuscular abundance were significantly decreased in mycorrhizal roots, but vesicles were still be formed. [Conclusion] In this study, 25 AM fungi belonging to 7 genera that might be resistant to high phosphate concentration were identified from root-zone soils containing (88.2±17.6) mg/kg phosphate in cultivated land in Nansha district of Guangzhou city. The isolated strains such as C. etunicatum and A. mellea could be used as experimental strains in further studies on the high phosphate inhibition and the production of high-quality AM fungal inocula resistant to high phosphate.

Abstract:[Background] At present, the bacterial agents used to degrade lignin in landscaping waste are mostly liquid or solid bacterial agents, and there is few research on powdered bacterial agents. [Objective] To develop the lyophilized powder with high activity and high lyophilized survival rate and optimize the process, thus solving the existing problems in transportation, storage, and application of the liquid or solid bacterial agents. [Methods] Aspergillus nidulans, a lignin-degrading strain, was taken as the research object, and the lyophilized powder was prepared by vacuum lyophilization. The lyophilized survival rate of the strain was used as the evaluation index. The types and concentration gradients of protective agents suitable for the lyophilizing process of the strain were screened out through the single factor test, and the compound formula of protective agents of lyophilized bacterial powder was optimized through the orthogonal test. The rehydration and storage conditions of lyophilized powder were further explored after obtaining the formula. [Results] The influencing order of the four protective agents with a superior protective effect on the lyophilized survival rate after compounding was sucrose>glucose>skimmed milk powder>α-lactose. The optimum formula for protective agents was 15% sucrose, 1% glucose, 10% α-lactose, and 1% skimmed milk powder. The optimum rehydration condition was 30 min rehydration with normal saline as the solvent. The lyophilized survival rate of the strain of lyophilized powder prepared and applied under the above conditions reached 83.33% with 1.2×10¹⁰ CFU/g effective live bacteria. There was no significant decrease of the activity of lyophilized powder storing at the optimum temperature (-20 ℃) for 28 days. [Conclusion] The preparation and storage conditions of the lyophilized powder of A. nidulans obtained in this study have low strain loss rate and can be stored for a long term time, which plays a positive role in promoting the application of lignin-degrading bacteria in practical production.

Abstract:[Background] Amid large-scale production, the environmental management of cowshed is the key to the milk production of dairy cows. [Objective] To explore the characteristics of soil bacterial communities in different cowsheds and thereby lay a theoretical basis for healthy production of dairy cows. [Methods] Soil samples were collected from 8 different cowsheds: new calf born area (NCA), weaned calf cowshed area (WCA), rearing heifers cowshed area (RCA), low-yield lactating cows cowshed area (LCA), high-yield first-calf lactating cows cowshed area (HFCA), high-yield multiparity lactating cows cowshed area (HMCA), dry cows cowshed area (DCA), and sick cows cowshed area (SCA), with 6 replicates from each cowshed and a total of 48 samples. Through the sequencing of 16S rRNA gene amplicons, the structures and diversities of the bacterial communities were analyzed and the functions of different communities were predicted. [Results] The bacterial community composition of soil samples was different among the cowsheds. Among the 8 cowsheds, HFCA boasted the highest bacterial community diversity, and NCA showed huge difference in the bacterial community of soil at phylum level from other cowsheds. Moreover, LCA, HFCA, and HMCA respectively showed high similarity in the community structure among soil samples. At the phylum level, Bacteroidetes, Proteobacteria, Actinobacteria, and Firmicutes dominated all the soil samples. At the genus level, the haloalkaliphilic Halomonas, Fermentimonas and Aequorivita with potential degrading ability, and the pathogenic Ornithobacterium were dominant in the soil samples collected from cowsheds for calves. Haloalkaliphilic Truepera dominated RCA, and the pathogenic Acinetobacter and Parapedobacter, and the drug-resistant Pedobacter were the dominant genera in the soil samples collected from cowsheds for lactating cows. [Conclusion] Pathogenic bacteria and bacteria involved in nitrate respiration are mainly distributed in LCA and haloalkaliphilic bacteria are mainly found in WCA and RCA. Methanogenic bacteria are mainly discovered in HFCA. This study analyzed the diversity of soil bacterial community in different cowsheds to lay a theoretical basis for the healthy production of dairy cows.

Abstract:[Background] Soil bacteria are very sensitive to environmental changes and thus are important index of soil environmental quality. [Objective] To study the vertical distribution of soil bacteria in purple soil of winter-flooded paddy fields in different seasons and reveal the relationship between soil bacterial community structure, species diversity and soil environmental factors. [Methods] Purple soil of winter-flooded paddy fields were taken as the research object in this study. Soil samples at different depths were collected in August 2020 (summer) and January 2021 (winter) separately. Illumina MiSeq high-throughput sequencing of 16S rRNA gene of soil bacteria was performed to analyze the vertical distribution of bacterial community composition and diversity in different seasons. [Results] The bacterial ACE index, Chao1 index and Shannon index were higher in summer than in winter, and decreased with the increase of soil layers. The dominant bacterial phylum in purple soil of winter-flooded paddy fields were Proteobacteria, Chloroflexi, Acidobacteria, Nitrospirae, Actinobacteria and Planctomycetes, and the dominant bacterial genera were Desulfobacca, Haliangium, Anaeromyxobacter, Candidatus_Omnitrophus and Defluviicoccus. The relative abundances of Chloroflexi, Actinobacteria, Anaeromyxobacter and Candidatus_Omnitrophus were higher in summer, while those of Proteobacteria, Nitrospirae, Desulfobacca, Haliangium and Defluviicoccus were higher in winter. The redundancy analysis (RDA) and correlation analysis of environmental factors showed that total nitrogen (TN), soil organic matter (SOM) and soil redox potential (Eh) were the main factors that significantly affected the bacterial community structure in purple soil. [Conclusion] This study enriched the understanding of bacterial community composition and diversity in purple soil of winter-flooded paddy fields, and confirmed that there were differences in different seasons.

Abstract:[Background] Bacterial soft rot caused by Erwinia beijingensis results in serious economic losses to enterprises. The biological function of the glycosyltransferase gene wbnH2 in E. beijingensis remains unclear. [Objective] To explore the effect of wbnH2 gene on the pathogenicity of E. beijingensis. [Methods] Homologous recombination was employed to construct the E. beijingensis LMG 27579^T wbnH2 -deleted mutant Δ -wbnH2. The biological characteristics such as pathogenicity, growth rate, motility, biofilm formation, and adhesion of Δ -wbnH2 were studied. Further, we used the broad-host-range plasmid pBBR1MCS2 to construct the complementation strain C -wbnH2 for excluding the polarity effect-caused phenotypic change of the mutant. [Results] Compared with that of the wild type, the growth rate of Δ -wbnH2 had no significant change. However, the deletion of wbnH2 resulted in significant decreases in polysaccharide secretion, biofilm formation, adhesion and pathogenicity. [Conclusion] The glycosyltransferase gene wbnH2 affects the polysaccharide secretion, biofilm formation, adhesion, and pathogenicity of E. beijingensis, playing a role in the pathogenic process. This study provides a theoretical basis for the prevention and control of bacterial soft rot.

Abstract:[Background] Walnut blight, caused by Xanthomonas campestris and Pantoea agglomerans, is a bacterial disease resulting in severe damage to walnut tree and substantial economic loss. [Objective] To isolate and screen out the actinomycete strains with inhibitory activity on these two pathogens from the rhizosphere soil of walnut tree for the development of biocontrol agents. [Methods] The actinomycetes were isolated by spread plate method and further screened by plate confrontation method and improved Oxford-cup test. The target strain was identified based morphological, physiological, and biochemical properties, as well as 16S rRNA gene sequence. The antibacterial spectrum and biocontrol effect in vitro of the cell-free fermentation broth were evaluated. [Results] An actinomycete strain YNF36 with strong antagonistic effect on both pathogens was screened out and identified as Streptomyces arenae. The strain YNF36 showed antimicrobial activity to seven indicator bacterial species: Staphlococcus aureus, Escherichia coli, Aspergillus niger, Monilia albicans, Bacillus subtilis, Pseudomonas aeruginosa, and Bacillus cereus. Furthermore, it had inhibitory activity against seven plant pathogens: Alternaria alternata, Valsa mali, Colletotrichum gloeosporioides, Botrytis cinerea, Fulvia fulva, Colletotrichum capsica, and Fusarium solani. The strain YNF36 had the largest yield and the strongest antimicrobial activity on SYP medium. In addition, the biocontrol experiment in vitro showed that the cell-free fermentation broth of the strain YNF36 had the biocontrol effects of 75.69% and 62.39% against X. campestris and P. agglomerans, respectively. [Conclusion] The strain YNF36 could be considered as the actinomycete resource with biocontrol potential against walnut blight and demonstrates a wonderful prospect for development and application.

Abstract:[Background] Silicon, which improves the stress resistance, yield, and quality of crops, has been widely reported. However, the conventional neglect of silicon fertilizer results in the continuous decrease in the content of available silicon in arable land. Therefore, it is an urgent task to raise the content of soil available silicon. [Objective] The content of available silicon in soil affects the absorption and utilization of silicon by crops. Silicate-solubilizing microorganisms can continuously activate available silicon in soil, which helps improve crop yield and quality and reduce the application rate of chemical fertilizer. [Methods] Selective media were used for the enrichment, isolation, and purification of soil silicate-solubilizing strains, and the silicate-solubilizing ability, saline-alkaline tolerance, low-oxygen tolerance, phosphate-solubilizing ability, potassium-releasing ability, and cellulose-degrading ability were determined. With malachite green as the primary stain and safranin as counterstain, the spore yield of high-efficiency silicate-solubilizing strains was measured. Finally, the maize growth-promoting ability of efficient silicate-solubilizing strains was investigated through pot experiment. [Results] Two efficient silicate-solubilizing strains were obtained, MB22 and MB35-5. They were identified as Priestia aryabhattai by 16S rRNA sequencing, and the concentration of available silicon in the culture of them was 1.5 and 1.7 folds that of the blank control, and 1.1 and 1.2 folds that of the control Paenibacillus mucilaginosus 3016, respectively. MB22 and MB35-5 showed high saline-alkaline tolerance, particularly MB35-5. Specifically, under the conditions of pH 10, 10% NaCl, and 10% KNO₃, the concentration of available silicon in the culture of MB35-5 was 0.28–0.37 mmol/L. The spore yield of MB22 and MB35-5 was 68% and 55%, respectively, 1.69 folds and 1.37 folds of the control strain, respectively. MB35-5 had phosphate-solubilizing ability, potassium-releasing ability, and cellulose-degrading ability. Pot experiment showed that the two strains significantly increased the plant height and root dry weight of maize plants, especially MB35-5. To be specific, the average height of plants treated by MB35-5 was 1.39 times that of blank treatment and 1.14 times that of plants treated by control strain. The root dry weight of plants treated by MB35-5 was 1.37 times that of the blank treatment and 1.24 times that of plants treated by control strain 3016. [Conclusion] The silicate-solubilizing strains MB22 and MB35-5 were screened out. Among them, MB35-5 with high spore yield also showed phosphate-solubilizing ability, potassium-releasing ability, cellulose-degrading ability, and growth-promoting function, as well as the saline-alkaline tolerance and low-oxygen tolerance. Thus, this strain should be further tested and developed.

Abstract:[Background] Biocontrol is a nature-based solution that meets the requirements of ecological civilization and sustainable development. Clarifying the biocontrol effects and antibacterial characteristics of the bacterial strain are the basis of biocontrol of technology research. [Objective] To develop biocontrol agent for the control of anthracnose in Cunninghamia lanceolata, we studied the antimicrobial activity and growth-promoting effect of strain T1-3-2 isolated from C. lanceolata. [Methods] Strain T1-3-2 was identified based on morphological, physiological and biochemical characteristics, and the 16S rRNA gene sequence-based phylogenetic analysis. The antimicrobial effects of the bacterial strain, volatile gas and secondary metabolites of the strain were determined by plate confrontation method, plate culture method, and colony growth inhibition test. The plant growth-promoting effect of the strain was determined by pot experiment indoors. [Results] Strain T1-3-2 belongs to Pseudomonas, forming a monophyletic group with Pseudomonas eucalypticola. It had stronger antagonistic effects on 10 target strains, with the inhibition rates over 80% on 6 strains of Colletotrichum, Pestalotiopsis, Nigrospora, and Botryosphaeria. The pot experiments showed the fermentation broth of T1-3-2 in Kings Medium B controlled the occurrence of anthracnose in C. lanceolata, with the control effect of 74.20%. Moreover, it improved the growth and increased the biomass accumulation of C. lanceolata seedlings. [Conclusion] Strain T1-3-2 is belonging to the Pseudomonas genus and it is beneficial to the biocontrol and growth-promoting on C. lanceolata. Thus, it is a great potential biocontrol strain.

Abstract:[Background] Tetragenococcus halophilus is a halophilic lactic acid bacterium detected in fermented food. Investigating the arginine (Arg) metabolism of this bacterium is of great importance for disclosing the mechanism of the accumulation of ethyl carbamate (EC) precursors during food fermentation and ensuring food safety. [Objective] To identify the key genes of arginine deiminase (ADI) pathway in the genome of T. halophilus strains isolated from soy sauce moromi mash, and to reveal the roles of these genes in arginine metabolism and utilization and accumulation of citrulline (the EC precursor). [Methods] The key genes involved in ADI pathway were identified for different strains by PCR amplification and sequencing. The transcription levels of key genes of ADI pathway and the activities of corresponding enzymes were compared to reveal the effect of environmental factors on amino acid metabolism and the role of each gene. [Results] The genes associated with the ADI pathway in the genome of T. halophilus included two categories. One contained intact arc operon genes and had the most copies, as represented by strain R23, and the other lacked arcA and arcB but had multiple copies of arcB and arcC, as represented by strain C3. Only the strains with arcA in their genomes can utilize arginine to produce citrulline. Whether the intermediate product citrulline was accumulated with the utilization of arginine by T. halophilus was affected by the content of arginine and the presence of ethanol and fatty acids in the medium. T. halophilus accumulated citrulline when arginine was more than 5 g/L or ethanol and fatty acids were present in the medium. In the presence of fatty acids and ethanol, the activities of ADI, ornithine transcarbamylase (OTC), and carbamate kinase (CK), the key enzymes of ADI pathway, were significantly reduced by 41.0%, 46.4%, and 60.0%, respectively. The transcription level of arcB in T. halophilus was 10.5 and 29.8 times as high as that of arcB ₁ and arcB ₂, respectively, and the transcription level of arcC was 17.6, 20.3, and 23.9 times as high as that of arcC ₁, arcC ₂, and arcC ₃, respectively. The results demonstrated that arcB and arcC played major roles in citrulline metabolism. [Conclusion] Arginine content and the presence of ethanol and fatty acids are key environmental factors influencing whether citrulline can be accumulated by T. halophilus when metabolizing arginine. Among the multiple copies of arc operon genes in T. halophilus, arcB and arcC play key roles in citrulline metabolism.

Abstract:[Background] Riemerella anatipestifer (RA) is the pathogen that causes infectious serositis in a variety of domesticated and wild birds such as ducklings, geese, and turkeys. With wide distribution all over the world, it endangers the development of poultry industry and causes serious economic losses. [Objective] To learn the epidemic status and biological characteristics of RA in China and guide the prevention and control of RA-caused disease. [Methods] PCR, biochemical tests, and serotyping were carried out for the suspected RA strains isolated from Shandong province, Hebei province, Guangdong province, and Shanxi province from 2020 to 2021. The drug resistance of the strains was evaluated by drug sensitivity test, and the pathogenicity was analyzed based on median lethal dose. [Results] A total of 78 RA strains were identified, including 4 strains of serotype 1, 21 strains of serotype 2, 11 strains of serotype 10, 3 strains of serotype 6, and 17 strains of serotype 7, 1 strain with cross agglutination, and 21 strains not serotyped. The 78 strains had the strong drug resistance to polymyxin B, fosfomycin, and clindamycin and were sensitive to cefradine, doxycycline, furazolidone, and florfenicol. In addition, all the 78 strains had multiple drug resistance, among which 77 strains had resistance to more than 5 antibiotics. The animal test results showed that the RA strains generally had strong pathogenicity, which varied among different strains of the same region and among strains of different regions. The LD ₅₀ of 14 RA strains ranged from 10⁴ to 10⁷ CFU. [Conclusion] RA has demonstrated strong pathogenicity and drug resistance in China. The results of this study provide a basis for the prevention and control, clinical medication, and subsequent research on the pathogenic mechanism of RA.

Abstract:[Background] Vibrio mimicus is a pathogenic bacterium that severely impacts aquaculture. The application of antibiotics to the control of vibriosis caused by this pathogen leads to drug resistance, environment pollution, and food safety problems. [Objective] To explore whether overexpression of the antimicrobial peptide Hepcidin (CiHep) from Ctenopharyngodon idella affects V. mimicus growth and NF-κB pathway at the cellular level. [Methods] The recombinant plasmids pEGFP-N1- Cihep and pcDNA3.1(+)- Cihep were constructed for eukaryotic expression. The expression of the fusion protein EGFP-CiHep was detected by immunofluorescence assay and Western blotting at different time points post transfection of pEGFP-N1- Cihep in C. idella kidney (CIK) cells. Then, the effect of the supernatant of the cells transfected with pcDNA3.1(+)- Cihep on the growth of V. mimicus was examined. The transcription levels of the key genes for NF-κB pathway activation and the downstream immunity-related genes in the transfected cells were measured. [Results] EGFP-CiHep was well expressed in the CIK cells and reached the highest expression level 48 h post transfection. The supernatant of the transfected cells significantly inhibited the growth of V. mimicus. CiHep overexpression significantly altered the transcription levels of key genes for NF-κB pathway activation and downstream immunity-related genes. [Conclusion] CiHep can inhibit V. mimicus growth and activate NF-κB pathway. Our results provide a theoretical basis for developing fish-derived Hepcidin and a new perspective on regulation of antimicrobial peptide expression by activating NF-κB pathway for prevention and treatment of fish vibriosis.

Abstract:[Background] In recent years, bumblebee species and number have been decreasing worldwide due to habitat reduction, abuse of pesticides, and infection of pathogens. The pathogens can be effectively killed by the antimicrobial substances produced by microorganisms during their growth. [Objective] Bombus breviceps lives in the wild for a long time and has rich microbial resources in the intestinal tract. We screened antagonistic strains from the intestinal tract of B. breviceps and studied their antibacterial properties. [Methods] The Oxford cup double-layer method was used to screen out the antagonistic strains and determine the stability of antibacterial substances and the inhibition spectrum of the fermentation broth of the strains with strong inhibitory activity. Further, cell membrane permeability and flow cytometry were employed to investigate the inhibition mechanism. [Results] Five antagonistic strains with significant antibacterial effect were obtained, among which Fructobacillus tropaeoli CZ01 demonstrated strong inhibitory effect on all the five indicator bacteria: Staphylococcus aureus, Salmonella choleraesuis, Escherichia coli, Shigella flexneri, and Streptococcus agalactiae. It showed the strongest inhibitory effect on Staphylococcus aureus, with an inhibition zone diameter of (21.21±0.25) mm. Moreover, the inhibitory activity was still 67.36% after treatment at 121 ℃ and 78.16% after the medium was adjusted to pH 10.0. [Conclusion] B. breviceps carries rich microbial resources in intestinal tract. In particular, F. tropaeoli CZ01 with high antibacterial activity, good stability, and wide intestinal spectrum, has good killing effect on S. aureus and demonstrates good application potential.

Abstract:[Background] Enterovirus G (EV-G), commonly existing in pigs, can cause skin injury, muscle paralysis, pneumonia, fever, diarrhea and asymptomatic infection of pigs. [Objective] To establish a real-time quantitative polymerase chain reaction (RT-qPCR) for EV-G detection and carry out molecular epidemiological investigation in Sichuan province. [Methods] On the basis of primer design referring to the EV-G 5ʹUTR gene, SYBR Green I RT-qPCR (RT-qPCR for short) detecting all genotypes of EV-G was established and its sensitivity, specificity and repeatability were evaluated. Epidemic investigation of EV-G in Sichuan province was made with the established PCR method. [Results] The results showed that the C _t value of the RT-qPCR had a good linear relationship with the standard sample template in the concentration range of 1.89×10²-1.89×10⁸ copies/μL. In the specificity test, nine other swine viruses (PDCoV, PEDV, TGEV, JEV, PSV, PPV, PCV, PRV and PRRSV) could not be detected with the method. The minimum detection limitation was 1.89×10¹ copies/μL, and the intra-assay and inter-assay coefficients of variation were lower than 1% and 2%, respectively. A total of 431 samples with diarrhea in Sichuan province were detected by RT-qPCR, and the total positive rate of EV-G was 31.1%, indicating the virus was widespread in Sichuan province. Nine EV-G positive samples from Sichuan province were randomly selected to amplify VP1 gene for sequencing analysis. It was found that the prevalent genotypes of EV-G in Sichuan province were G1, G3, G4, and G9, but EV-G1 was the dominant genotype. [Conclusion] In this study, a RT-qPCR method for detecting EV-G genotypes is established, and the epidemic status of EV-G in Sichuan province is preliminarily mastered, which lays a foundation for further research on the virus.

Abstract:[Background] Xiamenmycin A, which shows significant anti-fibrotic activity and can serve as a potential medicinal candidate, is a major secondary metabolite of Streptomyces xiamenensis 318. However, the yield of xiamenmycin A in the wild-type strain is only 14 mg/L. Thus, it is urgent to improve the production of this compound in the strain. [Objective] Random mutagenesis and resistance reporter-based selection was used to develop a high xiamenmycin A-producing strain and further improve the yield of the compound through medium optimization. [Methods] An antibiotic resistance gene was fused at the 3' end of the last gene (ximE) of xiamenmycin A biosynthesis gene cluster, and thus the expression of the entire gene cluster could be indicated by antibiotic resistance level. After atmospheric and room temperature plasma (ARTP) treatment, the high-yielding strain was selected as the mutant with high antibiotic resistance level. The yield of xiamenmycin A in the mutant was further enhanced by medium optimization. [Results] A neo -labeled strain MT-XN was constructed as a starting strain. After one round of ARTP mutagenesis, a mutant MA-8 with resistance to 90 mg/L kanamycin and xiamenmycin A yield of 101.7 mg/L was obtained. Moreover, the yield of xiamenmycin A in MA-8 reached 134.2 mg/L with the medium optimized by response surface methodology, 845.1% higher than that in the wild-type strain. [Conclusion] Random mutagenesis combined with reporter-based selection can be used to identify high xiamenmycin A-yielding strain, which lays a foundation for drug development of this compound.

Abstract:[Background] Nocardia belongs to aerobic actinomycete. Being widely distributed, Nocardia can cause local or disseminated infection in human, especially in those with low immune function. The Nocardia infection is difficult to be clinically identified, and novel Nocardia strains are constantly being discovered. Different types of Nocardia from different regions have different prevalences and antibiotic sensitivities, which hinders the treatment. The treatment of Nocardia infection by the phage isolated from the host bacteria at the lesion has attracted great attention in recent years. [Objective] To isolate the virulent phage against Nocardia from the environment that can be used in clinic and explore the genomic characteristics. [Methods] The target phage was isolated by the double-layer plate method, and the plaque morphology was observed. The phage was purified, and the characteristics were observed through the transmission electron microscope. The DNA of the phage was extracted, and the whole genome was sequenced, annotated, and compared with the known phage genomes in the database. The phylogenetic tree was constructed for genetic evolution analysis. [Results] The virulent phage vB_Ncarnea_KYD1 with Nocardia carnea as the host, isolated from the environmental samples, formed transparent and uniform plaques with a diameter <2 mm on the double-layer plate. Genome analysis showed that the DNA of vB_Ncarnea_KYD1 was circular with a size of 66 621 bp. A total of 102 proteins and 1 tRNA-Ser were found in coding sequences (CDS). According to the transmission electron microscope observation and phylogenetic tree analysis, vB_Ncarnea_KYD1 was a new phage in the Siphophages, which experienced complex gene recombination in the evolution. vB_Ncarnea_KYD1 had practical value, and no virulence factor-related genes or antibiotic-resistance genes were found. [Conclusion] The novel virulent phage vB_Ncarnea_KYD1 of N. carnea was isolated from the environmental water sample. The transmission electron microscope observation and genome analysis showed that vB_Ncarnea_KYD1 belonged to the Siphophages. Since no relevant genes unfavorable to clinical application were found in the genome, vB_Ncarnea_KYD1 was a relatively safe virulent phage of Nocardia The findings of this study provided references for the follow-up treatment of Nocardia infection, and enriched the domestic phage resources.

Abstract:[Background] As one of the common non-tuberculous conditionally pathogenic mycobacteria, Mycobacteroides abscessus is a major clinical challenge because of its natural multi-drug resistance. Mycobactin (MBT) and carboxymycobactin (cMBT), the crucial systems for mycobacteria to acquire iron, one of the limiting nutrients, are closely associated with virulence and drug resistance. [Objective] To reveal the structure of MBT in M. abscessus and explore the evolution of MBT in pathogenic mycobacteria. [Methods] The structures of MBT and cMBT were analyzed by MALDI-TOF-MS and FT-MS/MS. Further, the biological activities of MBT and cMBT were determined. The MBT biosynthesis gene clusters were compared between M. abscessus and several representative mycobacteria. [Results] M. abscessus and M. marinum had similar modification patterns of MBT and cMBT core structure. In particular, the modifying groups at R1, R2, R3, and R5 were exactly the same, and the fatty acid chains were both located at R4. However, the MBT and cMBT of M. abscessus are a new structure because of the different lengths of the fatty acid chains (10-17 C for MBT and 4-8 C for cMBT). The growth of M. abscessus in iron-deprived media could be significantly recovered by supplying with Fe-cMBT in a dosage-dependent manner, and M. abscessus can much efficiently absorb Fe-cMBT than FeCl₃, which proved that MBT-cMBT system was vital for M. abscessus to acquirie iron from the environment. Synteny and phylogenetic analysis of the MBT biosynthetic gene cluster mbt-1 showed that M. abscessus was closely related to M. marinum rather than M. tuberculosis and M. smegmatis (referring to 16S rRNA gene phylogenetic tree). This result is consistent with that based on the structure of MBT. Further analysis revealed that the variation range of the fatty acid chain length of pathogenic mycobacteria such as M. marinum, M. tuberculosis, and M. bovis was only 4 C, while that of conditionally pathogenic and non-pathogenic bacteria such as M. abscessus, M. fortuitum, M. avium, and M. smegmatis was 7-11 C, which suggested that the range variation of fatty acid chain length of MBT might be associated with the lifestyles and habitats of mycobacteria. [Conclusion] As an important system for obtaining iron with unique structure, M. abscessus MBT deserved further study, especially its roles in pathogenesis and drug resistance, as well as its evolution in pathogenic mycobacteria.

Abstract:Biometric traces are regularly utilized as a breakthrough in criminal investigations, such as the forensic challenges concerning personal identification, suspect tracking, or postmortem interval (PMI), and biogenic evidence is frequently used. In spite of the numerous conventional techniques in forensic medicine, scholars have been sparing no efforts to create new instruments in light of the limitations of available methods or for the benefit of practitioners in this field. Thanks to the advancement of sequencing technology, it has been found that the microorganisms inside and outside the body far outnumber the cells, which have high value in location tracing, personal identification, inference of PMI, biocrime, and environmental protection. In this paper, the research progress in this area is described.

Abstract:Cholesterol is a major sterol compounds accumulating in animals, which has important biological significance and medical application value in maintaining cell membrane function, synthesizing steroid hormones, and producing steroid drug intermediates. Traditionally, cholesterol is obtained by animal tissue extraction, but the whole process is time-consuming and laborious, accompanied by severe environmental pollution. Due to the complex and delicate structure, steroids are difficult to be produced by chemical total synthesis. Most recently, microbial cell factories constructed by synthetic biology have been successfully applied on the biosynthesis and development of natural products, such as terpenoids and sterols. This paper summarized the research progress on cholesterol microbial cell factories, including characterization of cholesterol biosynthesis pathway, selection of chassis strains, mining and optimization of heterologous gene components, and regulation of metabolic pathway, as well as discussed the problems faced by the current research, hoping to provide references for the effective biosynthesis of cholesterol.

Abstract:Single-cell sequencing is becoming an essential tool for the basic research of biology, which brings revolutionary insights for us to understand biological phenomena. Many infectious diseases involve diversified functions of immune cells with great heterogeneity. Compared with high-throughput sequencing, the emerging single-cell transcriptome sequencing enables researchers to explore immune cell heterogeneity during infection, to fully excavate molecular information from precious clinical samples, and to obtain the genetic information of unculturable pathogenic microorganisms. Herein, we review the application status of single-cell sequencing in the research on infectious diseases and pathogenic microorganisms and make a brief prospect of this technology.

Abstract:Atopic dermatitis (AD) is an intractable and relapsing skin disease that has become a concern in the public health field due to its complex etiology and increasing prevalence year by year. With the application of high-throughput sequencing, metagenomics, metabolomics, and other technologies, it has been found that the occurrence and development of AD are closely related to the microbial community, and the “microbiota-skin-gut” axis and the crosstalk mechanisms between them have also been gradually verified. The “microbiota-skin-gut” axis has played an important role in allergic skin inflammation. This article reviews the relationship between the “microbiota-skin-gut” axis and AD, and the signaling molecules and potential pathways it may communicate with. Significant attention has been paid to the potential mechanisms involved in AD alleviation by microorganisms such as probiotics, flora transplantation, antimicrobial peptides, and so on, providing a new perspective for targeting microbiota to treat allergic skin inflammation.

Abstract:Influenced by the epidemic of Covid-19 pneumonia, online and offline blended teaching has gradually become a new normal teaching mode. How to improve the teaching effect, ensure the substantive equivalence between online teaching and offline teaching, and realize effective teaching is the key. Adhering to the teaching philosophy of student-centered, result-oriented and continuous improvement, and aiming at the problems existing in the current Microbiology teaching, the course team used the effective teaching mode of O-AMAS to reconstruct the teaching process of Microbiology course based on the analysis and grasp of students’ learning characteristics and cognitive style, construction of online teaching resources and optimization of teaching content. Our team adopted a diversified teaching mode, implemented effective evaluation and feedback, promoted deep learning through simple summary, and fully mobilized students’ enthusiasm for autonomous learning, thus enhancing the teaching effect of the course.

Abstract:[Background] A disturbance in the gut microbiota is an important influencing factor in the development of ulcerative colitis, and is a focal point for many researchers. [Objective] This study aimed to comprehensively analyze the frontiers, as well as developmental research trends in the association between the gut microbiota and ulcerative colitis over the past 20 years. The data from this study will provide guidance for future research. [Methods] Relevant publications between 2000 and 2020 were retrieved from the Web of Science Core Collection (WoSCC) search engine, and analyzed using VOSviewer 1.6.17, CiteSpace 5.8.R1, as well an online bibliometric analysis platform. [Results] An extensive online search yielded a total of 3146 publications. Our analysis revealed a positive trend in the annual number of publications, and the frequency of citations. The United States was the country that excelled terms of publication output, H index and international collaboration. The most influential research institution was Harvard University, and the most influential author was Xavier RJ. Journal co-citation analysis demonstrated that Gastroenterology, a highly-rated specialized journal in the field of gastrointestinal tract, had the highest number of publications, and was the journal most likely to contain relevant publications. Research hotspots included the keywords “expression,” “probiotics,” and “ Escherichia coli ”. Keyword burst detection analysis found “dysbiosis,” “microbiome,” and “fecal microbiota transplantation” were the most current research frontiers. [Conclusion] The current status, and developmental trends in the field of gut microbiota and ulcerative colitis yielded insightful results. The results from this study will aid researchers in selecting suitable journals and collaborators. It also provides an overall picture of the research that has been done, and an in-depth analysis of hotspots in this field.

Abstract:[Background] In recent years, more and more studies have focused on Rhodococcus, and few researchers have conducted a comprehensive review of the existing literature. [Objective] To investigate the research hotspots, frontiers and future development trends in the field of Rhodococcus in China and abroad. This study provided reference for subsequent research. [Methods] This study aimed to conduct statistical analysis and bibliometric analysis of the papers on Rhodococcus published in Web of Science in the last decade. VOSviewer was used for overlay visualization of the authors and keywords co-occurrence networks of Rhodococcus. [Results] The results showed that the overall number of publications in the field of Rhodococcus worldwide has been increasing year by year, and most of the published journals were specialist journals in microbiology. The number of publications and citations of articles in China and America far exceeds that of other countries, and the research content of Rhodococcus mainly focuses on biocatalyst, biodegradation, and non-ribosomal peptide synthases. [Conclusion] Rhodococcus has been paid more and more attention in the world and has gradually become a research hotspot. Countries and research institutions should continue to strengthen cooperation to promote the continuous development of the field of Rhodococcus.

Abstract:[Background] Pseudomonas aeruginosa, one of the main pathogens causing clinical infections, is resistant to a variety of antimicrobial agents, which is difficult to be treated in clinical practice. Increasing attention has been attached to the research on the drug resistance of P. aeruginosa. [Objective] With the visualization function of CiteSpace, we explored the status, hotspots and trend of the research on the drug resistance of P. aeruginosa. [Methods] We performed a bibliometric analysis involving 8 996 articles about the drug resistance of P. aeruginosa, which were published in Web of Science, CNKI, and Wanfang Data from 2014 to 2021. CiteSpace was used to analyze the number of articles published, author cooperation network, cooperation network of affiliated countries and institutions, and literature co-citation. Furthermore, we used CiteSpace to perform journal analysis, keyword clustering, and burst detection, aiming to reveal the hotspots and trends of this research field. [Results] The growth rate of the articles published in English was higher than that in Chinese. China ranked second only to the United States and India in the number of articles, showing great contribution to research achievements in this field and strong academic influence. The articles published in both Chinese and English concerned nosocomial infections and carbapenem-resistant P. aeruginosa. The difference was that papers published in Chinese focused more on the clinical prevention and treatment of drug resistance of P. aeruginosa, while those published in English mainly involved the basic research of the drug resistance. [Conclusion] The research on drug resistance of P. aeruginosa at home and abroad pays the highest attention to the production and control of new drug-resistant bacteria and nosocomial infection, suggesting that the above research topic is the research hotspot and trend in this field.