Abstract:[Background] Saccharomyces cerevisiae is widely used to produce industrial enzymes and pharmaceutical proteins. However, low protein production level and poor secretion efficiency are the major bottlenecks for its industrial applications. [Objective] To improve protein production by the recombinant yeast strains, and provide basis for development of robust yeast cell factory for heterologous protein secretion. [Methods] The cell wall protein encoding gene CWP2 was disrupted through CRISPR/cas9-based genome editing in the recombinant strain S. cerevisiae Y294-BGL, which can secret β-glucosidase. [Results] At 96 h of fermentation, the extracellular activity of β-glucosidase in the mutant Bcwp2△was improved by 53%, and intracellular activity was improved by 208%. No negative effect on cell growth was observed, and no significant change was observed in the tolerance to acetic acid and ethanol in the mutant yeast strain Bcwp2△. In addition, no difference in growth of the mutant in the presence of the endoplasmic reticulum stress inducers dithiothreitol and tunicamycin was observed. Further studies showed that less reactive oxygen species was accumulated inside the cells of the mutant. Disruption of CWP2 decreased transcription of genes associated with protein trafficking and secretion, as well as cell wall biosynthesis genes. [Conclusion] Disruption of the cell wall protein encoding gene CWP2 promotes extracellular β-glucosidase activity, and can be employed as a target in metabolic engineering of yeast protein production.

Abstract:[Background] By implementing multiple cycles of microbial oil recovery, the reservoir produced liquid bacteria concentration reached more than 106 CFU/mL, forming a stable microbial fermentation field in the reservoir. To further improve the effect of microbial oil recovery, the crude oil-emulsifying and hydrocarbon-degrading bacteria from the reservoir was selected to cultivate for microbial oil recovery application in the reservoir. [Objective] The strains with good performance of crude oil-emulsifying and hydrocarbon-degrading capacity are screened out to carry out multiple taxonomic identification and performance evaluation. [Methods] Crude oil was chosen as substrate for strains selection. Through morphological observation, biochemical and physiological characteristics and 16S rRNA gene sequence, the taxonomic position of the strain BLG74 was identified. The crude oil-emulsifying and hydrocarbon-degrading capacity of the strain BLG74 were investigated by emulsifying ability and degrading rate. [Results] strain BLG74 was selected from the production water sample of Huabei oil field. 16S rRNA gene sequence analysis showed that the strain belonged to the genus Compostibacillus and most closely related to Compostibacillus humi with identity of 99.6%. Its growth environment has a pH of 6.5 to 9.5 (optimum at pH 7.0), a temperature of 30 °C to 60 °C (optimum at 45 °C) and a salinity of 0% to 7%. Strain BLG74 was cultured in crude oil degradation medium. The surface tension of the fermentation broth was 56.3 mN/m, and the emulsifying capacity was about 95%. It was cultured for 20 days under the initial crude oil mass concentration of 0.5% and temperature of 45 °C, resulting in the degradation rate of up to 40.8%. [Conclusion] Strain BLG74 is a new member of bacteria capable of emulsifying and degrading crude oil, presenting a remarkable potential in microbial enhanced oil recovery with its crude oil-emulsifying and hydrocarbon-degrading capacity under hot salt conditions.

Abstract:[Background] The degradation of pyrethroid pesticides is important for food safety and environmental health, and biodegradation is considered to be a green effective solution. [Objective] The strain with high ability to degrade deltamethrin (DM) was isolated from strawberry rhizosphere contaminated soil by pyrethroid pesticides for a long term, and the degradation rate of DM degrading strain was improved by optimizing the medium and degradation conditions. [Methods] DM degrading strain was screened by enrichment domestication, isolation, purification and identified by morphological, physio-biochemical, 16S rRNA sequence analysis. The degradation conditions were optimized by Plackett-Burman design, steepest ascent path design and Box-Behnken design. [Results] Strain LH-1-1 was identified as Acinetobacter junii, it could degrade 53.43% DM (100 mg/L) within 96 h in initial conditions. The optimized conditions were DM concentration 75 mg/L, tryptone 3 g/L, pH 6.8, (NH4)2SO4 1.5 g/L, FeCl3 0.01 g/L, inoculation biomass 5%, strain age 12 h, culture temperature 30 °C. The degradation rate of DM under these conditions reached 82.36% within 96 h, which was 28.93% higher than initial conditions. [Conclusion] A. junii LH-1-1, a highly efficient DM degrading strain, can be used as excellent microbial resources for bioremediation of environment polluted by DM or pyrethroid pesticides.

Abstract:[Background] Cordyceps sensu lato was an important entomopathogenic fungus. [Objective] The entomopathogenic fungi and its allies from Southwest China were investigated. [Methods] Specimens were collected from Xishui and Huaxi in Guizhou province, and the target strains were isolated on PDA media with antibiotics in the laboratory. Then these strains were identified by the morphological characters and phylogenetic analysis of ITS rDNA sequences. [Results] Five target strains were obtained. Strains GY1113, GY90809 and GY90810 were very similar to the original description of Beauveria malawiensis in morphology and clustered into a subclade with the sequences of B. malawiensis in phylogenetic tree; strain A1997 was consistent with Cordyceps lepidopterorum in morphology and it was closely related with the type sequence of C. lepidopterorum in phylogenetic tree; strain A1972 was very similar to the original description of B. caledonica in the morphology and clustered into a subclade with the sequences of B. caledonica in phylogenetic tree. Therefore, these strains were identified to three species, B. malawiensis, C. lepidopterorum and B. caledonica, respectively. [Conclusion] B. malawiensis and C. lepidopterorum are new to China.

Abstract:[Background] In November 2013, verticillium wilt of melon was observed in greenhouse in Gaolan county, Lanzhou city, Gansu province and its incidence was about 1%. [Objective] The present study was to identify the pathogen of the melon disease. [Methods] Pathogen was isolated by diseased tissue isolation method. Koch’s procedures were used to verify the disease pathogen; morphological and molecular biological methods were used to identify the pathogen. [Results] Eight fungal isolates of Verticillium were isolated from diseased plant samples; the isolation rate was 100% in diseased plants. Under test conditions (temperature: 18?24 °C; photoperoid: day/night=11.5 h/12.5 h), artificial inoculation with two representative isolates GLGT-2 and GLGT-5 with similar in microscopic features, but different in colony morphology and growth rate, caused dwarfing and wilting on melon seedlings; the incidences of wilting plant were 70% and 40% respectively after 40 days inoculation; BLASTn analysis showed that the rDNA-ITS sequences of GLGT-2 had a 99.78% similarity with V. dahliae isolate MRHf7, and GLGT-5 had a 100.00% similarity with V. dahliae isolate MRHf7 and Vd414. [Conclusion] The isolates causing verticillium wilt on melon were both identified as V. dahliae by morphological and molecular biological identification. This is the first report of verticillium wilt of melon caused by V. dahliae in China and Asia.

Abstract:[Background] Some nitrogen-fixing bacteria can secrete exopolysaccharide, which was closely related to the host plants, and plays an important role in providing nitrogen and promoting the growth of plants. [Objective] To analyze the gene sequence of nitrogen-fixing bacteria Klebsiella variicola GN02, and the structural characteristics of the genes and proteins relating to the secretion of extracellular polysaccharides were investigated. [Methods] The whole genome of GN02 strain was analyzed by the combination of the second and third-generation sequencing techniques, and the physicochemical and structural characteristics of its secreted extracellular polysaccharides were also analyzed. [Results] The genome of GN02 strain contains many genes and proteins relating to nitrogen metabolism and polysaccharide synthesis and secretion. The yield of exopolysaccharide extracted by GN02 strain was 6.90 g/L, molecular weight was 1.8×103 Da, specific rotation [α] 25D 75° and the viscosity [η] was 80.28. The polysaccharides consist of glucose, galactose and mannose, which were β-configuration polysaccharides linked by (1→3) and (1→6) glycoside bonds, and had the characteristic absorption peak in infrared spectrum. [Conclusion] The genomic sequence of K. variicola GN02 strain was provided, and the gene-relating proteins, physicochemical properties and structural properties of extracellular polysaccharides were analyzed, which helped to further understand the mechanism of exopolysaccharides secreted by nitrogen-fixing bacteria and to provide the basis for the correlation research of plant growth promoting effect.

Abstract:[Background] Arbuscular mycorrhizal fungus (AMF) is the most widely distributed fungi in mycorrhizal fungi. They can form mycorrhizal symbiosis with more than 90% of the plants, and enhance the resistance of plants by regulating metabolic activities in the host. [Objective] To reveal the structure and composition of AMF in soil of main potato producing areas in Inner Mongolia, and analyze the effects of different development stages of potato and continuous cropping on AMF groups. [Methods] Using root and rhizosphere soils of potato collected from Dajing, Xumayao and Honggeertu villages in central of Inner Mongolia as material, though PCR amplification and establishing 18S rRNA gene library, try to study the effect on AMF composition and diversity in potato rhizosphere soils and root at different locations, different growth stage and continuous croping plot. [Results] AMF diversity of rhizosphere soil in Dajing village and Honggeertu village was more than that in Xumayao village, the dominant strain was Glomus in both Dajing village and Honggeertu village, while Diversispora was the dominant strain in Xumakuo village. In roots, the results of AMF diversity showed that there was no significant difference among three areas, but the proportion of AMF population was different. Diversispora was the dominant strain in Dajing village, Rhizophagus was the dominant strain in Honggeertu village and Xumayao village. The AMF diversity of potato rhizosphere soil in seedling stage and tuber swelling stage were more than that in tuber formation stage at the same area, Glomus was the dominant strain in the three stages, while in root the Rhizophagus was the dominant strain in the three stages. After 2015 and 2016 continuous cropping potato in Dajing village, the types of AMF populations in rhizosphere soil and root were identical, but the relative abundance of different AMF populations changed significantly. Among the rhizosphere soil samples, the relative abundance of Glomus and Archaeospora population increased with continuous cropping, and the relative abundance of Entrophospora and Diversispora decreased. But the dominant population of AMF in root samples changed from Glomus to Rhizophagus with continuous cropping. This result indicated that the effect of continuous cropping on AMF diversity in rhizosphere soil was significantly higher than that in root. [Conclusion] The diversity of AMF population varies with soil composition, potato growth period and cropping effect.

Abstract:[Background] p-cresol is an off-odor in a variety of fermented foods. With low threshold, ppm’s p-cresol can adversely affect the flavor of Chinese liquor. [Objective] Recombinant lactic acid bacterium with p-cresol reduction capability was constructed and tested in Chinese liquor fermentation. [Methods] The genes (creI and creH) coding 4-methylbenzyl phosphate synthase were cloned from Corynebacterium glutamicum and ectopically expressed in Lactobacillus brevis. The reduction capability of creIH overexpression strain was determined in Chinese liquor fermentation. [Results] The creIH overexpression strain eliminated p-cresol up to 2 130 μg/L in liquid medium and 530 μg/kg in simulated solid-state fermented grains. The reduction rate in simulated solid-state fermentation reached 37.9%. [Conclusion] This is the first report that lactic acid bacterium with p-cresol reducing capability was constructed. It provides a novel strategy to eliminate p-cresol in Chinese liquor fermentation.

Abstract:[Background] Pickled pear is a popular pickled fruit in Yunnan Province with delicious taste and unique flavor. It has been eating in Yunnan for more than 100 years. However, the systematic analysis of the microbial population in this product and the study of the fermentation mechanism are still lack. [Objective] The purpose of this study was to analyze the distribution of lactic acid bacteria in pickled pears, and to elucidate the effects of lactic acid bacteria on flavor substances in pear fermentation. [Methods] Lactic acid bacterial strains were isolated from twelve samples of pickled pears collected from four different regions of Yunnan Province. Bacterial strains were identified by colony morphology, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The isolated lactic acid bacteria were used in pickled pears preparation and the resulting products were evaluated by sensory evaluation and flavor substances analyses by GC-MS. [Results] Altogether, 79 strains of Lactobacillus plantarum, 3 strains of Lactobacillus paraplantarum, 1 strain of Lactobacillus pentosus, 1 strain of Lactobacillus casei, 2 strains of Lactobacillus paracasei and 1 strain of Lactobacillus brevis were isolated from pickled pear samples, with Lactobacillus plantarum being the dominant strain in pickled pears. Using isolated strains in the pickled pear fermentation, the product got better flavor and shorter process time than natural fermentation about 5 days. The composition of flavor substances analyses showed that in lactic acid bacteria fermented pears, ester and alcohol were much more abundant than those in natural fermented pears. [Conclusion] Lactic acid bacteria are abundant in Yunnan pickled pear. Using these bacteria in pickled pear fermentation could produce picked pear with better color and taste, more abundant flavor substances, in shorter time. This study confers great significance for the improvement and standardized preparation of Yunnan pickled pears producing.

Abstract:[Background] Neurospora crassa LY03 was the main fermentation strain isolated from traditional “Red fungus tofu” in Wuping. [Objective] The genomic information of Neurospora crassa LY03 strain was studied to reveal the fermentation characteristics of traditional “Red fungus tofu” in Wuping. [Methods] Morphological observation, ITS identification, resequencing and frame diagram sequencing were used to identify the isolated LY03 strains and analyzed the genome information. [Results] The main fermentation strain LY03 isolated from traditional “Red fungus tofu” in Wuping was identified as Neurospora crassa, and preserved in the China General Microbiological Culture Collection Center with preservation number CGMCC 3.1923. The total number of reads in LY03 strain compared to the reference genome was 95.85%, and the site of sequencing corresponding depth accounted for 76.13% of the whole genome. There was no variation in the intronic region of each variant type, and the main variation existed in the exon region of the genome, and the specific number of variation was as follows: The total number of SNP site variants was 203 128, InDel mutations was 26 859, copies increased and decreased by CNV was 1 039, variation in SV comments was 777. The genome sequence length of LY03 strain was 30 538 737 bp, (G+C)mol% was 52.24%, encoding 5 550 genes, accounting for 24.3% of the coding genes, which was involved in the metabolic pathway transformation of amino acid metabolism, carbohydrate metabolism, energy production and conversion and other substances. [Conclusion] The identification and genome information analysis of the main fermentation strains of traditional “Red fungus tofu” in Wuping are helpful to reveal the fermentation characteristics of the products and the essence of the genetic information of the fermentation strains, and to provide a theoretical basis for the improvement of the fermentation performance and the popularization of the products in the future.

Abstract:[Background] Bovine tuberculosis is a second-class animal disease in China, and it was listed as a legally reported animal disease by Word Organisation for Animal Health (OIE). Cattle are mainly infected by aerosols produced by respiratory secretions and coughs in Mycobacterium bovis-infected cattle; people are mainly infected with meat or milk from diseased cattle that have not been treated with high temperature. Therefore, the rapid detection of suspected diseased milk or slaughter tissue samples by pathogen PCR detection can minimize the economic loss of dairy farming industry, which is of great significance. [Objective] To study and determine the suitable PCR amplification primers and parameters of Mycobacterium bovis, providing reference for rapid and accurate diagnosis of Bovine tuberculosis. [Methods] For the five pairs of PCR primers reported, the appropriate annealing temperature (Tm) was determined by touch down PCR; to determine the sensitivity of PCR methods with different primers, we used the genomic DNA of the Mycobacterium bovis C68001 strain (the domestic strain for bovine tuberculin production) and the artificial liquid with different bacterial contents in simulate clinical samples (lymph nodes, lungs, and milk); and then 6 common bacteria infecting bovines (B. abortus 2308, B. melitensis Rev.1, M. bovis C68001 and AN5, M. avium C68202, M. paratuberculosis C68681 and M. intracellulare) were uesed to determine the specificity. [Results] All primers contained the target band at 53?63 °C, and the best suitable Tm was 60 °C. The primers No. 1 and No. 3 had the highest sensitivity detecting nucleic acid of C68001, reaching 10?10 ng/μL; followed by No. 2 and No.5, up to 10?5 ng/μL. For artificially simulated infection samples, primers No. 1, 3, and 4 were most sensitive to detection in lymph nodes and lungs, followed by No. 2; and primers No. 2, No. 3, No. 4, and No. 5 were the most sensitive to milk samples. For specificity tests, primers 2 and 5 have better specificity which can detect significant M. bovis specific bands. It is weak when detecting M. avium that do not normally cause bovine tuberculosis but interfere with immunological diagnosis, and it had no bands in brucella, M. bovis, M. paratuberculosis and M. intracellulare. [Conclusion] The PCR method with primer No. 2 had the best sensitivity and specificty, so it is suitable for rapid and accurate diagnosis of bovine tuberculosis.

Abstract:[Background] Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic pathogen that causes a variety of food-borne diseases. The biological function of ybiH gene in S. typhimurium has not been determined. [Objective] In order to define the pathogenicity of ybiH gene to S. typhimurium，the ybiH gene mutant and complementary strain of S. typhimurium were constructed and characterized. [Methods] In this paper, the mutant STΔybiH of S. typhimurium CVCC541 standard strain was successful constructed by λ-Red homologous recombination system. At the same time, its complementary strain STΔybiH/pybiH was successfully constructed. Then we analyzed the biological characteristics of growth characteristics, motility, biochemical properties and virulence. [Results] The results showed that the growth rate of STΔybiH was slightly faster than that of the standard strain and the complementary strain, but there was no significant difference in motility and biochemical characteristics. However, the deletion of ybiH gene significantly increased the adhesion and invasion of S. typhimurium to IEC-6 cells and RAW 264.7 cells. The results of qRT-PCR showed that the expression of InvH gene was significantly increased in the deletion strain. The results showed that the deletion of ybiH gene increased the invasiveness of S. typhimurium. In addition, the intracellular growth rate of the standard strain and the deletion strain did not change significantly in the intracellular survival experiment, indicating that the deletion of the ybiH gene had little effect on the survival of Salmonella in RAW 264.7 cells. [Conclusion] The ybiH gene mediates the adhesion and invasiveness of S. typhimurium, which provides a theoretical basis for further elucidating the function of ybiH gene.

Abstract:[Background] Studies have shown that the NADH oxidase (NOX) of Mycoplasma not only functions as an enzyme in cytoplasm, but also exists on the cell membrane as an adhesin. [Objective] To study the enzymatic activity and subcellular localization of the NOX of Mycoplasma synoviae (MS), analyze its potential role in the MS pathogenesis. [Methods] The recombinant MS NOX (rMSNOX) protein was prokaryotic expressed and purified. Then the enzymatic activity of purified rMSNOX and the factors affecting enzymatic activity were studied. In addition, the enzymatic specific activity, the maximum reaction rate (Vmax) and the Michaelis constant (Km) of the rMSNOX were determined. The MS positive chicken serum was used for Western blotting analysis of the immunogenicity of the rMSNOX. The Mycoplasma whole-cell, membrane and cytoplasmic fractions were prepared for Western blotting analysis to determine the subcellular localization of MSNOX using the rabbit anti-rMSNOX serum. [Results] The rMSNOX protein was successfully expressed in E. coli BL21(DE3) and purified. The relative molecular mass was about 53 kD. The enzymatic specific activity of the rMSNOX protein was determined as 14.17 IU/mg, the optimum enzymatic temperature was 37 °C, and the optimum pH was 7.5 by an enzyme activity analysis. The Km (NADH) of rMSNOX was determined as 244.0 μmol/L, and the Vmax as 21.8 μmol/(L·min) using the double-reciprocal method. The rMSNOX presented a specific binding to MS positive chicken serum, which proved that it had good immunogenicity. Subcellular localization analysis indicates that NOX protein distributed in MS cytoplasm and cell membrane. [Conclusion] This study demonstrates for the first time that MSNOX not only possesses NADH oxidase activity, but also is an immunogenic membrane protein, which provides a molecular basis for further exploring the role of NOX in the MS pathogenesis.

Abstract:[Background] Biofilm is a form of bacterial self-protection, which can enhance the resistance of bacteria to antibiotics and host immune response, and causes bacterial resistance and persistent infection. [Objective] To provide a reliable theoretical basis for the development of new natural antibacterial drugs, the inhibition mechanism of carvacrol on the biofilm formation of Staphylococcus aureus was explored. [Methods] Crystal violet staining was used to detect the inhibitory effect of carvacrol on biofilm formation and the clear effect on mature biofilm of tested strains. We used congo red agar to detect the influence of carvacrol on polysaccharide intercellular adhesion (PIA) formation, and used spectrophotometer to detect the influence of carvacrol on extracellular DNA (eDNA) release. RT-PCR analysis was used to determine the effect of carvacrol on the transcription levels of icaA, cidA and sarA. [Results] Carvacrol has strong effects on the inhibition of biofilm formation and the removal of mature biofilm. The inhibition of PIA synthesis and eDNA release by carvacrol at 256 μg/mL was significant. Carvacrol can inhibit the formation of biofilm by inhibiting the transcription of related genes. When carvacrol was 64 μg/mL, the transcription level of sarA decreased by 60.44%±2.91%, cidA by 76.48%±1.67%, and icaA decreased by 70.00%±1.94%. [Conclusion] Carvacrol had significant inhibitory and removal effects on the biofilm of Staphylococcus aureus 25923. Its mechanism is to inhibit the synthesis of PIA and the release of eDNA by reducing the transcription levels of icaA, sarA and cidA genes.

Abstract:[Background] Chlorella sp. contains 20 amino acids needed for animal growth and rich in protein, polysaccharides, unsaturated fatty acids, carotenoids, astaxanthin and various vitamins. It can be used as high-quality natural bait for fish, shrimp and shellfish. [Objective] Chlorella TX was isolated from cultured environment. The method of mutagenesis was used to breed high biomass and protein content mutants in order to obtain excellent algae resources for aquaculture natural bait production. [Methods] Chlorella TX, a relatively fast-growing and high protein content algae, was screened from the aquaculture environment as the starting strain. 18S rRNA gene sequence analysis was employed to identify strain Chlorella TX, and then ultraviolet mutagenesis, ethyl methane sulfonate (EMS), compound mutagenesis, 96-well plate high-throughput screening and progressive repeated screening were used to breed high biomass and protein content mutants. [Results] TX was identified as Chlorella sorokiniana by 18S rRNA gene sequence analysis. Eight mutants with genetic stability and growing fast were screened out from 540 mutants after progressive repeated screening. The total protein, soluble protein content and dry weight content of mutant H10 were 64.2%, 0.44 g/L and 0.72 g/L respectively, which were 3.4%, 15.8% and 26.2% higher than original algae strain TX. [Conclusion] The advantageous characteristics of H10 with high protein and growing fast can be used for natural bait production.

Abstract:[Background] Shenchi was obtained through co-fermentation with the soybean (Glycine max (L.) Merr.), Ginseng (Panax Ginseng C.A.Mey.), and the fermentation probiotics (Bacillus subtilis subsp. subtilis), that was obtianed from the Sojae Semen Praepartaum in our laborary. It has obvious regulation effect on blood index of rats with qi deficiency and blood stasis, and the effect on intestinal micro-ecology is still unclear. [Objective] In order to investigate the effection, that the intestinal micro-ecology for Shenchi on of rats with qi deficiency and blood stasis. [Methods] The rat model of qi deficiency and blood stasis was constructed by the method, that was “exhaustive swimming+restricted diet” with a long-term. Animal models were treated with the high, medium and low doses of Shenchi and Buyang Huanwu Decoction by intragastric administration for 60 days. After 60 days’ treatment, analyzed change in the number of six resident bacteria; and the carbon sources metabolism was determined by Biolog-ECO microbial identification system. [Results] The results showed that the Shenchi could promote the proliferation of beneficial bacteria including Bacteroides fragilis, Lactobacillus and Bifidobacterium in the intestinal flora of rats with qi deficiency and blood stasis, the shenchi could regulate the number of Enterobacter and Enterococci to normal levels, and inhibit proliferation of Clostridium perfringens. Biolog results showed the AWCD value of the high dose group, which was similar to the normal group. At 48 h cultivation period, the value of Shannon index, Shannon evenness, Simpson index and Mclntosh index were significantly higher in the model group than in the normal group, (P<0.05 or P<0.01). The Shannon index, Shannon evenness, and Simpson index of the high-dose group were similar to the normal group (P>0.05), and the values were significantly difference compared with the model group (P<0.01). Cluster analysis and principal component analysis showed that different dose groups of Shenchi were distinct from the normal group, model group and Buyang Huanwu Decoction group, which may be related to the regulation of intestinal microbes in rats with qi deficiency and blood stasis. [Conclusion] Shenchi had a significant improvement on the intestinal flora of rats with qi deficiency and blood stasis.

Abstract:[Background] AMP-17 is a specific and highly expressed gene screened from the database of housefly transcriptional group induced by microorganisms. Its recombinant AMP-17 protein was obtained by utilizing the prokaryotic expression system and proved to have significant antimicrobial effect, especially for Candida albicans. [Objective] To investigate the inhibitory effect of AMP-17 on the hyphae of Candida albicans. [Methods] The minimum inhibitory concentration (MIC) of AMP-17 on 11 strains of Candida albicans were determined by microdilution method; Three strains of Candida albicans were selected to plot the growth curve according to their sensitivity to AMP-17; The blastospore production rate and germ tube formation rate of Candida albicans after AMP-17 were observed and counted by optical microscope; The transformation of Candida albicans yeast phase to mycelial phase and AMP-17 promoted the transformation of mycelial phase into yeast phase with mycelial phase as the starting point were observed by fluorescent inverted microscope. [Results] The MIC of AMP-17 to the strain isolated from BALF 16105 was 10 μg/mL, the MIC to the strain isolated from fecal 16214 was 40 μg/mL, and the MIC to the other 9 strains of Candida albicans were 20 μg/mL. After the action of AMP-17 at different concentrations, the rate of blastospore formation at each time point was significantly lower than that of the control group, especially in the AMP-17 group of 40 μg/mL, the rate of blastospore formation was only 15%, obviously lower than that of fluconazole, a positive drug. The rate of germ tube formation in each experimental group were obviously lower than that in the control group, and the germ tube formation was slow. After 6 h of culture, the germ tube formation rate was only 6%, and the germ tube was short, which was only 1?2 times of thallus. Low concentration of AMP-17 could completely inhibit the growth of Candida albicans hyphae, and inhibit the growth of already formed hyphae to some extent, while high concentration of AMP-17 could transform some formed hyphae into yeast phase by microscopes. [Conclusion] Housefly antimicrobial peptide AMP-17 can inhibit the growth of Candida albicans hyphae.

Abstract:[Background] Acinetobacter baumannii is an important pathogen of nosocomial infection, which has attracted much attention due to its high drug resistance rate and difficulty in treatment. However, the cross-resistance and the factors related to drug resistance of this bacterium have not been fully elucidated. [Objective] In order to study the cross-resistance and the differences of respiratory oxygen consumption between Acinetobacter baumannii resistant to meropenem or tigecycline and Acinetobacter baumannii sensitive to them, Acinetobacter baumannii was intermediated in vitro with meropenem or tigecycline. [Methods] Meropenem and tigecycline susceptible Acinetobacter baumannii ATCC19606 was induced resistance in vitro, and sequenced PCR products of 16S rRNA of strains before and after drug induction. The minimal inhibitory concentrations (MIC) of meropenem, imipenem, tigecycline, amikacin, cefepime and levofloxacin were determined by a broth microdilution method. Bacterial respiration before and after drug induction, expressed as oxygen consumption rate, was quantified using Seahorse XFe96 extracellular flux analyzer. [Results] Through a 88 d in vitro induction experiment, strain of Acinetobacter baumannii ATCC19606 resistant to meropenem or tigecycline was obtained, respectively. Meropenem-resistant Acinetobacter baumannii ATCC19606 was still sensitive to tigecycline, imipenem, amikacin and levofloxacin, but was cross-resistant to cefepime. Tigecycline-resistant Acinetobacter baumannii ATCC19606 was still sensitive to all antimicrobial agents tested. After Acinetobacter baumannii ATCC19606 was induced by meropenem or tigecycline respectively, the oxygen consumption rate decreased, and the difference was statistically significant. [Conclusion] The use of meropenem may not only induce resistance to meropenem in Acinetobacter baumannii ATCC19606, but also may cause cross-resistance of the bacterium to one or more other antimicrobial agents. The oxygen consumption rate of Acinetobacter baumannii ATCC19606 decreased after its resistance to meropenem or tigecycline, suggesting that the decrease in oxygen consumption rate may be one of the factors contributing to the resistance of this bacterium.

Abstract:[Background] With the widespread use of medical implants, hospital-acquired infections caused by Staphylococcus epidermidis biofilm are increasing. For now, there are few reports about the effect of surfactant on the biofilm of Staphylococcus epidermidis. [Objective] To demonstrate the effect of anionic surfactant sodium dodecyl benzene sulfonate, including scavenging ATCC 35984 biofilm, bacterial metabolism and formation of polysaccharide intercellular adhesion (PIA). To provide reliable theory and practical evidence for SDBS, and prevent related infections caused by Staphylococcus epidermidis biofilm in clinic. [Methods] XTT reduction method was used to explore the effect of SDBS on the clearance of ATCC 35984 biofilm and bacterial metabolism in the biofilm. Laser scanning confocal microscope (LSCM) and Congo red medium was respectively used to observe the effect of SDBS on biofilm and PIA. [Results] SDBS on the concentration of 256 mg/L, 128 mg/L, 64 mg/L, 32 mg/L, 16 mg/L has significantly clearance on ATCC 35984 in the 6 h, 12 h, 24 h, respectively (P<0.01). It is significantly inhibition effect of SDBS with 32 mg/L on bacterial metabolism in the biofilm (P<0.05). 256 mg/L, 128 mg/L, 64 mg/L SDBS has satisfied clearance. 64 mg/L and 32 mg/L SDBS has no inhibition effect on PIA formation under the LSCM observation. [Conclusion] SDBS has significant inhibition effect on bacterial metabolism in the biofilm of Staphylococus epidermidis and damage the morphological structure of biofilm.

Abstract:[Background] The antimicrobial peptide merecidin can inhibit the clinical strain Pseudomonas aeruginosa PA03 biofilm. The PA4781 gene is a differentially expressed gene selected by bioinformatics analysis. As a phosphodiesterase, PA4781 has function to degrade c-di-GMP, which is a bacterial second messenger molecule. PA4781 plays a role in inhibiting biofilm in the antimicrobial peptide merecidin, while the mechanism of action is still unclear. [Objective] To study the role of the phosphodiesterase PA4781 gene, which degrades the bacterial second messenger molecule c-di-GMP, in the inhibition of Pseudomonas aeruginosa biofilm by the antimicrobial peptide merecidin. [Methods] The PA4781 gene was knocked out by approach of base editing and the sanger sequencing method was used to detect the correctness of knockout. Crystal violet staining was used to observe the growth of biofilm in PA03 strain, PA4781 overexpressing strain, PA4781 knockout strain for 24 hours, and the development of biofilm of each strain under the action of antimicrobial peptide mericidin 24, 48, 72 μmol/L. Dihydroxybiphenyl solution chromogenic method was used to detect alginate production under interference from antibacterial peptide mericidin 48, 72 μmol/L to the PA03 strain, PA4781 overexpressing strain and PA4781 knockout strain. Alginate is an exopolysaccharide polymer composed of mannituronic acid and guloruronic acid which produced by various bacteria. It is an important component of Pseudomonas aeruginosa biofilm. [Results] The results of sanger sequencing showed that the pnCasPA-BEC system successfully realized the single-base mutation at target position and terminated the transcription of PA4781 in advance. The results of crystal violet staining showed that under the treatment of 24 μmol/L antimicrobial peptide merecidin, there was no significant difference in the formation of biofilm between the three groups (p>0.05). Under the treatment of 48 μmol/L and 72 μmol/L antimicrobial peptide merecidin, there was a significant difference between the overexpression group with the normal group with the knockout group (P<0.05), the biofilm was significantly reduced, and the biofilm thickness of the knockout group was higher than that of the PA03 group (P<0.05). With the increase of the concentration of the antimicrobial peptide mericidin, the alginate content of each group decreased, and the overexpression strain had the highest inhibition rate of alginate production under the action of the antimicrobial peptide merecidin, which reached to 65%. [Conclusion] Antibacterial peptide merecidin can promote the expression of bacterial second messenger molecule c-di-GMP phosphodiesterase PA4781, which may provide a new research idea for the mechanism of antibacterial peptide merecidin inhibiting Pseudomonas aeruginomonas biofilm through the signaling pathway of bacterial second messenger molecule.

Abstract:Viable but non-culturable (VBNC) state is a dormant-like state of bacteria, and many species of bacteria can enter into this state when suffering stress conditions. VBNC bacterial cells can resuscitate to culturable state and recover their pathogenicity when conditions become favorable. Consequently, entering into VBNC state is considered as a survival strategy for bacteria to overcome the hostile conditions. Nowadays, VBNC bacteria pose a great potential threat to human health, food industry and agricultural production. Research on detection, induction, resuscitation and their mechanisms can provide theoretical basis for managing or reducing the risks of VBNC bacterial cells. Based on the research results of VBNC state of phytopathogenic bacteria in our lab and other research teams in recent years, we summarize all progresses of VBNC bacteria in this review, especially on the mechanisms of VBNC state formation and resuscitation. We hope that this paper will be of significant reference to the study on the survival mechanism of plant-pathogenic bacteria under stresses, the primary inoculum analysis for bacterial diseases in the field, as well as the role of VBNC cells in the disease cycle.

Abstract:Escherichia coli is a conditional pathogenic bacteria. Pathogenic E. coli is highly contagious and threatens human health. Rapid determination of the contaminant source of E. coli can help effectively control epidemic, to protect human health and reduce economy loss. Simple and efficient tracing techniques and typing methods are key to trace the source of pathengen. Common typing methods of E. coli include phenotypic typing and molecular typing. These typing methods have their own advantages and limitations, and have different application scopes. In this paper, phenotypic typing and molecular typing methods and the research progress of E. coli typing are reviewed. It will provide reference for the selection of traceability methods for pathogenic bacteria, and have the great significance for the prevention and control of epidemic spread caused by pathogenic bacteria.

Abstract:Microbial ecological agents are environmental friendly alternatives to antibiotics, which could be utilized as feed additives for preventing disease and promoting growth of animals. Bacillus subtilis could generate spores, which have strong resistance to environmental stress including heat in drying process, mechanical force in granulation process and acid in digestive tract. Therefore, Bacillus subtilis is one of the preferred strains for microbial ecological agents preparation. Sporulation rate and viable cell number determine quality of Bacillus subtilis microbial ecological agents significantly. Increasing sporulation rate is considered as one of the key methods to improve the quality of Bacillus subtilis microbial ecological agents. Molecular mechanism of sporulation process was presented and the factors regulating sporulation were discussed. Different fermentation methods for microbial ecological agents production were compared. Furthermore, a systematic description was given of the process optimization for enhancing effective cell number of Bacillus subtilis. At last, applications of Bacillus subtilis microbial ecological agents were presented and the future research directions were proposed.

Abstract:This review summarizes the biosynthesis pathway of lysine in fungi and the key gene in the pathway, saccharopine dehydrogenase. The de novo synthesis pathway of lysine was introduced in detail, and the mechanism and physicochemical properties of saccharopine dehydrogenase was described, which are aimed to provide information and ideas for exploring lysine biosynthesis pathway and genes in the pathway.

Abstract:In habitant niche the bacteriocinogeny facilitates the producer with survival advantages by two ways: 1) through elevating colonization capacity due to superficial hydrophobicity of bacteriocins aggregation, and 2) through inhibiting competitors by dysfunction of biofilm formation, wall synthesis, membrane integrity and essential genes expression. However, both approaches are demanding either metabolism-wise or energy-wise. In nature, the producer strains have evolved a panel of subtle induction mechanisms, such as auto-induction, co-culture induction and environmental induction. These unique mechanisms enable such a sophisticated regulation of biosynthesis, so that the global metabolic network and bacterocinogeny are well balanced. The bacteriocin tolerance or resistance is mostly associated with mutations on cell membrane fluidities, metabolic pathways and surface receptors. The use of bacteriocin should be urged to avoid the potential spread of resistance, just as the emergency of drug-resistant pathogens as found nowadays.

Abstract:The gut microbiota is a stable and complex ecosystem that forms a protective barrier by forming a membrane barrier and promoting proliferation and differentiation of intestinal epithelial cells, and plays an active role in maintaining and promoting immune homeostasis during intestinal pathogen infection and threats. The paper focuses on the mechanism of anti-pathogenic infection in host-intestinal microbial interactions and the involvement of intestinal microbes in the synthesis of antibacterial compounds against intestinal pathogens and threats, and to provide a theoretical reference for regulation of intestinal microbes to solve clinical gastrointestinal diseases and related symptoms.

Abstract:Efficient, cheap and environmental friendly method is necessary to treat heavy-metal wastewater. Microbial adsorption has the advantages of excellent adsorption, low price and environmental friendliness. Microorganisms such as bacteria, fungi and algae can bind heavy metals to their surface of cell walls through electrostatic adsorption, complexation and other processes. However, the adsorption effect of untreated microorganisms is often not satisfied. By physical and chemical modification, immobilization and other methods, the active sites on the microorganisms, can be significantly increased, to improve the removal rate of heavy metals. In this paper, the modification methods of microorganisms, the adsorption ability of modified microbial adsorbents for heavy metals in wastewater and the influencing factors are described. We also discuss the problems of microbial adsorbents, and the future research directions.

Abstract:Phytophthora infestans, which is responsible for potato and tomato late blight disease, belongs to Oomycetes, Peronosporales, Pythiaceae and genus of Phytophthora. Most research focus on P. infestans because it is destructive to potato production. Firstly, in this review, late blight symptoms caused by P. infestans, disease occurrence characteristics and epidemiology were illustrated, then the inheritance of sexual reproduction occurrence and variation of population structure with coexistence of various mating types, were also summarized. Since P. infestans genome sequenced in 2009, characteristics of genomics in various species among genus of Phytophthora were compared, and the research progress of effector clonging and mitochondrial genome were also introduced. Finally, the review demonstrated two significant technologies in functional genomics, high density genetic linkage mapping and genome-wide association study (GWAS), which were convenient for functional genes searching. This review can help to better understand the research highlight and further breakthroughs of P. infestans, which may provide a reference to further analysis on functional gene and pathogenic mechanism of P. infestans, and it is important to develop chemical control targets and epidemic trend forecasting.

Abstract:Methanotrophs can use methane as the only carbon source and energy sourcey to effectively remove nitrogen in the oxidation, which can be divided into aerobic methane oxidation coupled to denitrification (AME-D) and anaerobic methane oxidation coupled to denitrification (ANME-D), and thus, they are of vital significance in the researches of carbon and nitrogen cycles. In this paper, after summarizing the classification and distribution of methanotrophs in recent years, the basic principles, influencing factors and application of AME-D and ANME-D are then expounded, and in the end, the related future trend is analyzed and proposed, in an attempt to make a slightest contribution to the studies of methanotrophs-based applications in wastewater denitrification.

Abstract:The production of energy via fermentation is an important method to develop the renewable energy sources. Due to adverse environmental factors such as higher temperature, higher osmotic pressure and solvent toxicity in the process of industrial production, the physiological functions of producing strains were often changed, then reducing biotransformation efficiency. Therefore, obtaining excellent strains with higher yield and higher solvent resistance is of great significance for the current ethanol and butanol industrial fermentation. In this paper, we propose to discuss the ethanol and butanol producing strains, and the current breeding methods for improving ethanol and butanol fermentation performance were systematically reviewed. The opportunities and challenges in the production process of ethanol and butanol were also discussed.

Abstract:[Background] Cyanobacterial bloom caused by eutrophication has caused serious pollution to freshwater resources. The use of environmentally friendly alginolytic bacteria can effectively control the growth of cyanobacteria, which is one of the effective ways to prevent and control the formation of cyanobacteria bloom. [Objective] To optimize the alginolytic conditions of Microcystis aeruginosa by bacteria EHB01, so as to produce alginolytic agents for controlling the pollution of cyanobacterial bloom. [Methods] The concentration, temperature, light, C:N and N:P of alginolytic fermentation broth were analyzed by single factor test, and the carbon source, nitrogen source and pH of alginolytic bacteria EHB01 fermentation broth were optimized. Based on single factor test, the optimal quantity levels of key factors were determined by central composite design (CCD), and regression analysis was conducted by Desig-Expert 8.0.5. The parameters with the best alginolytic effect were obtained by response surface methodology (RSM). [Results] The effect of fermentation liquid concentration on the algicidal ratio was increasing continuously. The effect of temperature on the algicidal ratio was first increased and then decreased. However, the effects of light, C:N and N:P on the algicidal ratio of bacteria EHB01 fermentation broth showed a tendency of decreasing first and then increasing. The best carbon source for EHB01 fermentation liquid was sucrose, nitrogen source was potassium nitrate and pH was 7.5. Under the optimized conditions, the algicidal ratio was up to 86.97%, which was 21.72% higher than before optimization. [Conclusion] RSM was used to optimize the optimal culture conditions of alginolytic bacteria EHB01 fermentation broth, and the model fitting effect was good, which could provide an effective basis for industrial preparation of alginolytic agent.