Abstract:[Background] Antimicrobial resistance of bacteria has become more and more serious due to the abuse of antimicrobials. As an important foodborne pathogen, Vibrio parahaemolytics also exhibited a certain level of antimicrobial resitance. Quorum sensing system can regulate the antimicrobial resistance of bacteria, which provides a new pathway to study the mechanism and control technique of antimicrobial resitance of V. parahaemolytics. [Objective] To study the effect of signaling molecule autoinducer-2 (AI-2) on tetracycline resistance of V. parahaemolyticus strains from seafoods. [Methods] AI-2 was synthesized in vitro by a reaction using the critical enzymes, S-ribosylhomocysteinase (LuxS) and S-adenosylhomocysteine nucleosidase (Pfs), which were prepared by prokaryotic expression. The effect of AI-2 on tetracycline resistance of V. parahaemolyticus was determined by colonies counting method. The effect of AI-2 at different concentrations on the transcriptional levels of tetracycline resistance genes in V. parahaemolyticus was assayed by reverse transcription and real-time quantitative PCR. [Results] LuxS and Pfs enzymes were obtained by prokaryotic expression. The bioactive AI-2 could be synthesized in vitro by adding LuxS and Pfs to the substrate S-adenosylhomocysteine (SAH), with the fluorescence intensity of about 6 times as much as positive control. When treating with tetracycline at subinhibitory concentration, AI-2 could significantly promote the growth of V. parahaemolyticus strains, and AI-2 at the concentrations of 6, 15 and 30 μmol/L could increase the transcriptional levels of tetracycline resistance genes in V. parahaemolyticus strains to a certain extent. [Conclusion] AI-2 could enhance the tetracycline resistance of V. parahaemolyticus, which provided a reference for further study on the antimicrobial resistance mechanism of V. parahaemolyticus and developing control techniques targeting AI-2 on antimicrobial resistance of V. parahaemolyticus.

Abstract:[Background] The growth and activity of sulfate-reducing prokaryotes (SRP) that reside in petroleum reservoirs could generate large amount of H2S, resulting in various problems such as microbial souring and microbially-influenced corrosion. Unfortunately, knowledge on the diversity, physiology, and activity control of SRP therein is quite limited. [Objective] In order to further understand the phenotypic characteristics of SRP residing in the offshore high-temperature oilfield in the Bohai Sea area, China, and to explore the potential methods for addressing the SRP-mediated problems. [Methods] We first isolated, using Hungate techniques, SRP strains from the production water, and then evaluated the phenotypic features of the dominant strain. We also investigated the efficacy of each of five biocides in suppressing the sulfidogenic activity of the selected SRP strain. [Results] Cells of the isolated dominant SRP strain WJ1 are motile, rod-shaped, and approximately 2.0?5.5 μm in length. WJ1 exhibits a sequence identity of 99% for 16S rRNA gene with Soehngenia saccharolytica BOR-YT. Isolate WJ1 could survive at 60 °C (optimum at 37 °C), pH 5.0?10.0 (optimum pH 8.0), and in the presence of 0%?3% NaCl. Isolate WJ1 utilized a wide range of carbon substrates, including propionate, lactate, and acetate. Sulfate, sulfite, and thiosulfate could be utilized as electron acceptors, but not sulfur. Neither sodium hypochlorite (600 mg/L) or benzyltrimethylammonium chloride (600 mg/L) could inhibit H2S production by WJ1. By contrast, glutaraldehyde (30 mg/L), bronopol (10 mg/L), or THPS ((bis[tetrakis(hydroxymethyl)phosphonium] sulfate solution, 120 mg/L) could inhibited H2S production by WJ1 for at least 30 days. [Conclusion] The dominant SRP strain WJ1, isolated from the soured production water from this specific high-temperature offshore oilfield in the Bohai Sea area, shares the highest sequence identity of 16S rRNA gene with S. saccharolytica BOR-YT but presents distinct phenotypic traits from this phylogenetically close relative; bronopol, glutaraldehyde, or THPS could serve as potent agents for the mitigation of microbial souring by WJ1.

Abstract:[Background] The wastewater discharged by coal chemical industries contains a large number of refractory and highly toxic organic pollutants. It is an economically feasible strategy to treat the wastewater with bioaugmentation technology based on efficient degrading bacteria. Promoting the biofilm formation of degrading bacteria is proved to be beneficial to the performance of the biofilm wastewater treatment system. [Objective] To investigate the biofilm formation process and characteristics of a pyridine-degrading bacterium Pseudomonas sp. ZX08, and to identify the influence of different environmental factors such as temperature, pH, Na+, K+, Ca2+, Mg2+ on biofilm formation, and finally to provide references for regulating biofilm formation in wastewater treatment systems. [Methods] A modified microtiter dish biofilm formation assay was used to determine the biofilm formation and the planktonic bacteria growth in the 12-well plate under different conditions; the structural characteristics of biofilm was observed and analyzed by a confocal laser scanning microscope (CLSM). [Results] Pseudomonas sp. ZX08 showed good pyridine-degrading performance and biofilm-forming abilities. According to the CLSM analysis, the thickness of its biofilms formed at the surface reached 40?50 mm, and the proportion of live cells and protein/cell ratio were higher in the outer layer of the biofilms. A periodic variation was observed in the biofilm formation process in 72 h, and the biofilm biomass at 12 h and 48 h were relative peaks. The optimum temperature for ZX08 biofilm formation was 25 °C, and the optimum pH range was 7.0?9.0. Higher concentrations of NaCl (>0.6 mol/L) and KCl (>0.4 mol/L) significantly inhibited the biofilm formation of ZX08. Within a certain range (0?16 mmol/L), the increase of Ca2+ concentration could promote the biofilm formation at the solid-liquid interface of the 12-well plate bottom. Adding Mg2+ ranged from 0?16 mmol/L also resulted in a slight increase in the biofilm formation of ZX08. [Conclusion] The pyridine-degrading bacterium Pseudomonas sp. ZX08 can form thick and stable biofilms, and it needs to comprehensively consider the influence of various environmental factors on its biofilm formation in future applications.

Abstract:[Background] Aerobic denitrification is carried out under aerobic conditions, so that nitrification and denitrification can occur at the same time in the same reactor, which is the most competitive technology for nitrogen removal from wastewater. Mangrove wetlands are rich in microbial resources and a large number of aerobic denitrifying microorganisms are distributed. [Objective] In order to understand the denitrification mechanism of salt-tolerant microorganisms and provide a theoretical basis for the engineering practice of biological denitrification of salt-bearing wastewater, a salt-tolerant aerobic bacteria A63 was isolated from the mangrove wetland and its nitrate reduction ability was analyzed. [Methods] The species were identified by morphological characteristics and 16S rRNA gene sequencing. The nitrate reduction ability of the strain under different environmental factors was determined by single factor experiment, and its denitrification performance was optimized. [Results] It was preliminarily determined that the strain belonged to the Zobellella sp. The strain can carry out denitrification and dissimilatory nitrate reduction to ammonium (DNRA) action in the range of salinity 0%?10%, pH 5.0?10.0 and temperature 20?40 °C. The optimum growth carbon source is sodium citrate (1.2 g/L), and the optimum salinity 3.0%, the pH is 7.0?7.5, the temperature is 30?35 °C and C/N is 10. Under the optimum denitrification condition, the strain could reduce 208.8 mg/l NO3?-N to 0 in the medium within 12 hours, and only a small amount of ammonium nitrogen was produced. There was no accumulation of nitrite nitrogen, and the denitrification rate was as high as 99%. Furthermore, the strain had a significant effect on DNRA in adverse habitats such as high salinity, low C/N ratio, weak acidity and low temperature. [Conclusion] The strain A63 has a wide range of growth and remarkable nitrogen removal efficiency, so it is suitable for the treatment of mariculture wastewater. The present study lays a foundation for the development of high efficiency biological nitrogen removal process for salty wastewater in the future, and is of great significance to deepen the understanding of nitrogen transformation law and enrich the theory of biological nitrogen removal.

Abstract:[Background] As a type of mixed organic compound, crude oil can cause serious harm to humans and the environment once oil pollution occurs. [Objective] Crude oil degrading bacteria from contaminated soil in Xinjiang were isolated and screened to provide data support and technical reference for the bioremediation of crude oil contaminated soil. [Methods] Using crude oil as the sole carbon source, a total of 123 individual strains were isolated from crude oil through enrichment culture and screening. Thirty different strains were selected according to the morphology of the colonies, and their species were determined by 16S rRNA gene sequencing to construct a phylogenetic tree. Highly efficient crude oil degrading bacteria were screened out through crude oil degradation experiments, and the naphthalene, a representative compound of aromatic hydrocarbons, as the sole carbon source to screen high efficient degradation strains. [Results] Five strains of highly efficient crude oil degrading bacteria were isolated; these strains showed degradation rates higher than 85%. The strains capable of degrading naphthalene, salicylic acid, and catechol were obtained and applied for naphthalene degradation at a 1:1:1 inoculation ratio. The degradation rate of naphthalene increased from 60.74% to 89.40%, thus proving that division of labor cooperation between strains could improve their degradation efficiency for organic matter. [Conclusion] The strains obtained from screening enrich the crude oil degrading microbial species bank, and the observed division of labor cooperation among different microbial strains provides new ideas for the degradation of crude oil pollutants. The results also offer a reference for further research on crude oil pollution control.

Abstract:[Background] Acinetobacter indicus JL-1 can convert the insoluble phosphorus into soluble phosphate, which can be absorbed by plants, but the mechanism has not been known clearly. [Objective] Acinetobacter indicus JL-1 was used to study the mechanism of phosphate solubilization primarily. [Methods] To determine the optimal phosphorus source, the phosphorus solubility of strain JL-1 in different insoluble phosphorus sources was detected by using Mo-blue colorimetric method, particle-size distribution, ultrasonic crushing, HPLC detection were used to study the dissolving effect on calcium phosphate, the storage of phosphorus, and the produced organic acids and phosphatase activity during fermentation. [Results] Acinetobacter indicus JL-1 showed the best phosphate-solubilizing ability in the liquid medium with calcium phosphate, the solubilizing phosphate concentration peaked at 118.04 μg/mL at 48 h incubation. Strain JL-1 had the dissolving effect on calcium phosphate and could absorb a part of the released phosphorus. The dissolved phosphorus was released by the combined action of organic acids and phosphatase. Organic acids contained gluconic acid, propionic acid, acetic acid, lactic acid, etc. and propionic acid content was up to 118.11 mg/mL; the highest acid phosphatase activity was 22 901.32 μmol/(L·h), and the highest alkaline phosphatase activity was 23 826.02 μmol/(L·h). [Conclusion] Strain JL-1 had the erosion effect on calcium phosphate, the soluble phosphate was released by the organic acids and phosphatase produced during fermentation, some of the phosphate was absorbed by strain cells. The results improve the feasibility that Acinetobacter indicus can be used in agricultural production and provides data reference.

Abstract:[Background] Transmembrane transporters play important roles in the transport process of various substances by microorganisms. [Objective] By comparing the difference of genes encoding phosphotransferase system (PTS system) and ATP-binding cassette transporter (ABC transporter) in the prokaryotic microbiome between cyanobacteria and moss biocrusts, it will reveal the potential change trends in the biological processes of transmembrane transport along with the developmental succession of the biological soil crusts. [Methods] The metagenomic sequencing was performed for twelve samples of cyanobacteria and moss biocrusts collected from the southeastern of the Tengger desert. The sequencing data was compared with PTS system and ABC transporters metabolic pathway in KEGG database, then the related genes were selected and analyzed for the comparison between biocrusts. [Results] The gene diversity of PTS system and ABC transporter is consistent among cyanobacteria and moss biocrusts. A total of sixteen PTS system transporter genes were detected in the biocrusts, five of which had significant difference between cyanobacteria and moss biocrusts. 106 ABC transporters-encoding genes were detected, 46 of which were significantly changed. Then we have detailed descriptions of the substrates and shift trends of these 46 transporters. [Conclusion] In the process of the development and succession of biological soil crusts, the microbiome had a tendency to reduce substances uptake from the environment that can increase the osmotic potential and increase the potential of amino acids, cell membrane and cell wall components transportation, while there was no significant changes in the transport of mineral ions, auxiliary factors, sugars and carbohydrates. It should be noted that the relationship between the diversity and difference of these transporter coding genes and biological soil crusts remains to be proved and explained experimently.

Abstract:[Background] Plant endophytic fungi begin to play an important role in litter decomposition with the death of plant tissues into the saprophytic process. However, this effect may vary with plant species and endophytic fungi species. [Objective] in order to analyze the effects of endophytic fungi with different dominance on litter decomposition and associating microbial activity. [Methods] Leaf litters of Cunninghamia lanceolata were selected in this study as the decomposition substrate with litterbag method. [Results] The colonization of endophytic fungi or their combinations almost significantly accelerated litter decomposition at the former stage of decomposition process, but at the later stage this acceleration effect was weakened, and even their colonization inhibited the decomposition process except Irpex lacteus and Colletotrichum sp. The responses of each variable of microbial activity to endophytic colonization were not completely consistent with the mass loss, and these responses depended on decomposition stage. CO2 releasing showed a poor correlation to mass loss at the former stage of decomposition, but a close correlation at the later stage. Carboxymethyl cellulose (Cx enzyme) greatly contributed to mass loss at the former stage of decomposition, but the contributions of laccase and peroxidase to mass loss were improved at the later stage. In a word, the colonization of endophytic fungi had a great influence on litter decomposition and associating microbial activities. [Conclusion] The colonization effect of endophytic fungi will contribute to the understanding of the mechanism on supporting soil carbon pool balance and nutrient cycle of forest ecosystem, and is of great significance for studying the restoration of soil fertility in barren plantation.

Abstract:[Background] Complete and incomplete nitrification driven by Nitrospira plays a key role in global nitrogen cycle, but little is known about the niche differentation of Nitrospira, and associated environmental driving forces for Nitrospira’s niche separation and as well as possible functions like complete nitrification driven by Nitrospira in Xilin river basin. [Objective] To reveal the niche differentiation of Nitrospira and associated environmental driving forces, and deciphering the possible functions like complete nitrification driven by Nitrospira. [Methods] Based on high throughput sequencing of 16S rRNA gene with bioinformatics analysis, the compositions and abundance of sediment/soil Nitrospira populations were analyzed. Meanwhile, their niche differentiation and associated environmental driving forces were characterized so as to predict their potential complete nitrification. [Results] Nine Nitrospira genera were detected, of which Nitrospira 1, 2 and 4 were dominated in xeric environment, which were negatively correlated with sand, water content, pH and ammonia nitrogen (AM) while positively correlated with nitrate nitrogen (NR), dissolved salt (DS), total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), silt and clay contents. In comparison, Nitrospira 5, 6, 7, 8 and 9 were dominated in aquatic and hygric environments, positively correlated with sand content (except Nitrospira 9 negatively correlated with AM), etc. and negatively correlated with silt content, etc. Interestingly, Nitrospira 3 was preferentially distributed in the center of the river bed with aquatic environment, only positively correlated with sand and silt contents, but negatively correlated with the other environmental factors. [Conclusion] There was obvious niche differentiation of Nitrospira populations with a wide range of habitat adaptability in Xilin river basin. Nitrospira 1, 2 and 4 were most suitable to colonize xeric environment with relatively rich nutrients, positively driven by silt content, etc. Whereas Nitrospira 5, 6, 7, 8 and 9 were most suitable to inhabit relatively oligotrophic aquatic and hygric environments, positively driven by sand content, etc. Nitrospira 3 preferred silt, free water and low ammonia in oligotrophic aquatic environments, positively driven by only sand and silt contents. Briefly, silt content, dissolved salt (DS) and ammonia nitrogen (AM) were the main environmental driving forces for niche differentiation of Nitrospira groups. So i) Nitrospira 3 was presumed as probable comammox; ii) the other Nitrospira populations like 1, 2, 4, 5, 6, 7, 8 and 9 belong to comammox remains to be further discussed.

Abstract:[Background] Small RNA EsrE affects cell growth by regulating the expression of succinic acid dehydrogenase in Escherichia coli, the exploration of its regulatory mechanism is conducive to deepening the understanding of EsrE on cell growth. [Objective] To explore the transcriptional regulation mechanism of small RNA EsrE in E. coli. [Methods] Transcriptional regulators were screened by using a dual plasmid reporting system; the interaction between RpoH and PesrE was verified by electrophoretic mobility shift assay (EMSA); qRT-PCR shows effects of the transcriptional regulators on EsrE. [Results] The results of reporting system demonstrated that RpoH up-regulated PesrE, and FabZ down-regulated PesrE. The results of EMSAs showed that RpoH bound the PesrE fragment directly, however FabZ did not. [Conclusion] RpoH is involved in the regulation of esrE transcription by binding the PesrE promoter directly, while FabZ is involved in the transcriptional regulation of EsrE indirectly by other ways.

Abstract:[Background] Cecropin is extensively studied and which is an effective antimicrobial peptide. It will lay the foundation for the practical application of cecropin in aquaculture and agriculture to realize the commercial production of cecropin. [Objective] To obtain a genetically engineered strain for efficient production of cecropin AD. [Methods] The recombinant vector pGAPZαA-CAD was firstly constructed and then transformed to P. pastoris X33 strain by electric shock. Cecropin AD gene was successfully expressed and X33/GCAD strain was obtained. Secondly, the recombinant vector pUCGAP-CAD was constructed and transformed into X33/GCAD strain. The pGAPZαA-CAD plasmid was integrated into the GAPDH promoter region of P. pastoris X33 with Zeocin as resistance screening label, while pUCGAP-CAD plasmid was integrated into the non-translated rDNA region of P. pastoris X33 with Geneticin as resistance screening label. Finally, a recombinant X33/GUCAD strain with an efficiently expressed cecropin AD gene was obtained. [Results] The antimicrobial compound of X33/GUCAD was cecropin AD which was identified by mass spectrometry. Afterward optimizing its fermentation conditions, we found that the X33/GUCAD strain having higher potential to express cecropin AD by consuming glycerol as a sole carbon source while peptone and yeast extract for organic nitrogen source. [Conclusion] The higher copy number is more beneficial to increase the yield of cecropin AD, and the engineering strain is more stable in the later fermentation process and suitable for industrial production.

Abstract:[Background] Streptomyces lavendulae gCLA4 is an actinomycete strain isolated from cucumber. Preliminary researches have showed that the strain was prominent in antagonism against a variety of pathogenic bacteria, and has potential biocontrol value. [Objective] In-depth study of the function of necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4, and to clarify its mechanism of action in improving plant resistance. [Methods] The necrosis-inducing protein 4955 gene was cloned and expressed in Escherichia coli BL21(DE3). Activity and stability of the protein were tested in tobacco. Then, the basic properties and tertiary structure of the protein were analyzed with Protparam, PredictProtein, NCBI CDD, and SWISS-MODEL. Tobacco defense-related enzymatic activities (CAT, SOD, POD, PAL) were detected after treatment with protein 4955. Additionally, altered gene expression (NPR1, PR1-b, PAL, LOX, PR1-a) was detected by qPCR. [Results] The protein 4955 retained its activity at temperatures up to 40 °C and pH 6.0 to 10.0. The protein has a molecular weight of 24 491.12 Da and consists of 225 amino acids. Its isoelectric point is 5.96, and the amino acid sequence alignment contains a conserved NPP1 domain. The activity of CAT, SOD and PAL increased in tobacco after two days of treatment with 4955, and the activity of POD did not change significantly. The expression of PR1-b and LOX genes was increased 1, 3, and 5 days after treatment with protein 4955, and the expression of PAL was up-regulated on the 4th day. [Conclusion] The necrosis-inducing protein 4955 in Streptomyces lavendulae gCLA4 does induce plant defense response in tobacco.

Abstract:[Background] Human Parvovirus B19 (B19 virus) is a member of the two Parvoviriade families that cause human diseases. The unique viral p6 promoter in B19 virus controls all the viral mRNA transcription. Previous research shows that some transcription factor binding sites (TFBSs) within p6 and nonstructural protein NS1 of B19 virus are both involved in regulation of p6 activity. However, effects of other TFBSs or viral proteins on p6 activtiy have not been reported. [Objective] Here, we investigate the roles of 11 kD protein in regulation of the activities of B19 p6 promoter as well as some cytokine promoters. [Methods] Based on bioinformatics methods, we predicted some new TFBSs and identified the CpG island in p6 region. In vitro methylation assay was carried out to study the effect of CpG on p6 activity. Meanwhile, EGFP and luciferase reporter system were employed to detect the activities of p6 and other cytokine promoters. [Results] Consistent with previous research, p6 shows similar high activity in different non-permissive cells. The truncated p6 mutation assay indicated that CTCF, YY1, STAT3, Sp1/3 and E2F7 binding sites at nt. 302?479 played significant roles in maintaining p6 promoter activity. In addition, in vitro methylation assay demonstrated that CpG methylation on p6 inhibited its promoter activity. Interestingly, though 11 kD protein did not regulate p6 activity, it increased the promoter activity of TNF-α, IL6, STAT3 significantly. [Conclusion] Our findings imply that the newly discovered TFBSs and CpG island in p6 are essential for the maintenance of p6 promoter activity. Meanwhile, the association between nonstructural protein 11 kD and cytokine promoter suggests that 11 kD may participate in B19 virus host interaction by regulating the cytokine factor expression.

Abstract:[Background] Botrytis cinerea is an important phytopathogenic fungus. In previous studies, it was confirmed that the BcKMO gene encoding kynurenine 3-monooxygenase (KMO) is involved in the regulation of the growth and pathogenicity of Botrytis cinerea. KMO is a key enzyme in the kynurenine pathway, but the presence of the kynurenine pathway in Botrytis cinerea and its function in the growth, development and pathogenesis of pathogens have not yet been related reported. [Objective] To identify the key enzyme genes in the kynurenine pathway of Botrytis cinerea, and to determine the presence of kynurenine pathway in Botrytis cinerea, lay the foundation for elucidating the molecular mechanism of growth and pathogenicity of Botrytis cinerea. [Methods] Bioinformatics methods were used to analyze the coding genes of key enzyme, kynureninase (KYN), indoleamine-2,3-dioxygenase (IDO), and kynurenine amino transferase (KAT), in the kynurenine pathway of Botrytis cinerea. Real-time PCR was used to detect the expression level of key enzyme genes of the kynurenine pathway in Botrytis cinerea wild-type BC22, BcKMO mutant BCG183, and the BcKMO complementing strain BCG183/BcKMO. The content of kynureninase in the BcKMO mutant was determined by using a fungal kynureninase assay kit. [Results] Botrytis cinerea contains 2 genes coding KYN, 3 genes coding IDO, and 10 genes coding KAT. The expression levels of Botrytis cinerea KYN, IDO, KAT coding genes in mutant BCG183 were significantly higher or lower than those in BC22 and BCG183/BcKMO. The content of kynureninase (KYN) in the mutant BCG183 was significantly lower than that of BC22 and BCG183/BcKMO. [Conclusion] Kynurenine pathway is present in Botrytis cinerea. BcKMO mutation affects the expression of KYN, IDO and KAT encoding genes and the content of kynureninase (KYN) in Botrytis cinerea.

Abstract:[Background] The bacterial community diversity affected the composting process and biochar affected the growth of bacteria, but the effect of biochar on the bacterial community structure of pig manure composting has not been reported yet. [Objective] According to the variation of bacterial community structure and composting temperature, adequate content of biochar was added to pig manure composting to improve the proportion of major bacteria and composting efficiency of pig manure composting, so as to provide reference for the joint application of biochar and pig manure composting. [Methods] The biochar content of 0%, 3%, 6% and 9% was set, and four levels of biochar were selected in the high-temperature period and the stable-temperature period of composting process, respectively. According to the Illumina MiSeq’s high-throughput sequencing results of bacterial 16S rRNA gene, the effects of biochar content and composting temperature on bacterial community structure of pig manure composting were analyzed. [Results] At the phylum level, the major bacteria with the highest abundance in pig manure composting were Proteobacteria, Actinobacteria, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Firmicutes, Acidobacteria and Deinococcus-Thermus. At the genus level, the major bacteria with the highest abundance in pig manure composting were Chryseolinea, Subgroup_6_norank, Steroidobacter, Anaerolineaceae, Nonomuraea, Longispora, Bacillus, Sporacetigenium, Luteimonas, Phyllobacteriaceae, Truepera, Rhodothermaceae and Aquamicrobium. The change of biochar content could promote or inhibit the growth of major bacteria in pig manure composting. As the content of biochar increased, the abundance of Bacillus, Streptomyces, Rhodothermaceae and Firmicutes increased, whereas the abundance of Chryseolinea, Longispora and Steroidobacter decreased. At the high-temperature composting period, the abundance of Firmicutes, Bacillus and Streptomyces was greater than that of the stable-temperature composting period; while Chloroflexi, Anaerolineaceae and Longispora were opposite. The number of bacterial community in high-temperature composting period was up to seventy, significantly greater than stable-temperature composting period of fifteen. Among them, the number of bacteria played a major role in pig manure composting was up to seven (Rhizobiales, Incertae_Sedis, Proteobacteria, Alphaproteobacteria, Xanthomonadales, Gammaproteobacteria and Steroidobacter) in the high-temperature period, while only three (Micromonosporales, Longispora and Micromonosporaceae) were found in the stable-temperature period. The bacterial diversity of pig manure composting in high-temperature period was significantly higher than that of stable-temperature period. After adding biochar to pig manure composting, environmental factors (electrical conductivity, water content, temperature and pH) had no significant effect on the major bacteria of pig manure composting. β-Proteobacteria, Rhodothermaceae, Phyllobacteriaceae and Bacterium were significantly affected by the water content, temperature and pH. [Conclusion] The content of biochar and composting temperature could change the bacterial community structure of pig manure composting, and significantly increase the number and diversity of bacteria in pig manure composting at the high-temperature period. The electrical conductivity, water content, temperature and pH of pig manure composting could affect the growth of composting bacteria, but the effect on the major bacteria with the highest abundance in pig manure composting was not significant.

Abstract:[Background] Compared with single microbial agent, microbial compound agent is more efficient and stable to promote crop growth. [Objective] To develop a microbial compound agent that can significantly promote the growth of rice, and build a mathematical model for the construction of microbial compound agent. [Methods] Bacillus amyloliquefaciens FH-1 was mixed with one of the seven plant growth promoting bacteria, in a biomass ratio of 1:1. The high-efficiency compound microbial agent was screened by rice pot experiment. Biochemical methods were used to determine the growth promoting characteristics of compound agent. Based on the analysis of the correlation between the plant characteristics and the growth promoting characteristics of the strains, a mathematical model for the construction of microbial compound agent was established by using the general linear equation. [Results] Compared with the control, microbial compound agent FN (Bacillus amyloliquefaciens FH-1 and Brevundimonas sp. NYM-3) significantly increased the rice shoot length, root length and fresh weight of rice by 20.79%, 26.67%, and 74.84% (P<0.05), respectively. The microbial compound agent FN was the best microbial compound agent among the seven microbial compound agents. Partial correlation analysis showed that the abilities to dissolve inorganic phosphorus, to produce siderophore and to secret ACC deaminase might play more important roles in promoting plant growth. According to the results of partial correlation between the plant growth promoting characteristics of microbial agents and the characteristics of plants, the mathematical model for the preparation of microbial compound agent was constructed and the prediction accuracy was over 97%. [Conclusion] We have successful developed a high-efficient microbial compound agent to promote rice growth, and constructed a mathematical model for the construction of microbial compound agent, which could provide scientific guidance for the development of the microbial compound agent.

Abstract:[Background] Many species of Bacillus with the merits of strong stress resistance and safety for human become a research hotspot in exploiting new active substances. [Objective] To isolate and screen antifungal bacteria and develop its active ingredients as a natural bio-antifungal agent of food. [Methods] These methods of plate culture, plate-confrontation and mycelia growth inhibition were used to isolate and screen antifungal bacteria from air and bamboo endophytic bacteria. Z21 was identified based on morphological, physiological, biochemical characteristics and 16S rRNA gene sequence. The growth conditions of Z21 were through orthogonal experiment. [Results] Z21 showed strong antifungal activity to 6 kinds of fungi. The 16S rRNA gene sequence of Z21 was consistent with that of Bacillus methylotrophicus CBMB205T, and the morphological characteristics and physiological and biochemical characteristics of Z21 were consistent with the CBMB205T strain. The optimum culture conditions for Z21 were 32 °C for 48 h with a medium of glucose 20.0 g/L, NaNO3 20.0 g/L and MgSO4 3.0 g/L. [Conclusion] The Z21 was identified as B. methylotrophicus, it showed strongly and stalely inhibitory activities against Aspergillus niger, Trichoderma koningii, Trichoderma viride, Rhizopus arrhizus, Mucor fragilis, and Penicillium ochrochloron. So it was an abroad-spectrum antifungal bacterium.

Abstract:[Background] Avian pathogenic Escherichia coli (APEC) is one of the main pathogens of poultry. ETT2 (Escherichia coli type III secretion system 2) regulates its pathogenicity through transcriptional regulators. Currently, the effect of the transcriptional regulators EtrA on its pathogenicity in APEC is unclear. [Objective] To study the effect of ETT2 transcriptional regulator EtrA on the pathogenicity of APEC. [Methods] APEC40-ΔetrA and APEC40-CΔetrA were constructed by the lambda Red recombinase system. Then the biological characteristics, including growth characteristics, biofilm formation ability, motility and sensitivity to serum was compared between APEC40 and APEC40-ΔetrA. Besides, the transcriptional levels of virulence genes, biofilm formation related genes and flagellin synthesis genes of wild and complementation strain were compared by RNA-Seq and real-time PCR. [Results] The growth characteristics did not significantly change between APEC40 and APEC40-ΔetrA (P>0.05). However, the biofilm formation and sensitivity to serum were significantly enhanced (P<0.001), while the motility was significantly decreased (P<0.01) compared with the wild strain APEC40. There was some recovery in the phenotype of the complemented strains. Furthermore, transcriptomics screened seven virulence difference genes, which showed that the genes related to biofilm formation were significantly up-regulated, and the genes involved in motility were significantly down-regulated. The results of qRT-PCR were consistent with those of RNA-Seq. [Conclusion] Loss of etrA can significantly affect the biofilm formation, motility, and sensitivity to serum in APEC. This study provides a reference for further study on the pathogenicity of ETT2 in APEC.

Abstract:[Background] Phenyllactic acid (PLA) is a natural broad-spectrum antibacterial substance with a great application potential. In the previous work, Gluconacetobacter sp. FBFS97, an acetic acid bacterium (AAB) strain with high-yield PLA, was isolated, but the specific species of this strain and the molecular mechanism of PLA production is unclear. [Objective] To determine the species relationship of FBFS97, explore the genetic information of FBFS97, especially the genes related to PLA biosynthesis. [Methods] The morphology of FBFS97 was characterized by light microscopy and scanning electron microscopy, while the classification was identified by 16S rRNA gene sequence alignment. The effect of phenylalanine on FBFS97 producing PLA was detected by high-performance liquid chromatography (HPLC). On this basis, the complete genome was sequenced by Illumina MiSeq sequencers, and genome assembly, gene prediction, functional annotation, GO/COG cluster, metabolic pathway and virulence were analyzed using the relevant software, and the biosynthetic pathway of PLA is predicted. [Results] The strain was identified as Gluconacetobacter tumulisoli by 16S rRNA gene sequence alignment analysis and morphological analysis. When 1 000 mg/L phenylalanine was added to the liquid medium of FBFS97, the maximum concentration of PLA in the fermentation broth reached 400 mg/L, which was 8 times higher than that of control group. The genome size of FBFS97 is 3 988 308 bp with 66.62% (G+C)mol% and 3 500 encoding genes, and no toxin-related gene was predicted by VFDB database in the genome, and whole genes relative for biosynthetic PLA by the shikimate pathway were found. [Conclusion] This is first attempt to describe the whole genome sequence of Gluconacetobacter tumulisoli sp. FBFS97, a high-yield strain of PLA. The genes related to PLA biosynthesis are found in the FBFS97 genome, which provides a basis for further investigation on the PLA biosynthetic pathway in FBFS97.

Abstract:[Background] CueR is involved in transcription al regulation of Cue copper-resistant system in model bacteria Escherichia coli. However, if a similar system is present in Acidovorax citrulli, a bacterial plant pathogen, remains unclear. [Objective] Analyzing the cueR gene and its protein in Acidovorax citrulli can facilitate the mechanism study of copper resistance for this destructive phytopathogen. [Methods] AcCueR of Acidovorax citrulli was identified and compared with CueRs from four representative bacteria, i.e. EcCueR of E. coli, PaCueR of Pseudomonas aeruginosa, SeCueR of Salmonella and VcCueR of Vibrio cholerae. The characteristics of the structure, physicochemical properties, subcellular localization, and interaction proteins of these CueRs were analyzed by bioinformatics methods. The cueR gene mutant was prepared through homologous recombination with Acidovorax citrulli strain FC440. The phenotypes of copper resistance of wild type strain, cueR gene mutant, and gene functional complementary strain were also assayed. [Results] Comparative sequence analysis of CueR proteins revealed that AcCueR and PaCueR have the highest sequence similarity. All these five CueR proteins belong to HTH-MerR-SF super family. The tertiary structure is mainly composed of alpha-helix and coiled-coil. The CueR proteins of different bacteria are similar. AcCueR can interact with P-type ATPase (CopA) and multi-copper-oxidase (CueO) in Acidovorax citrulli. In the promoter of copA, there is a typical palindrome motif that binds to CueR. When challenged with Cu2+, the cueR mutated strain FC440(?cueR) exhibited a significantly reduced in growth. Consistently, the growth capacity of the complementary strains completely recovered. [Conclusion] The cueR gene contributes to its copper resistance in Acidovorax citrulli. The AcCueR protein has a similar structure and function compared with CueR in E. coli and other bacteria. These results indicate that the Cue copper-resistance system works in Acidovorax citrulli.

Abstract:[Background] The secondary metabolites of endophytic bacteria are the important sources of new natural active substances. [objective] We aimed to isolate the bacterial strains and their secondary metabolites with antibacterial activity against Staphylococcus aureus from Paeonia lactiflora. [Methods] The antagonistic strains were screened against Staphylococcus aureus through the method of dual culture. The strains were indentified based on characteristics in morphology and DNA identification. The biosynthetic genes of lipopeptide were amplified using polymerase chain reaction, and the antibacterial activity of their fermentation and crude lipopeptides were detected using the method of Oxford cup. The antibacterial activity of the crude lipopeptides was isolated through Sephadex LH-20 gel chromatography, which was analyzed by MALDI-TOF mass spectrometry. [Results] A total of 13 antagonistic strains were screened from the endophytic bacteria isolates, which had different degrees of inhibition against Staphylococcus aureus. The isolates SY11 had the most obvious inhibition ability, and their fermentation and crude lipopeptides had strong inhibitory effect. The isolates SY11 was identified as Bacillus amyloliquefaciens based on characteristics in morphology and phylogenetic analysis of 16S rRNA gene sequences. Three biosynthetic genes of lipopeptide, fenA, ituD and srfkn were detected using polymerase chain reaction. The results suggested that the strain had the ability to synthesize lipopeptide. By further analysis of MALDI-TOF mass spectrometry. The main active substance was presumed as Bacillomycin D. [Conclusion] Bacillus amyloliquefaciens SY11 had significant inhibitory effect on Staphylococcus aureus, and their crude lipopeptides also showed inhibitory activity against Staphylococcus aureus in vitro, which laid the foundation for the further development and application of endophytes from Paeonia lactiflora.

Abstract:[Background] The YycFG two-component regulatory system plays a critical role in the Streptococcus pneumoniae response to the external environment. The response regulator protein YycF(VicR) is essential for the growth of Streptococcus pneumoniae, but the function of YycF in regulating bacterial virulence is unclear. [Objective] To analyze the effect of the response regulator protein YycF on biological characteristics and pathogenicity, the pcsB constitutive-expressing, yycF-deficient mutant strain of Streptococcus pneumoniae was constructed and characterized. [Methods] First, the pcsB constitutive expression strain (Pc-PcsB+) was constructed by janus cassette (JC) counter selection, and the yycF gene in Pc-PcsB+ was replaced with an erythromycin resistance gene (erm). The growth characteristics, the contents of capsular polysaccharide, cell adhesion and invasion abilities, and pathogenicity of D39rpsl41, Pc-PcsB+ and Pc-PcsB+DyycF were assessed. [Results] The yycF-deficient mutant strain (Pc-PcsB+DyycF) was derived from Pc-PcsB+. Compared with Pc-PcsB+, we observed slower growth, abnormal division, increasing amount of capsular polysaccharide in intracellular and smaller molecule capsular polysaccharide in the Pc-PcsB+DyycF. In vitro studies showed that the adherence ability of Pc-PcsB+DyycF was significantly reduced than that of Pc-PcsB+ (P=0.006). The virulence test suggested that all mice infected with D39rpsl41 died, while the mortality rates of mice challenged with Pc-PcsB+, Pc-PcsB+DyycF were decreased to 91.7% and 75% respectively, though no statistical significance between them was observed (P=0.183). The colonization study revealed that the bacterial burden of Pc-PcsB+DyycF in the lung tissue was significantly lower than that of Pc-PcsB+ (P=0.033). [Conclusion] In this study, the yycF-deficient mutant Streptococcus pneumoniae D39 was constructed successfully, and the biological characteristics and pathogenicity of Pc-PcsB+DyycF were identified, which provide a theoretical basis for further study on the regulatory mechanism of YycFG on the pathogenicity of Streptococcus pneumoniae.

Abstract:[Background] Staphylococcus aureus is a type of common food-borne pathogenic bacteria that can easily form biofilm on the surface of food and processing equipment, leading to food corruption and disease spread, and threatening food safety. [Objective] To study the inhibition of S. aureas biofilm formation by oridonin. [Methods] The inhibition of biofilm formation by oridonin was studied using crystal violet staining assay and scanning electron microscopy. The influence of oridonin on the formation of polysaccharide intercellular adhesion and the release of extracellular DNA (eDNA) was detected by Congo red agar and spectrophotometer. RT-PCR analysis was used to determine the effect of oridonin on the expression of genes, including icaA, cidA, agrA and sarA. [Results] Oridonin showed strong antimicrobial activity on S. aureas biofilm formation. Polysaccharide intercellular adhesion formation and eDNA release were greatly inhibited by oridonin. eDNA release decreased by 48.62% after incubated with oridonin at 1/4MIC for 16 h. Oridonin could significantly inhibit the expression of biofilm forming related genes in S. aureas. After incubated with of oridonin at 1/2MIC for 16 h, the relative expression of icaA, cidA, agrA and sarA of S. aureas were reduced by 91.6%, 94.7%, 77.6% and 70.4%, respectively. [Conclusion] Oridonin can significantly inhibit the biofilm formation of S. aureas via the reduction of icaA and cidA expression, so as to influence the synthesis of polysaccharide intercellular adhesion and the eDNA release.

Abstract:Sexual reproduction in fungi, including cell recognition, cell fusion, zygote production, meiosis and mitosis, and final formation of sexual spores, is one of the important reproductive cycles that provide the source of genetic recombination. The MAT loci determine the mating types in fungi and play a key role in fungal sexual reproduction. MAT loci differ in gene composition, sequences and arrangements in fungal species. In recent years, rapid progress has been made in the functions and regulatory networks of MAT loci and MAT genes. In the present paper, we review the advances in ascomycetes on the gene composition and distribution in MAT loci, the function of MAT genes, and the relationship between MAT loci and sexual signaling pathways.

Abstract:The 2-methylcitrate cycle is a widely distributed carbon metabolic pathway playing a crucial role in consuming propionate or propionyl-CoA in bacteria. We have been working on the field of microbial metabolic regulation, and made new progress in the transcriptional mechanism and physiological function of 2-methylcitrate cycle in Bacillus thuringiensis. In this paper, current researches on the composition and transcription regulation of the key genes in 2-methylcitrate cycle, and the physiological functions of 2-methylcitrate cycle are reviewed. Meanwhile, the existing scientific problems and future research topics about this cycle are discussed. In addition, the potential application of the key enzymes in 2-methylcitrate cycle as drug targets for prevention and treatment of bacterial infection is also proposed.

Abstract:Thermostable α-amylase is a group of important industrial enzymes famous for their earliest industrial usage tracing back to thousands of years ago. Thanks to their advantages such as thermal stability, high efficiency on starch liquefaction and the easy-to-stock nature, the thermostable α-amylase is widely used in starch-based sugar industries, food fermentation industries, beer and other brewing industries, and textile printing and dyeing associated industries, respectively. This review covered the advances in the studies on the thermostable α-amylase regarding the producer strains selection and construction, structure-function relationship of the enzyme, the ways for improving α-amylase enzyme activity and the approaches of heterologous expression of the enzyme, respectively. In addition, the status quo of thermostable α-amylase exploration worldwide was depicted in general, and the comparison between domestic achievements with the global leading establishments was made in the specific.

Abstract:Inflammatory bowel disease (IBD) is an intestinal chronic inflammatory disease which pathogenesis is not yet clear. However, the incidence of IBD is increasing, which brings great financial burden to patients and their families. We need to find positive and effective treatments to help them. The latest view is that the balance between the host and the intestinal microorganism is broken, which triggers an immune inflammatory response in genetically susceptible individuals. Imbalance of intestinal flora plays an important role in the pathogenesis and development of IBD. Clinical studies found that IBD patients had varying degrees of intestinal flora imbalance. Combined application of probiotics can improve theses patient's symptoms. More and more researchers are paying close attention to the relationship between intestinal flora and IBD, and have carried out in-depth basic and clinical research. This article reviews the physiological influence of intestinal flora on IBD and the therapeutic effects of probiotics and fecal bacteria transplantation on IBD.

Abstract:Fluoride had been widely used as an effective anti-caries agent for decades. However, the long-term use of fluoride might lead to the emergence of fluoride-resistant strains. The fluoride-resistant ability of microorganism could be induced by the phenotypic adaptation or genotypic changes, subsequently, the anti-caries effect of fluoride might decrease. Besides, long-term fluoride interference might disturb the balance of oral micro-ecosystem, leading to associated oral diseases. In general, fluoride played an important role in the prevention and treatment of oral diseases, while oral microbial homeostasis was crucial for oral and systemic health. Therefore, in this paper we reviewed the research progress on the fluoride-resistant mechanism of oral microorganisms.

Abstract:Experimental teaching is an important part of undergraduate teaching of life sciences. This paper takes the course of Microbiology Experiment as the research object, using interactive website to conduct on-line and off-line blended teaching, evaluating students’ learning effects and rules, exploring the effect of blended teaching in experimental courses of life sciences, the possibility of popularization of the blended teaching mode is also be discussed. In three consecutive years of teaching practice, statistical analyzing of data from various dimensions shows that under blended teaching mode, the vast majority of students have the ability to study on their own initiative, at the same time, the depth and breadth of experimental courses have been improved. The monitoring and comprehensive evaluation of learning rules show that the students who study in-depth are outstanding in all kinds of evaluation indexes. Consequently, under the blended teaching mode, how to guide students to conduct real and effective depth learning is an important task for teachers using the new teaching mode.

Abstract:In 1984, Harvard University professor Eric Mazur founded peer-instruction (PI) in physics teaching practice, PI guides students to explore actively, learn from each other and obtain good teaching effect. In the 21st century, with the rapid development of information technology and life science, the experimental teaching of life science is also moving towards the network platform. In the blended teaching of Microbiology Experiment, the PI method is applied. In the explanation of experimental principle, operation technology, analysis of experimental results and mutual evaluation of homework, the PI method keeps the learning process at the state of teaching to others all the time, that is, the retention rate of knowledge and skills is stable at 90% after two weeks. Comparing the classes with PI method and classes without PI method, the classes that implemented the peer teaching method have significantly improved in the statistics of various achievements. Therefore, PI method in experimental teaching is an effective teaching method worthy of further research and promotion.

Abstract:Innovation and entrepreneurship education has become an important orientation in the reform of national higher education. How to combine curriculum reform with innovation and entrepreneurship education is a crucial subject in Microbiology teaching. In that project, several measures were taken to cultivate students? innovative ability and entrepreneurial thinking. Firstly, participation in experimental preparation of interest group enabled some students to rapidly grow into experimental experts. This practice had become a good inheritance and characteristics of Microbiology curriculum, which had been widely used in other courses in our university. Secondly, the students? ability of comprehensive application of knowledge, scientific research thinking and innovation could be improved by the modular experiments with diversified designing, so that students could step on the scientific research road in the process of subtle influence. Thirdly, guiding students to participate in the teacher?s project and applying for the discipline competition project could cultivate students’ scientific research thinking and experimental skills, which was a beneficial extension of practical teaching. The above reform had achieved remarkable results. The activity of studying and participating in scientific research of the students had been significantly improved. The quality and number of scientific research projects applied by students remarkably increased. Those students also did well in academic competitions, scientific paper publishing, and patent applications. This mode played an important role in improving the undergraduates? innovation and entrepreneurship ability, as well as comprehensive capability, which could be applied in talent training in other local universities.

Abstract:[Background] Streptococcus thermophilus AR333, isolated from fermented dairy products, could produce a high-yield active exopolysaccharide. [Objective] To establish a high-efficiency electroporation method of S. thermophilus AR333. [Methods] Electroporation conditions were optimized by single factor experiment and Box-Behnken response surface methodology. [Results] The optimum condition was glycine content 8.3 g/L, cell density of 0.8 (OD600), buffer composed of 10% glycerol (V/V) and 0.5 mol/L sucrose, 80 ng pIB184 plasmid, electric field strength 14 kV/cm, the recovery LM17 media composed of 0.4 mol/L sorbitol, 2 mmol/L CaCl2 and 20 mmol/L MgCl2 and recovery time of 5 h. [Conclusion] The transformants reached 3.68×105 CFU/μg-DNA under the optimal condition, which was improved about 14-fold in comparison with that of initial condition. The optimized condition was used to achieve a high electro-transformation efficiency, which lays a foundation for genetic engineering of AR333.