Abstract:[Background] The acid tolerance of industrial strains is a significant challenge in the fermentation process. The bacterium Serratia marcescens is part of the Enterobacteriaceae family of eubacteria. It can produce 2,3-butanediol, acetoin, prodigiosin and other high value-added products. However, the molecular mechanism behind S. marcescens acid resistance is not properly understood. [Objective] By mining of the transcriptional regulator XrpA and studying its functions, the molecular mechanism of acid tolerance of S. marcescens was preliminarily investigated, and a new direction was provided for improving the acid-resistant ability of industrial strains. [Methods] A Tn5G transposon insertion mutant library was constructed by transposon insertion mutation of S. marcescens, and an acid-sensitive mutant strain was screened from the library for sequencing identification. Then, the transcription level of key genes related to acid tolerance and the changes of cell membrane permeability, cell membrane integrity and H+-ATPase activity in the mutant strain were detected. Finally, the mechanism of XrpA regulating the acid tolerance of the S. marcescens was studied by analyzing the experimental data. [Results] we screened for novel regulators that respond to acidic conditions and found mutations in a gene encoding for the HTH_XRE super-family regulatory protein member, here named xrpA. We showed that the xrpA disruption conferred pleiotropic phenotype changes, including highly decreased biomass, H+-ATPase activity, and deficiency of cell membrane permeability and integrity, compared with those of the parent (JNB5-1) strain at low pH. [Conclusion] These data revealed that the molecular mechanism by which xrpA affects acid resistance of S. marcescens is through positive regulation of cell membrane permeability, integrity, and H+-ATPase activity to maintain intracellular homeostasis at low pH. Meanwhile, these results indicated that XrpA regulates tolerance to low pH by transcriptional regulation of acid stress response genes to maintain cell membrane function in S. marcescens.

Abstract:[Background] Biogenic natural pigments have potential applications in industry, agriculture, and textile industries. [Objective] The isolation and screening of marine pigment-producing actinobacteria is aimed to lay a foundation for the applications of bacterial pigments in the textile and food industry. [Methods] Actinobacteria, producing extracellular soluble pigment, were screened, and 16S rRNA gene phylogenetic tree was constructed based on their 16S rRNA gene sequences. Subsequently, factors affecting pigment yield and stability were investigated. [Results] A blue pigment-producing actinobacterium Q2N-42 and a yellow-green pigment-producing actinobacterium X4C-5 were obtained. Phylogenetic analyses of the 16S rRNA gene sequences of Q2N-42 and X4C-5 showed that they have high similarity with Streptomyces coelicolor or Streptomyces violaceoruber and Streptomyces pratensis, respectively. Comparative analyses of the effects of carbon and nitrogen sources on pigment yields showed that glycerol and sodium nitrate can significantly increase the blue pigment yield of S. coelicolor Q2N-42, whereas, starch and sodium nitrate can significantly increase the yellow-green pigment yield of S. pratensis X4C-5. Both the blue and yellow-green bacterial pigments displayed fine stability when they were treated with different concentrations of the oxidant, reductant, salinity, and pH. FTIR spectral analyses of the pigments showed that the pigment molecules contain the functional groups of ?OH, ?CH3, and C=C. Different mordants coupled with the bacterial pigments were used to check the dyeing propriety on the cotton threads. The results demonstrated that copper sulfate combined with the bacterial blue pigment presented a excellent performance when the cotton threads were dyed with it, the cotton threads were stained in darker blue, and enough to withstand the water wash treatment. Whereas, ferrous sulfate combined with the bacterial yellow-green pigment gave a good performance for the cotton thread staining. [Conclusion] Those data indicate that both the blue pigment produced by Q2N-42 and the yellow-green pigment produced by X4C-5 are potential resources towards their industrial applications.

Abstract:[Background] The problem of heavy metal pollution caused by mining has caused great harm to the ecological environment and human health. Therefore, the problem of heavy metal pollution in mining areas needs to be solved urgently. Biological remediation method has low cost, wide resources and no secondary pollution. It is an effective way to remediate soil heavy metal pollution. [Objective] Using the soil around a coal gangue mountain in Qufu city, Shandong province as a material, a strain with high tolerance to lead, zinc, and chromium and strong adsorption capacity was separated and selected by the flat line method. [Methods] Morphological characteristics, physiological and biochemical identification, and molecular biology methods were used to identify the strain. The concentration of each heavy metal was measured by an atomic absorption photometer. [Results] The identified strain was Bacillus cereus, named Bacillus cereus MZ-11. The strain MZ-11 can grow normally when the pH was 5.5?8.5, the temperature was 15?45 °C, and the NaCl mass fraction was 2%?8%. The strain MZ-11 had the highest resistance to lead, zinc and chromium up to 1 000, 1 200 and 1 600 mg/L. The three heavy metal concentrations of lead, zinc, and chromium in the medium were the initial concentration (containing Pb2+, Zn2+, and Cr6+ at 50, 60, and 80 mg/L, respectively), and the initial concentration was 5 times, 10 times, and 20 times. As the concentration increased, the adsorption ratio of strain MZ-11 to Cr6+ and Zn2+ gradually increased, and the adsorption percentages were all above 98%?99%. Under the condition that the Pb2+ concentration was 5 times the initial concentration, the adsorption ratio of MZ-11 to Pb2+ reached the maximum 97.01%. [Conclusion] Strain MZ-11 has high tolerance to lead, zinc and chromium and good adsorption capacity, which provides strong theoretical support for bioremediation of heavy metal pollution in the mining area and ecological environment of the mining area.

Abstract:[Background] Halophilic microorganisms with unique physiological and metabolic characteristics for living in hypersaline environment have high application and research value in the fields of environmental pollution control, enzyme preparation and are one type of important microbial resources from extreme environment. [Objective] To uncover species diversity of cultivable moderate halophilic microorganisms in rock salt in China, to develop and utilize halophilic microbial resources and to accumulate strains of halophilic microorganisms, this research was conducted. [Methods] Moderate halophilic bacteria were isolated from brine and salt soil by using alkaline oligotrophic medium (AOM), neutral haloarchaeal medium (NHM), diluted modified marine agar (dmMA) and ISP3 medium (ISP3) under 5% and 10% salinity separately. Bacteria 16S rRNA gene amplification and sequencing were carried out by using bacterial common primers 27F and 1492R. EzBioCloud and Blast in NCBI web were used for the bacterial taxonomy and phylogenetic tree was constructed by Mega 5.0. [Results] In all, 78 strains were isolated from brine and salt soil in rock salt in Yexian county, Henan province. The results of 16S rRNA gene sequences indicated that they ranged three phylum in seven genus including: 26 strains in genus of Bacillus, 30 strains in Halobacillus, 10 strains in Oceanobacillus and one strain of Staphylococcus in phylum Firmicutes; 3 strains in Sphingomonas and 5 strains in Halomonas in the phylum Proteobacteria; 3 strains in Brevibacterium in the phylum Actinobacteria. The genus of Halobacillus and Bacillus had higher abundance in the rock salt from Yexian county, Henan province. At the most, five genus bacteria were isolated by using AOM medium and only by using AOM, Sphingomonas and Brevibacterium were isolated. In addition, Oceanobacillus were isolated from brine by using ISP3. By using the four media, Halobacillus and Bacillus were isolated. Bacillus, Halobacillus, Oceanobacillus, Sphingomonas and Staphylococcus were from brine, and Bacillus, Brevibacterium, Halomonas were from salt soil. Third, under the 5% and 10% salinity, Bacillus, Brevibacterium, Halobacillus and Sphingomonas were isolated and only under 10% salinity, Halomonas and Oceanobacillus were isolated, only one strain Staphylococcus was isolated under 5% salinity. [Conclusion] The community structure and diversity of rock salt in Yexian county, Henan province were uncovered and Halobacillu and Bacillus had higher abundance in the culturable Moderate Halophilic Bacteria genus. This research has provided rich Moderate Halophilic Bacteria resources for developing rock salt bacteria.

Abstract:[Background] Cytosine is one of the four basic bases of nucleic acids. Cytosine is firstly synthesized in the form of cytosine triphosphate. There is no specific primary pathway for the formation of free cytosine in nucleic acid primary metabolism. The biosynthetic pathways of blasticidin S and gougerotin both utilize free cytosine as the precursor. The biosynthetic gene cluster of blasticidin S contains a hydrolase BlsM that can hydrolyze cytidine monophosphate into cytosine, while gougerotin producer doesn’t encodes a homolog to BlsM. [Objective] To detect the existence of free cytosine in different bacteria, and to explore whether there is an isoenzyme or a new pathway to produce free cytosine. [Methods] blsM was knocked out in blasticidin S heterologous producer Streptomyces lividans WJ2, its fermentation products of the mutant strain and WJ2 were detected by high performance liquid chromatography. Free cytosine in the supernatant of 10 fractionated cell lysates was measured by LC-MS. [Results] The mutant WJ2?blsM strain still synthesizes blasticidin S, but the yield of each component is significantly lower than that of WJ2. In addition to Streptomyces lividans, free cytosine was detected in Staphylococcus aureus, Amycolatopsis mediterranei and Bacillus subtilis. [Conclusion] WJ2?blsM still produces blasticidin S, indicating that wild-type Streptomyces lividans has an uncovered pathway for synthesis of free cytosine to meet blasticidin S synthesis. The content of free cytosine varies in different microorganisms.

Abstract:[Background] Commensal bacteria can affect host’s physiology, metabolism and neural behavior. Drosophila melanogaster is an excellent genetic model to investigate the mechanism of interaction between host and commensal bacteria. [Objective] To isolate and identify the commensal bacteria from D. melanogaster, and investigate their effects on the growth and development of D. melanogaster. [Methods] The YG agar medium was prepared to isolate bacteria from the intestinal tract of D. melanogaster. Gram-staining, biochemical identification and 16S rRNA gene blast were used to identify the bacteria strain. The commensalism between the bacteria and D. melanogaster was verified by colonization and generation transfer experiments. The sterile and gnotobiotic animal models were established to verify the growth promotion effect of E. hormaechei through the development timing and growth rate. RT-PCR was used to examine the molecular markers; immunofluorescence staining monitored proliferation of intestinal cells in fruit flies. [Results] A bacterial strain that could robustly promote the growth of the flies was isolated from the gut of D. melanogaster and identified as E. hormaechei. It stably colonized the gut of D. melanogaster and medium, and could be delivered to descendant. E. hormaechei promoted the growth of the flies obviously via increasing PTTH secretion and promoting the proliferation of intestinal cells in fruit flies. E. hormaechei decreased the level of glucose compared to germ-free flies. [Conclusion] E. hormaechei is a beneficial commensal bacterium of D. melanogaster to promote the growth and development of D. melanogaster.

Abstract:[Background] Phytophthora capsici is a worldwide soil borne disease, which has serious impact on pepper production all over the world and brings huge economic losses. Pectate lyase (PL), as a kind of important cell wall degrading enzymes, is an important pathogenic factor of the disease. [Objective] The gene of PL was cloned and its bioinformatics characteristics were analyzed to further elucidate its mechanism. [Methods] According to the whole genome sequence of P. capsici, the full-length cDNA of PL101 gene was amplified with high pathogenic strain SD33 as template, and its physical and chemical properties, transmembrane regions, hydrophobicity, structural domain and other biological characteristics were analyzed. [Results] In addition to the analysis of biological characteristics of PL101, the three-dimensional structure modeling of PL101 was also carried out to obtain protein structure with high reliability, and the possible catalytic sites of PL101 were determined as Asp183, Arg212 and Arg272 amino acids. [Conclusion] The cloning and bioinformatics analysis of PL101 will provide a reference for further elucidating the functional characteristics of PL.

Abstract:[Background] Polyethylene mulch film is widely used for agricultural production in China. Because it is very difficult to be naturally degraded in the fields, it finally accumulates in the soil and negatively affects the growth of crops and the ecological environment. Therefore, it is of great significance to explore the microbial resources for biodegradation of such “white pollution”. [Objective] Bacteria were isolated and screened from the intestinal flora of plastic-eating insects such as Galleria mellonella and Tenebrio Molitor from different sources, and their degradation efficiency of agricultural plastic film was characterized. [Methods] The larvae of Galleria mellonella and Tenebrio molitor were domesticated by polyethylene membrane, and the bacteria with polyethylene as the sole carbon source were isolated from intestinal bacteria by collecting intestinal fluid, enriching culture, co-metabolic domestication and selecting culture medium. The strain was inoculated into the medium with polyethylene membrane as the only carbon source for co-culture. The degradation effect of the strain on polyethylene mulch film was evaluated by measuring cell growth, regularly detecting the weight loss rate of the film, combined with high-resolution field emission scanning electron microscope observation, infrared scanning analysis and determination of mechanical properties of the film. The strains with good degradability were identified by 16S rRNA gene amplification and sequence analysis. [Results] Most polyethylene-degrading bacteria were isolated from the intestines of the indigenous Galleria mellonella in the honeybee hive in Xinjiang, and the degradation efficiency of polyethylene was higher than that of the isolates from other sources. Three strains XJDLM-3, XJDLM-8 and XJDLM-12, with high degradation ability were selected to grow using polyethylene membrane. scanning electron microscope observation showed that obvious erosion holes and cracks appeared on the surface of degraded polyethylene film after 30 days, and the infrared scanning pattern changed. The mechanical properties such as tensile strength, elongation at break and elastic modulus decreased significantly, and the weight loss rate of polyethylene diaphragm reached 8.06%, 5.66% and 5.39%, respectively. A bacterial strain with good degradation effect was isolated from the intestines of the native Galleria mellonella in the honeybee hive in Xinjiang. XJDLM-8 and XJDLM-12 were identified as Bacillus cereus, XJDLM-3 and Enterobacter bugandensis. [Conclusion] Strains were isolated with high ability to degrade polyethylene in the intestines of the native Galleria mellonella in the honeybee hive in Xinjiang, to provide the potential of development and application in the degradation of polyethylene mulch film.

Abstract:[Background] The lack of protein feed has promoted the research and application of yeast single-cell protein with high protein content and good safety performance. [Objective] In order to screen strains with strong ammonia nitrogen utilization ability, provide excellent strains for single-cell protein fermentation. [Methods] Yeasts were isolated and identified from the soil, dairy products and fruits based on morphology and molecular biology. Then, with ammonium sulfate as the sole nitrogen source medium, the bacterial colony size, cell dry weight and protein content were determined, and the yeasts with high ammonia nitrogen utilization rate were rescreened. The enzyme activities related to ammonia assimilation of the rescreening strains were determined. [Results] By morphological, molecular biological identification and ammonia nitrogen utilization ability evaluation, three strains of high ammonia nitrogen utilization were obtained, they were Rhodotorula mucilaginosa, Saccharomyces cerevisiae and Torulaspora delbrueckii. S. cerevisiae has the highest activities of glutamate dehydrogenase, glutamate synthetase, and glutamine synthetase, followed by R. mucilaginosa. [Conclusion] R. mucilaginosa N5 and S. cerevisiae J1 isolated from cheese and watermelon have strong ammonia nitrogen utilization ability and enzyme activities, and can provide excellent strains for single cell protein fermentation.

Abstract:[Background] Hymenobacter is dominant in bacteria in adverse environment (e.g. desert soil with nutrition poor). The research on this group has focused on the isolation and identification, yet until now there no the plant growth promotion is studied. [Objective] To isolate and identify bacteria from the soil of Desert Hunshandake, and evaluate the effect on the growth of potato rapid propagation plantlet. [Methods] On selective media, spread and streak plating was employed to isolate. For the primary taxonomic identification, 16S rRNA gene was PCR amplified and sequenced and the similarity and phylogeny were analyzed, combined with the basic morphological, physiobiochemical characteristics analysis. The plant growth-promoting traits were characterized on selective media or colorimetry. MS media was used to assay the effect on the growth of potato rapid propagation plantlet. [Results] A bacterial strain, designated as L28, was obtained in this study. Strain L28 showed the highest 16S rRNA gene sequence similarity of 96.46% with Hymenobacter koreensis GYR3077T. The strain possessed multiple plant growth-promoting traits, including nitrogen fixation, Ca3(PO4)2-P dissolving, phytate-P degrading, indole-3-acetic acid (IAA) producing (7.51 mg/L), siderophore producing (D/d=2.47) and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase active. Compared to the uninoculated, the inoculation of L28 markedly increased the plantlet’s node number, height, root length, stem length, dry weight of root, and dry weight of stem with the increments of 28.57%?234.94%; for transplanted plant, the inoculation of L28 significantly elevated the number and weight of potato tuber seed with the increments of 40% and 181.87%, respectively. [Conclusion] Bacterial isolate L28 from the soil of Desert Hunsandake belongs to the genus Hymenobacter, and has multiple plant growth promoting traits. The strain markedly promotes the growth of rapid propagation potato plantlet and the development of potato tuber. It can be used as a plant growth promoting agent and has important prospect in potato production.

Abstract:[Background] Microbial degradation lignin has attracted more attention due to its characteristics of high degradation efficiency and environmental protection. [Objective] Screening highly efficient lignin degradation fungi and optimizing their degradation conditions. [Methods] High efficiency lignin degradation strains were screened by guaiacol-PDA and aniline blue plate methods, and the culture conditions were optimized by single-factor screening and response surface experiment. [Results] An efficient lignin degradation bacteria BYL-7 was screened and initially identified as Trametes versicolor by morphological and multi-sequence analysis. The single factor test proved that the initial pH, temperature and inoculum amount were the significant influencing factors for lignin degradation, and the response surface test determined that the optimal conditions for degradation lignin were initial pH 6.7, temperature 25 °C, and inoculum amount 8%. Under these conditions, the alkaline lignin degradation rate was 36.5%, which was 54.0% higher than before; the degradation rates of rice straw lignin, hemicellulose, and cellulose were 32.8%, 21.5%, and 13.2%, respectively. Among them, the lignin degradation rate was enhanced by 36.1% compared with before. Laccase activity peaked at 120.0 U/L in the 6th day, which was 25.0% higher than before; the lignin peroxidase activity reached a peak of 1 343.8 U/L in the 6th day, which was 36.0% higher than before; the manganese peroxidase activity peaked at 463.8 U/L in the 5th day, which was 31.7% higher than before. [Conclusion] The experimental results provided a useful bacteria resource for lignin degradation, but also accumulated relevant data for subsequent research on lignin.

Abstract:[Background] At present, verticillium wilt of cotton seriously hinders the stable and high yield of cotton and hinders the development of cotton industry. Endophytic bacteria have great potential in biological control, but the variation of endophytic archaea in verticillium wilt of cotton is rarely reported. [Objective] In order to provide theoretical support for the following studies, the quantitative changes of endophytic archaea in cotton plants with verticillium wilt and healthy plants in different growth periods and different planting areas were studied. [Methods] Using MiSeq high-throughput sequencing and TaqMan probe real-time fluorescence quantitative PCR technology, the endophytic archaea of verticillium wilt and healthy strains in Xinjiang cotton were quantitatively analyzed at different growth stages and in different typical ecological regions. [Results] The community composition of endophytic archaea was similar to that of verticillium wilt and healthy cotton plants at different sampling sites and different growth stages in Xinjiang. At different growth stages, the number of endophytic archaea in verticillium wilt and healthy cotton plants in Xinjiang increased first, then decreased, then tended to be flat, and reached the highest value in bud stage. In different regions, the number of endophytic archaea of verticillium wilt cotton in Xinjiang was the highest in the northern region, followed by the eastern region and the southern region. Health plant is the highest in the southern region, followed by the eastern region, the lowest in the northern region. [Conclusion] The quantity of endophytic archaea in cotton plants with verticillium wilt and healthy plants in Xinjiang was significantly different in different growth periods and different spaces, and the overall variation trend was significant.

Abstract:[Background] Nisin is a small antibacterial peptide mainly produced by Lactococcus lactis. It is the only natural food preservative approved for wide applications. However, its low biosynthesis yield, and lack of simple and efficient detection methods limit its research and applications. [Objective] To develop a simple and efficient method to detect nisin, the nisin controlled gene expression (NICE) system and a visible red fluorescent protein were adopted for construction of a whole-cell nisin biosensor. The biosensor was used to quickly screen for nisin-producing candidate strains. [Methods] A Golden-Gate cloning method was used to construct a vector which contains a nisin-inducible promoter (Pnis) for regulating a red fluorescent protein gene (rfp). Two rfp genes, identical in protein sequences but different in DNA sequences, were tested. The constructed biosensor plasmids were transferred into Lactococcus lactis, generating the whole-cell nisin biosensors and further used for the screening. [Results] Both of the two whole-cell nisin biosensors could specifically and quantitatively respond to nisin varied from 2 to 200 ng/mL. Moreover, the biosensor was applied to screen candidate strains and identified a nisin-producing strain Lactococcus lactis ATCC 11454. [Conclusion] The developed whole-cell nisin biosensor can specifically respond to nisin, and can be used to simply and quickly identify nisin-producing strains.

Abstract:[Background] Aging and some diseases of organism are mostly related to oxidation, with the further research of antioxidant, the safety of synthetic antioxidants is called into question. So the research for natural antioxidants has become a hot topic. [Objective] To screen for Lactobacillus with high antioxidant activity from air dried mutton and to analyze their antioxidant capacity in vivo and in vitro. [Methods] Taking 24 strains of meat-derived Lactobacillus as research objects, the ability of scavenging free-radical, chelating ferrous ion, reducing and anti-lipid peroxidation were used as the indexes for analyzing the antioxidant capacity in vitro. The 16S rRNA gene sequence homology analysis was used to identify the species, and its antioxidant activity in vivo was studied in mice. [Results] All of strains that we tested had anti-oxidation ability in vitro, among them, strain of TR13 had the strongest anti-oxidation ability, its scavenging activity of ·O2? was 54.29%, and scavenging activity of hydroxyl radical was 90.84%, the scavenging activity of DPPH radical was 99.38%, the chelation rate of ferrous ion was 55.85%, the ability to reduce was 1.345, the rate of detecting anti-lipid peroxidation was 39.99%. The strain of TR13 was identified as Lactobacillus helveticus according to the 16S rRNA gene identification. Feeding D-galactose-induced aging mice with TR13 bacterial solution verified that TR13 could promote the activity of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) in liver, kidney, and blood tissues of mice significantly, and the GSH-Px activity of TR13 group was significantly higher than that of vitamin C (Vc) group. [Conclusion] The strain of TR13 can effectively prevent oxidative aging for body, and the result can provide reference for researching Lactobacillus antioxidants.

Abstract:[Background] Salmonella is an important zoonotic foodborne pathogen. Therefore, controlling of Salmonella infection in animals and pollution in the food production chain has great significance for livestock production, public health and food safety. [Objective] Studying the infection and epidemic of Salmonella from a parental breeder farm of white broiler and its commercial chickens in northern Jiangsu will provide a reference for the prevention and control of Salmonella. [Methods] A total of 360 samples of cloaca swabs were collected from farms in Taizhou, Suqian, Yancheng and Lianyungang from 2018 to 2019, including 120 samples from parental breeders, their 1 or 2 days old weak commercial broilers and 10 days old commercial broilers, respectively. Then, the Salmonellas were isolated and identified, their drug resistance phenotypes and genotypes were characterized by Kirby-Bauer and PCR methods, and the genetic relationship of the isolates was analyzed by multilocus sequence typing (MLST). [Results] In total, 120 Salmonella strains were isolated, including 3 strains from the parental breeders, 25 strains from the 1 or 2 days old weak commercial broilers and 82 strains from the 10 days old commercial broilers. By using Kirby-Bauer test, all strains were found to be sensitive to antibiotics polymyxin B, imipenem, doxycycline, trimethoprim, tigecycline, florfenicol, cefotaxime and enrofloxacin, and resistant to macrodantin, penicillin, rifampicin, linezolid and vancomycin. Moreover, 80 isolates (72.73%) were sensitive to antibiotic amoxicillin while the other 30 strains (27.27%) were resistant to it. The drug-resistant genes analyzed by PCR showed that blaTEM existed in all isolates, but not other common resistance genes. The ST type of all Salmonella isolates in this study was identified as ST11 based on the MLST typing and sequencing results of 7 pairs of housekeeping genes. Collectively, out data demonstrate that the phylogenies of these Salmonella isolates are very similar. [Conclusion] Salmonella infections occurred in the commercial broilers seemed to be caused by vertical transmission from the same herds of parental breeders. This study provided a reference for the prevention and control of Salmonella in white broiler farms in this area.

Abstract:[Background] CFP10 and ESAT6 are important virulence factors of Mycobacterium tuberculosis (Mtb), and can cause the apoptosis of macrophages. [Objective] To investigate the effects of CFP10 and ESAT6 on cell apoptosis and AIM2/ASC/Caspase-8 pathway in RAW264.7 macrophages. [Methods] E. coli expression system was used to express and purify recombinant CFP10 and ESAT6 proteins of Mtb. Then, the RAW264.7 cells were treated with the recombinant proteins with different concentrations. The cell viability of RAW264.7 cells after incubation with CFP10 and ESAT6 at different concentrations for 24 h was measured by CCK-8 assay, expression of apoptosis related proteins as well as AIM2 and ASC inflammasomes were determined by Western blotting analysis, and the cell apoptosis was detected by flow cytometry. [Results] The results of SDS-PAGE and Western blotting showed that we have successfully purified the recombinant proteins of CFP10 and ESAT6 from E. coli. After treatment of RAW264.7 cells with different concentrations of CFP10 and ESAT6, the cells proliferation was significantly inhibited. When CFP10 and ESAT6 were treated separately at the concentration of 5 μg/ml, the cell viability rate decreased significantly as compared with the control group (P<0.001), and the cell viability rate decreased significantly in a dose dependent manner (P<0.001). Western blotting results further showed that both of CFP10 and ESAT6 with the concentration of 5 μg/ml could lead to RAW264.7 cells apoptosis after 24 h treatment. When RAW264.7 cells were treated with CFP10 and ESAT6 together at a final concentration of 5 μg/ml respectively, the expression of apoptosis-related proteins of BAD, CHOP, Caspase-8 and Caspase-3 decreased significantly as compared to ESAT6 treatment group (P<0.001), an indication that the co-treatment of RAW264.7 cells with CFP10 and ESAT6 significantly reduced the apoptosis of macrophages caused by ESAT6 treatment alone. Furthermore, the results of Western blotting showed that ESAT6 treatment could activate the expression of AIM2 and ASC inflammasomes. [Conclusion] CFP10 and ESAT6 of Mtb alone at the concentration of 5 μg/ml could cause apoptosis of RAW264.7. The co-treatment of RAW264.7 cells with CFP10 and ESAT6 could reduce the apoptosis of macrophages caused by ESAT6 treatment alone. Further studies show that ESAT6 may cause RAW264.7 cell apoptosis by activating AIM2/ASC/Caspase-8 signaling pathway. The results of this study will lay a foundation for fully understanding the immunoregulatory roles of CFP10 and ESAT6 on cell apoptosis and its molecular mechanisms in macrophage defense against Mtb infection.

Abstract:[Background] Atypical porcine pestivirus (APPV) as a new virus, it has been detected in various provinces in China in recent years. At present, most of the research reports on APPV focus on genomics and epidemiology. The pathogenic mechanism after infection of the host is unclear. [Objective] To investigate the pathogenicity of APPV in the cerebellum of infected piglets by high-throughput sequencing technology. [Methods] Three cerebellum of APPV-infected piglets of experimental group excluded from pathogens which can cause congenial tremors (CT) or neurological symptoms were sequenced with 3 cerebellum of healthy piglets of control group, and compare the differences in transcription levels between experimental and control groups. [Results] Two cDNA libraries were constructed in this study: APPV infected cerebellum tissue library and healthy cerebellum tissue library. Under the screening criteria of P≤0.05, |log2(FC)|≥0.263, a total of 381 differentially expressed genes (DEGs) were screened, including 163 up-regulated genes and 218 down-regulated genes; 8 differentially expressed genes were randomly selected. The gene was detected by RT-qPCR and β-actin was used as an internal reference gene. The expression trends of eight differential genes were consistent with high-throughput sequencing. [Conclusion] A transcriptome study was conducted on the cerebellum samples of clinically infected piglets with APPV. Based on the GO and KEGG, we performed the functional annotation and classification of DEGs. It was found that the transcriptional levels of Pak4, Robo4, Sema3f, Wnt5a and other genes involved in axon guidance signaling pathways had significantly decreased. This result indicates that APPV infection may inhibit the axon guidance process in piglets. Furthermore, it inhibits the development of the central nervous system, which provides direction and guidance for further research on the pathogenic mechanism of APPV.

Abstract:[Background] Campylobacter is one of the most common zoonotic pathogens and is a leading cause of human gastroenteritis through food chain. [Objective] To study the phenotypic resistance and molecular type of Campylobacter isolated from pig source in ten intensive pig farms of Jiangsu province. [Methods] The minimal inhibitory concentrations (MICs) was determined by agar dilution method and the resistance genes were detected by polymerase chain reaction (PCR). Seven Campylobacter housekeeping genes including aspA, glnA, gltA, glyA, pgm, tkt and uncA were amplified and sequenced, then the sequences of genes were analysed through multilocus sequence typing (MLST). [Results] A total of 22 isolates were identified from 100 samples (22%), the detection of Campylobacter was independent of breeding scale and age (P>0.05). 81.82% of the isolates were resistant to three or more than three drugs, resistance rates to ten drugs were under different degrees, gentamicin (36.36%), streptomycin (50%), clindamycin (27.27%), chloramphenicol (13.64%), tetracycline (40.91%), ciprofloxacin (18.18%), nalidixic acid (63.63%), telithromycin (59.09%), erythromycin (100%), azithromycin (81.82%). The resistance-genes detection rates to cfr, adE-Sat4-aphA, ermB and Tet(O) were 4.5%, 59.1%, 9.1% and 100%, MLST results showed that 11 sequence types were found and CC-828 was the prevalent clonal complex (100%), Evolutionary tree results showed that all strains were belonged to two groups, which had two branches and nine branches, respectively. χ2 test and Logistic regression analysis showed that three sequence types (STs) were associated with their corresponding antimicrobial agents. [Conclusion] The high resistance to macrolides antibiotics and the high detection of Tet(O) were found in Campylobacter coli from swine feces, C. coli population showed diverse genetic nature.

Abstract:[Background] Pseudorabies virus (PRV) is an important pathogen to pig industry, a large-scale of pseudorabies (PR) has swept many Bartha-K61-vaccined farms in China since 2011. [Objective] In order to investigate the epidemiological characteristics of PRV strains prevalent in Sichuan province, China, 384 suspected PRV infected samples were collected from 86 pig farms in 2018?2019. [Methods] According to the amplification of PRV-gE gene, 384 samples were detected by PCR. The positive rates of PRV in different seasons and regions were counted, and the correlation between PRV infections and clinical symptoms was statistically analyzed. We selected some PRV positive samples for virus isolation through BHK-21 cells, followed by gC, gE and TK genetic informatic analysis of isolated strains. [Results] The individual positive rate of PRV was 9.9% (38/384); the group positive rate of PRV was 16.3% (14/86); the positive rate in aborted fetuses was 32.1% (27/84); the positive rate in semen of breeding boars was 2.0% (4/198); the positive rate in nervous symptoms pig was 11.4% (4/35); the positive rate in respiratory symptoms pig was 4.5% (3/67). Statistical analysis indicated that PRV infection was associated with pig reproductive failure (P<0.01). The highest PRV infection rate was detected in winter (December, January, February), with a positive rate of 33.0% (31/94); Spring (April, March, May) ranked the second highest positive rate of 9.1% (3/33); Summer and autumn was about 1.5% (2/130) and 1.6% (2/127) respectively. Totally, we isolated 3 PRV strains between 2018?2019, named PRV-SN, PRV-DJY, PRV-CD respectively. PRV-XJ, a strain isolated by our laboratory in 2016 in Sichuan province was also included, these strains were used for gC, gE, TK gene sequencing. Sequence alignment shows that Sichuan isolates were similar to those Chinese isolates, except for sporadic mutations and deletions. [Conclusion] Farms should strengthen the purification of pseudorabies in breeding herds.

Abstract:[Background] Salmonella is a kind of Gram-negative intestinal pathogen that relies primarily on type III secretion systems (T3SSs) to produce pathogenicity-associated effector proteins. The Salmonella pathogenicity island (SPI) is a key genetic region. The conditions of high-salt can induce the expression of effector proteins on SPI-1. [Objective] In order to explore the differential expression of glycoprotein of Salmonella enterica serovar Typhimurium under high-salt concentration, and to find meaningful effector glycoprotein. [Methods] Salmonella enterica serovar Typhimurium was cultured in common medium and high-salt medium, and then the cells were collected. The glycoproteins were enriched by hydrazine coupling method after ultrasonication and protein extraction. After the enzymatic hydrolysis, the glycoproteins were quantified by dimethyl-labeled quantitative method, and the labeled proteins were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry. [Results] Mass spectrometry results showed that the expression of 19 glycoproteins in Salmonella significantly changed in the high salt environment. Among these, 10 glycoproteins were up-regulated, the most notably one was the outer membrane porin encoded by ompC. Nine glycoproteins were down-regulated, the most notably one was translation initiation inhibitor encoded by yjgF. [Conclusion] The expression levels of many important glycoproteins in Salmonella have significant changes at high-salt environments. This study has important significance in investigating the expression of effector glycoprotein produced by Salmonella SPI-1 and searching the pathogenic mechanism of Salmonella.

Abstract:[Background] Collagen is widely used in daily-use chemical industry and biomedicine. Genetic engineering method for preparing collagen exhibits several advantages, such as avoiding virus infection and high expression level, which has attracted substantial attention in recent years. [Objective] A human-like collagen gene was obtained to achieve heterologous expression in E. coli. [Methods] Using human type III collagen α1 chain as template and (Gly-X-Y) as the smallest research unit, preferably selecting hydrophilic amino acids, designing and synthesizing gene kit, and constructing recombinant E. coli pET-28a(+)-kit/BL21(DE3), and characterizing it. [Results] The human-like collagen gene kit was successfully expressed in E. coli with expression level of about 0.53 g/L. After fed-batch fermentation on a 7 L fermenter, the maximum expression level was increased to 3.02 g/L. Purification by affinity chromatography was performed, which obtained the human-like collagen with a purity of about 91%. Human-like collagen was analyzed by N-terminal sequencing, amino acid analysis, mass spectrometry, and circular dichroism analysis to determine the successful expression of human-like collagen. [Conclusion] The successful expression of human-like collagen would lay the foundation for future preparation and its practical application in the daily chemical and biomedical industries.

Abstract:[Background] Compared with traditional genetic modification, molecular dynamics simulation of cyclodextrin glycosyltransferase can effectively improve the transformation efficiency and reduce blindness. [Objective] To explore the catalytic specificity mechanism of cyclodextrin glycosyltransferase, and to provide an efficient mutation method for obtaining cyclodextrin glycosyltransferase with higher specificity for producing γ-cyclodextrin. [Methods] Through molecular docking and molecular dynamics simulation, the docking simulation structures of three product types of CGTase and substrate were obtained, and verified by site-specific saturation mutation experiment. [Results] The results of molecular dynamics simulation showed that α- and β-CGTase and decarbose chains appeared closed in S1 region, while γ-CGTase and decarbose chains appeared more open in S1 region. There are seventeen corresponding sites in the three CGTase and ten sugar chain amino acids with hydrogen bonds at the same position, of which the amino acid types at fourteen sites are consistent. The corresponding α-CGTase sites of the three inconsistent amino acids were Y89, D234 and Y262, respectively. This study conducted site-directed mutagenesis and product specificity experiments on the Y262 locus. Y262L predicted by molecular dynamics was helpful to increase the specificity of γ-CD production, from 13.7% of the wild enzyme to 39.9%, and the percentage of γ-cyclodextrin products increased by three times. [Conclusion] The results of molecular dynamics simulation have positive significance for guiding the specificity mechanism of cyclodextrin glycosyltransferase.

Abstract:[Background] Due to the short time of discovery of methylotrophs, only a few genomes of methylobacterium strains that can produce PQQ were sequenced. It increases the difficulty of study the genomics and biological metabolic pathways of methylobacterium. [Objective] The PQQ-producing bacteria was screened and treated with various mutagenesis methods to improve the yield of PQQ. Whole genome analysis of high-yield mutant strains was performed to provide sequence background information for studying the molecular mechanism of PQQ synthesis and subsequent molecular breeding of methylobacterium. [Methods] The wild-type PQQ production strains were subjected to ultraviolet rays mutagenesis, nitroso-guanidin mutagenesis, ethylmethylsulfone mutagenesis, diethyl sulfate mutagenesis, and ultraviolet rays-lithium chloride compound mutagenesis. The whole genome sequence of the mutant strain obtained by mutagenesis was sequenced using the PromethION sequencing platform and the MGISEQ-2000 sequencing platform. The assembled whole genome sequence was compared with the model strain Methylobacterium extorquens AM1. [Results] A mutant strain NI91 was obtained after 11 rounds of mutagenesis with PQQ yield 19.49 mg/L, which was 44.91% higher than the original strain. The genome of the mutant strain NI91 consists of a chromosome of 5 409 262 bp, encoding 4 957 proteins. Compared with the model strain M. extorquens AM1, it was found that the pqqF and pqqG genes that relate to shear processing during PQQ biosynthesis were deleted. Meanwhile, the pqqL was first discovered in methylotrophic bacteria which has a similar function to the pqqF, and the sequences of pqqC/D between the two strains were quite different. [Conclusion] This study provides basic data for functional genomics research of the methylotrophic bacterium and the study of PQQ synthesis mechanism. Comparative genomics between NI91 and the model strain M. extorquens AM1 provides a molecular basis for revealing different mechanisms of PQQ synthesis.

Abstract:[Background] EV-D68 belongs to the enterovirus D group of the Enterovirus genus of the small RNA virus family. Between August 2014 and January 2015, the infection caused by the virus increased significantly in North America, and also appeared in China. Compared with the original Fermon strain, the epidemic strain has almost one or two deletions in the 5′ UTR region, and there are two repeated ataaca sequences before the translation start codon. [Objective] We explored the effect of the deletion of 5′ UTR region of epidemic strains on downstream gene expression and the function of ataaca repeat sequence. [Methods] Sequence comparison was used to analyze the differences and conserved regions of 5′ UTR between the current epidemic strains and the original Fermon strain. The above regions were deleted by using molecular clone methods and then the dual-luciferase reporter system was used to analyze the effect on downstream luciferase reports genes. [Results] Sequence alignment analysis found that the current epidemic EV-D68 strain has a 23 base deletion in the region corresponding to the 685?707 of the 5′ UTR of the Fermon strain genome, while some strains have an additional deletion in the 718?729 regions. The luciferase assay showed that only the first deletion can greatly increase the expression of downstream genes, while the two deletions occur at the same time are almost equivalent to the wild type, while the deletion of the second sequence only reduces the expression of downstream genes slightly. In addition, we also found that the ataaca sequence in the first deletion may have a suppressive effect on downstream gene expression, and the role of the ataaca sequence near the start codon is not yet clear. [Conclusion] At present, most of the epidemic EV-D68 strains have one or two deletions in the 5′ UTR region. The first deletion greatly enhances the expression of downstream reporter genes, while the second deletion has the opposite function. The phenomenon may be related to the repeated ataaca sequences before the start codon.

Abstract:[Background] methicillin-resistant Staphylococcus aureus (MRSA) can exist in the state of biofilms, which results in multiple resistance and persistent infections. [Objective] By investigating the inhibition and clearance of thymol and oxacillin separately and concurrently on the biofilm formation of MRSA, the effects of combined medications on MRSA biofilms were explored to provide the theoretical basis for clinical combined application of anti-MRSA drugs. [Methods] Broth microdilution method was used to measure oxacillin minimal inhibitory concentration against USA300 strain. The effect of thymol and oxacillin separately and concurrently on the inhibition and clearance of biofilm formation in USA300 was evaluated by crystal violet staining method and colony counting method. [Results] Thymol and oxacillin inhibited the formation of USA300 biofilm at subinhibitory concentrations. At high concentrations, thymol had good removal effects on the biofilm formed in 24 and 72 hours, while oxacillin had no removal effect. The inhibition and clearance effect of the combination of the two drugs on biofilms was further enhanced, and it had a better inhibition and clearance effect at low concentrations. [Conclusion] Compared with thymol and oxacillin alone, the combination of thymol and oxacillin has an enhanced inhibitory and clearance effect on the biofilm of USA300. The combination of the two drugs has a synergistic antibacterial effect.

Abstract:[Background] Pseudomonas aeruginosa is a common opportunistic pathogen, most of them can form biofilms which show the characteristics of high mutation rate and strong antibiotic resistance. Non-homologous end joining (NHEJ) is one of the major DNA double-strand break repair pathway which leads to DNA mutations. [Objective] To study the effects of NHEJ on biofilm-forming ability, mutation rate and drug resistance within biofilm in P. aeruginosa. [Methods] The ku gene deletion mutant strain Δku was constructed by in-frame deletion and its complementary strain was also obtained. We studied the biofilm-forming ability of each strain as well as the mutation rate and drug resistance within their biofilms. The transcription level of ku gene in P. aeruginosa biofilm was determined by qPCR. [Results] Compared with the wild-type strain, the ku gene deletion strain did not show significant difference in biofilm-forming ability but displayed a slight reduction in mutation rate and minimum inhibitory concentration (MIC) to ciprofloxacin and gentamicin within biofilms. The qPCR results showed that the transcription level of the ku gene was increased when biofilms were formed. [Conclusion] NHEJ affects the mutation rate and antibiotic resistance of P. aeruginosa biofilm. This study will provide a basis for exploiting the mechanisms of antibiotic resistance in P. aeruginosa.

Abstract:[Background] Tongue coating refers to the substances covered on the tongue, which are composed of food residues, microorganisms and epithelial keratinocytes of tongue coating. According to the thickness and color of the tongue coating to identify the patient’s physical condition, is one of the components of “Look” diagnosis, is one of the main basis for traditional Chinese medicine (TCM) experts to treat patients with syndrome differentiation. However, there are few studies on the relationship between the formation of tongue coating quality and microbial flora. [Objective] To discover the difference of microbial flora between yellow greasy and thin white in tongue coating, and the relationship between the flora and the quality of tongue coating. [Methods] The thin white tongue coating and yellow greasy tongue coating of the young students were used as the research samples. The surface of the young students’ tongue coating was scraped with a disinfectant cotton swab, and the tongue coating samples were obtained, and sent to Shanghai Meiji company for DNA extraction. Then, 16S rRNA gene was amplified by PCR, and 16S rRNA gene was obtained by high-throughput sequencing systems, and then analyze the bacterial species in the sample by interactive sequence analysis software. [Results] There were significant differences in the composition of the main flora between the yellow greasy tongue coating and the thin white tongue coating. At the level of genus and species, the composition of Actinomyces was significantly different, The content of Actinomycetes in yellow and greasy tongue coating was significantly higher than that in thin and white tongue coating (P<0.05), while the content of Porphyromonas and Moraxella in thin and white tongue coating was significantly higher than that in yellow and greasy tongue coating (P<0.05). [Conclusion] The thick, yellow and peculiar smell of the yellow greasy tongue coating may be caused by the high content of Actinomyces; Moreover, some Actinomycetes can cause aggressive bacterial diseases, called actinomycosis. Therefore, the occurrence of yellow greasy moss in healthy people may indicate the potential early warning of inflammation in the body. Moraxella and Porphyromonas were significantly more than those on yellow and greasy tongue coating, indicating that these two normal flora might played an important role in maintaining normal tone and tongue coating function.

Abstract:[Background] Sujiang pig is a new breed of high-quality lean meat pig. The growth performance of weaned piglets will be different under various stress. [Objective] To study the differences and similarities of intestinal flora structure of Sujiang piglets with different growth performance. [Methods] 100 healthy piglets with similar body weight at the same age were selected and fed under the same feeding conditions for 42 days. At the end of the experiment, three weaker and heavier piglets were selected for the further analysis. The average body weight of the three weak and healthy piglets was 18.43±2.37 kg and 27.27±1.36 kg, respectively. After slaughtering, the contents of jejunum and cecum were collected, and the microbial genomic DNA was extracted and analyzed by high-throughput sequencing. [Results] There were significant differences in microflora abundance and diversity between jejunum and cecum of Sujiang piglets (P<0.01). The predominant strain of jejunum flora of Sujiang piglets is the non-pathogenic strain of Proteobacteria Pseudomonas, accounting for more than 90%. The dominant bacteria in the cecum of Sujiang piglets were Bacteroidetes, Firmicutes and Proteobacteria in turn. There were differences in the composition and structure at the phylum level, the abundance of Firmicutes in weak piglets was lower than that in healthy piglets, and the abundance of Proteobacteria was higher than that in healthy piglets. Prevotella, Ruminococcaceae_UCG-005, Alloprevotella and other bacteria related to fiber digestion were abundant in the cecum of Sujiang pigs. There were significant differences in cecal flora between weak piglets and healthy piglets at genus level (P<0.05). Prevotella_9, Escherichia-shigella, Ruminococcaceae_UCG-005 and norank_f_Prevotellaceae in cecum of weak piglets were higher than those of healthy piglets, while Lactobacillus, Prevotella_2, Terrisporobacter and Clostridium_sensu_stricto_1 in cecum of healthy piglets were higher than those of weak piglets. Lactobacillus and Terrisporobacter are related to the health of piglets, Escherichia-shigella is associated with disease of piglets. [Conclusion] There were significant differences in the composition of intestinal microflora among piglets with different growth performance. The results provided a basis for further study on the function of intestinal microflora in Sujiang pigs.

Abstract:Acetic acid is a promising carbon source, as a common by-product in microbial fermentation. It widely exists in non-food fermentation feedstock media such as lignin-cellulose hydrolysates. However, high concentrations of acetic acid or acetate in media inhibit cell growth, biomass, yield and productivity. It is significant to transform acetic acid into value-added chemicals efficiently through engineered strains with improved acetic acid tolerance. This paper reviews the state-of-the-art progresses in the strategies of improving Escherichia coli acetic acid tolerance via metabolic engineering, adaptive laboratory evolution, global transcription machine engineering and CRISPR traceable genome engineering. Furthermore, we summarize the acetic acid tolerance response mechanisms in E. coli, including acetic acid assimilation metabolism, amino acid-dependent metabolism, ion transport system regulation, and cell membrane component modification in E. coli. Finally, we discuss the application of improved acetic acid tolerance strains and the future direction in this field.

Abstract:Traditional anti-corrosion methods are costly or may cause secondary pollution. Microbiologically influenced corrosion inhibition (MICI) is a new green anti-corrosion technology. With the discovery of variety anti-corrosion microorganisms and the development of the studies on beneficial bacterial films, many mechanisms of microbial inhibition of metal corrosion have been found. Microorganisms can inhibit or slow down metal corrosion by biocompetitive exclusion, secretion of corrosion inhibitors, generation of extracellular polymeric substances, reduction of dissolved oxygen, formation of biofilm barriers, secretion of biosurfactants, phage control, non-biofilm barriers and other processes. Microbial corrosion inhibition of metal is usually not caused by a single mechanism but rather a combination of different mechanisms. In-depth study of its inhibition mechanism is helpful to understand how to slow down the corrosion behavior of metals.

Abstract:The type VI secretion system (T6SS) of bacteria is a newly discovered secretion system. It plays an important role in the adhesion and invasion of bacteria pathogens to their host and destruction of their host. Currently, most studies focused on the functions of T6SS in bacteria pathogenesis, bacterial competition, et al, but the research on its regulatory factors is still in the preliminary stage. For most bacteria, the expression of T6SS is not steady, which could be regulated by environmental factors such as temperature, osmotic pressure, antibiotics and ions. At the molecular level, H-NS protein, RpoN transcription factor and c-di-GMP may also help regulate T6SS. Under the regulation of these multiple factors, the expression of T6SS can be on and off so as to better adapt to the environment. Hence, it is very important to understand comprehensively the regulatory factors of T6SS to fully understand the pathogenicity of bacteria. This review summarizes the environmental factors and regulatory factors that regulate T6SS.

Abstract:The microbial immobilization technology is widely used in food processing, light chemical engineering and environmental protection for its high microbial density, excellent bioactivity, strong environmental adaptability and reusability. In this paper, the microbial immobilization technology is reviewed. Then, the application progress is emphatically explained by analyzing practical cases in the treatment of polluted water, soil and atmosphere. The studies found that the carrier can improve system loading and efficiency of water treatment by providing the microenvironment and suitable niche to different microorganisms in the water environment. In the soil environment, it is important to improve the bioavailability of pollutants for improving the efficiency of treatment. In the air environment, the mechanical strength and mass transfer capacity of the carrier are required. Finally, the characteristics and advantages of the microbial immobilization technology are compared and summarized to guideline its future application in the treatments of polluted environment.

Abstract:Microbiology as one of the most important subjects of the modern biology is the core curriculum for Biology Science, Food Science, Marine Science, Medical Science and related disciplines. A sound understanding of microbiology is essential for students who are engaged in research or industrial work in the above disciplines. The challenges associated with microbiology learning are the student’s incomplete knowledge network and the lack of initiatives and creativities. We have introduced teaching reform and practice into the teaching of microbiology for inspiring students’ pluralistic interests and individualized talents. By integrating microbiology curriculum with activities that promote student’s interests, such as comics, music, poetry and handcraft etc. to diversifying the course content in order to guide students developing personal interests and understanding on Microbiology. It has been proven that the implementation of the current teaching reformation has aroused the student’s interests and knowledge on Microbiology.

Abstract:Mind maps have been used as a mind tool in various fields. This plan focused on variations from hand-painted to software drawing; from designated topic to free-choice topics; from independent work, pair work to group work; thus gradually applied mind map to Microbiological courses teaching and learning. The observation and investigation methods were used for evaluation by teacher and students. Mind maps could be helpful in students’ understanding, and knowledge structure construction; thinking process and communication. Finally, we paid attention to the value of mind maps in connecting biological branches. Mind map is a tool for graphical thinking, teamwork, and knowledge management that is worth practicing.

Abstract:[Background] Lactobacillus plantarum has a lot of natural plasmids. Analyzing the sequence characteristics of these plasmids was beneficial to research the genetic information of them. [Objective] To identify a new plasmid isolated from L. plantarum PC518 and analyze conservation and diversity of plasmids belonging to a family. [Methods] Plasmid DNA was isolated from L. plantarum PC518, and two plasmid DNA libraries were constructed after enzyme digestion. New sequences in the libraries were identified by sequencing and BLAST alignment. The whole sequence of the plasmid was completed by reverse PCR. the new plasmid was annotated. The phylogenetic tree of Rep protein was built by MEGA X, and analysis of binding sequence was carried out. [Results] Plasmid pLP224 was a 1 766 bp circular molecule with a (G+C)mol% content of 41.39%. The maximum sequence similarity with known plasmids was 86.85%. pLP224 belonged to the pMV158 family of rolling-circle replication. The results of phylogenetic tree based on Rep protein showed that the closer evolution distance was the higher similarity of binding sequence, and the farther evolution distance was the lower similarity of binding sequence. [Conclusion] pLP224 was a new member of the pMV158 family. The nick sequences of dso site belonging to the pMV158 family were conservative, and the binding sequences belonging to the pMV158 family were diverse. The Rep protein varies with the binding sequence. The difference of Rep protein was conducive to the coexistence in a host, which was the basis for the continuous existence and stable evolution of a family.

Abstract:[Background] Lactobacillus is commonly found in fermented foods which associated with food quality and safety. Quantifying the real-time dynamic and tracking the composition of viable Lactobacillus is substantial to explore metabolic function in fermentation and even intestinal microorganism system. [Objective] To establish and employ a real-time and rapid approach to detect 5 kinds of viable Lactobacillus at species level by quantitative PCR combined with propidium monoazide (PMA) treatment, and then assess its applicability. [Methods] Using Lactobacillu plantarum, Lactobacillus fermentum, Lactobacillus brevis, Lactobacillus acidophilus and Lactobacillus casei which are the common Lactobacillus strains in fermented foods as the target, the specific primers for quantitative PCR (qPCR) analysis were searched and screened. We also optimized PMA conditions and ascertained the specificity, sensitivity and reliability of the method. Then 5 kinds of viable Lactobacillus during Chinese rice wine fermentation were quantified by PMA-qPCR. [Results] The optimal process included that 20 μmol/L PMA for 15 min incubation followed by 15 min photoactivation can eliminate 99.89% amplified signal of non-viable bacteria. This method exhibited good specificity to accurately identify 5 kinds of Lactobacillus, and possessed strong linear relationship (R2>0.98) and high sensitivity (limit of detection=101.8? 103.2 CFU/mL) and the variation coefficients of Cq values were less than 1%. Additionally, it had no statistically significant (p>0.05) difference compared with plate count. Furthermore, the PMA-qPCR method was applied during Chinese rice wine fermentation, indicating that Lactobacillus fermentum, Lactobacillus casei and Lactobacillus brevis were the dominant Lactobacillus (59%?89%), which was consistent with known Lactobacillus composition in Chinese rice wine brewing system. [Conclusion] The established PMA-qPCR method could quickly and accurately quantify 5 kinds of viable Lactobacillus, which provided a feasible approach on tracking the real-time composition of viable Lactobacillus and detecting viable but nonculturable Lactobacillus in samples.

Abstract:[Background] The relationship between gut microbiota and human health has attracted much attention and became a popular research area. [Objective] To explore the feature of gut microbiota of obese people based on the American Gut Project. To provide a theoretical basis for the intervention of obesity based on gut microbiota by constructing machine learning models to predict the status of people obesity. [Methods] Total of 1 665 normal samples (18.530) obese samples were downloaded from the website of the American Gut Project (AGP). The Wilcox rank-sum analysis was performed to explore the alteration of alpha-diversity between the obese and normal group. In addition, the logistic regression was performed to explore the correlation between alpha-diversity of gut microbiota and obese. For beta-analysis, we performed the principal component analysis (PCA) to explore the difference in the structure of gut microbiota between obese and normal groups. For the phylogenetic profiles, we performed the Wilcox rank-sum analysis to detect any significantly different taxa between the two groups. The PICRUSt analysis was used to predict the pathway based on the 16s rRNA gene sequences. Then, the Wilcox rank-sum analysis was used to detect the significantly different pathway between the two groups. To find the correlation between these significantly different pathways and genus, we performed the correlation analysis. Finally, we used the Scikit-Learn packages in python to construct the machine learning model and used the AUC value as the standard to justify the performance of each model. [Results] The decreasing trend of alpha-diversity in the obese population compared to the healthy population was observed after the Wilcox rank-sum analysis. In addition, the correlation between the alpha-diversity and the statues of obese was confirmed using the logistics regression. As for the beta-diversity, we did not observe the significant difference of the structure of gut microbiota after PCA based on three beta-diversity distance matrix including Weighted Unifrac, Unweighted Unifrac and Bray-Curtis. For the phylum, the high relative abundance of Bacteroidetes and the low relative abundance of Firmicutes was observed in the obese group. Besides, a total of 57 genera was significantly different between the two groups after the Wilcox rank-sum analysis. The genus of Ruminococcus increased in the obese groups, but the genus of Prevotella, Akkermansia and Methanobacteriales decreased in the obese group. All the pathway which predicted by the PICRUSt analysis were performed the Wilcox-rank-sum analysis between two groups and a total of 63 significantly different pathways was observed. The gradient boosted regression tree (GBDT) had the best performance with the AUC value (0.769) and test precise (0.725) among other models. [Conclusion] This study revealed the feature of gut microbiota of obese population based on a large-scale data sets. Besides, this study also constructed the machine learning models based on gut microbiota to predict the status of obese, which provide the new idea and theory basis of personalized medicine and diet.

Abstract:[Background] The colonization of plant growth-promoting rhizobacteria (PGPR) in rhizosphere is the basis of its function. Direct and effective tracking techniques and quantitative methods are important tools to study the distribution of PGPR in rhizosphere. [Objective] To establish a real-time fluorescence quantitative PCR system for rapid detection of antagonistic bacteria QHZ11 against the pathogen, Rhizoctonia solani and to detect the dynamic changes of antagonistic bacterium QHZ11 in potato rhizosphere. [Methods] The specific primers were screened according to the genetic sequence difference between the Paenibacillus and proximal strain gyrB in GenBank. The reaction conditions of fluorescence quantitative PCR were optimized. A soil pot experiment of potato was arranged to detect the number of antagonistic bacteria QHZ11 in potato rhizosphere, with three treatments: T1: CK (sterile water was irrigated); T2: QHZ11 bacterial suspension was irrigated (QHZ11); T3: the biological organic fertilizer, secondary solid fermented with antagonistic bacteria QHZ11 was applied (BOF11). [Results] A pair of special primers gyrB-F/gyrB-R for antagonistic QHZ11 were screened. The established of antagonistic bacteria QHZ11 real-time fluorescence quantitative PCR method could detect the antagonistic bacteria of 1×103?1×1010 copies/g-soil, with such advantages as very good specificity, high sensitivity and better reproducibility, and the linear correlation coefficient was 0.999 8, the coefficient of variation within the detection group was less than 1%, the amplification efficiency was 0.9, characterized by a lower detection limit and higher amplification efficiency. The results of pot experiment showed that the number of antagonistic bacteria QHZ11 in potato rhizosphere of T3 was one order of magnitude higher than that of T2 treatment from the 10th day after inoculation, and reached the peak at the 60th day after inoculation, indicating that the survival rate and reproduction rate of antagonistic bacteria in rhizosphere soil were increased by secondary solid fermentation. [Conclusion] The established real-time fluorescence quantitative PCR system is sensitive and efficient, which provides a convenient and effective method for understanding the distribution of antagonistic bacteria in potato rhizosphere and the interaction between antagonistic bacteria and pathogens.

Abstract:[Background] Common biological agents such as emulsifiable concentrate (EC), wettablepowders (WP), dustpowder (DP) and other biological agents contain benzene and derivatives as organic solvents and dust, which could cause pollution to the environment. Water dispersible granule is considered as one of the most promising dosage forms owing to its environmental friendliness, high added value, and large market potential. However, there are few studies on the compound Bacillus sp. water dispersible granules. [Objective] To prepare a compound water dispersible granule using Bacillus amyloliquefaciens BZ6-1 and Bacillus pumilus SC-12, and evaluate its antimicrobial effects on suppression against bacterial wilt in pepper. [Methods] The effects of different carriers and auxiliaries on the spores of two kinds of Bacillus were investigated in a biocompatibility experiment to select optimal carriers and additives. The effects of different sieve diameter, drying temperature, drying time on the quality of water dispersible granules investigated in a quality inspection experiment to optimize the granulation conditions. Field pepper experiment was conducted by spraying different dosage after planting to field. [Results] The best formula included 4% sodium dodecyl sulfate (wetting agent), 6% sodium carboxymethyl cellulose (dispersant), 4% ammonium sulfate (disintegrating agent), 4% polyethylene glycol (binder), and 82% diatomite (carrier). The optimum conditions for granulation were mesh size 0.8 mm, drying temperature of 40 °C, drying time of 45 min. The optimal water dispersible granule, which meets the national standards for wetting powder, was developed with bacteria content of 2.52×108 cfu/g, suspension rate of 79.3%, pH 6.8, water content of 4.5%, wetting time of 19.6 s, disintegration time of 86.4 s, and moderate particle strength. [Conclusion] Compound Bacillus sp. water dispersible granules effectively controlled pepper bacterial wilt and improved the yield and quality of pepper. The most suitable application rates of as-prepared dispersible granules were recommended to be 3.0 kg/hm2.

Abstract:[Background] Lincomycin is a lincomamide antibiotic that plays an important role in clinical application, few studies can be found on regulating the parameters of third-stage seed fermenter to optimize the lincomycin fermentation process. [Objective] The fermentation process of lincomycin was optimized in order to improve the fermentation titer and market competitiveness. [Methods] The medium and inoculation amount of the third-stage seed fermenter and the transplanting age of third-stage seeds in lincomycin production were optimized. [Results] The concentrations of glucose, starch, corn pulp, soybean meal and ammonium sulfate in the third-stage seed fermenter’s medium were 64.0, 5.0, 15.0, 14.5 and 3.5 g/L, respectively. The inoculation amount of third-stage seed fermenter and the transplanting age of third-stage seeds were 25% and 60 h, respectively. Under the optimized conditions, the fourth-stage fermenter’s titer of lincomycin was up to 7 883 U/mL, which was 10% higher than that of the before optimization. [Conclusion] Regulating the parameters of third-stage seed fermenter in lincomycin production, the fermentation process was preliminary optimized and the fermentation titer was improved. It provides a new idea for optimization of lincomycin fermentation process.