Abstract:[Background] hereditary stability of recombinant strain is the prerequisite for high-level and stable expressing, which determines the success of industrial applications. [Objective] hereditary stability of Pichia pastoris recombinant strain containing heterologous Thermomyces lanuginosus lipase gene was studied. [Methods] recombinant strain was subcultured for 15 generations and its stability judged from the following aspects: colony morphology, cell morphology, hydrolytic activity of lipase, DNA sequence and integrated copy number of the target gene. [Results] colony and cell morphology of the 1st, 5th, 10th and 15th generations of strains remained the same, as well as the molecular weight of the target protein and the target gene sequence. It was also found that after pass-generation culture for 5 times, the integrated copy number of lipase gene was greatly reduced and then stabilized at about 7 in the 10th and 15th generations, while the relative hydrolytic activity of lipase reached up to 90%. [Conclusion] high-level expression of the lipase is achieved by moderate gene copy number. this recombinant strain has a good hereditary stability and is feasible for large-scale production.

Abstract:[Background] Lactobacillus is intestinal probiotics with the frequent observation of high angiotensin converting enzyme (ACE) inhibitory activity in its fermented milk. The marine is an optimal source for biological flora, including abundant types of Lactobacillus. [Objective] Lactobacillus resources were distributed in sediments of the Bohai Sea through the results of high-throughput sequencing. In order to exploit the sources for producing ACE inhibitory peptide and increase the ACE inhibitory activity in fermented milk of Lactobacillus, the present study was performed by using Lactobacillus helveticus GY-3 selected from sediments of the Bohai Sea. [Methods] Lactobacillus resources were detected by using high-throughput sequencing in sediments of the Bohai Sea. After the enrichment separation, 16S rRNA gene identification and genome sequencing analysis were then done on the selected strain. We also measured the ACE inhibitory activity of the fermented milk of the selected strain, and optimized the fermentation conditions of the selected strain through orthogonal test. [Results] Lactobacillus helveticus bacterium (GY-3) was identified and high ACE inhibitory activity was detected in its fermented milk. The optimal conditions for producing ACE inhibitory peptides by strain GY-3 are as follows: 37 °C, 3% inoculation, and 1.0% glucose, 0.6% soy protein, 1.0% yeast extract and 0.04% MnSO4·4H2O in skim milk medium. The maximal ACE inhibitory activity could reach up to 79.52%. On the basis of genome sequence data of the bacterium, it was found that the ACE-inhibiting peptide were involved in the protease system, the polypeptide transport system and the peptidase system. [Conclusion] The present study provides a broader source and foundation for the development of lactic acid bacteria that are capable of producing ACE inhibitory peptide. Studying the genome lays a foundation for the study of biological characteristics and mechanism of ACE inhibition activity in the future. Additionally, this study is of great significance for the development of antihypertensive products.

Abstract:[Background] Luteimonas abyssi XH031 is one kind of marine bacteria with strong starch degradation ability. Previous studies showed that LamA, a cold-adapted α-amylase identified from strain XH031, kept high activity under low temperature. LamA will have great application prospects if the high temperature tolerance is improved. [Objective] To determine the calcium ion-dependent thermo-stability enhancement mechanism of LamA, site-directed mutagenesis of key amino acids in the calcium ion binding region was constructed. [Methods] The thermo-stability of LamA was measured in the presence of different chemicals. The amino acid sites that affect calcium ion binding and thermal stability were searched by bioinformatics analysis. Furthermore, the mutant proteins were constructed by the site-directed mutagenesis method and then overexpressed and purified. [Results] Under the calcium-free conditions, LamA was completely inactivated after incubation at 65 °C for 30 minutes. However, in the presence of 5 mmol/L calcium ion, LamA still had 36% of the activity after incubation at 65 °C for 30 min. The result showed that calcium ions can significantly improve the thermo-stability of LamA. The D200R and H233D/M234C mutant proteins completely lost starch degradation activities. Moreover, the activities of N120D, Q185E and T224D mutant proteins were decreased. However, the mutant proteins kept similar stability compared with the wild-type enzyme under the high temperature and calcium-free conditions. N120D mutant protein preserved only 27.8% residual activity compared with the wild-type enzyme at 65 °C supplemented with the calcium ion. Through molecular biology experiments and protein structure simulation, we speculated that the calcium ion binding to the Asn120 site stabilized the structure of LamA under high temperatures. [Conclusion] This study preliminarily clarified the mechanism of calcium ion-dependent thermo-stability enhancement of the cold-adapted α-amylase LamA and provided a theoretical basis for the engineering transformation of related enzymes.

Abstract:[Background] Chlorinated benzenes (CBs) are widely used for the production of many important industrial compounds. High toxicity and environmental persistence of CBs bring serious threats to human health and the environment. Developing new methods for efficiently degrading CBs has become hot topics in this field. [Objective] The objective of this study was to construct a novel synthetic system, containing nanoscale zero-valent iron (NZVI) and Pseudomonas sp. JS100 (JS100), to explore its degradation efficiency of pentachlorobenzene (PeCB) and the potential degradation mechanism under aerobic conditions. [Methods] The NZVI and JS100 reaction system was constructed to degrade PeCB. The concentrations of PeCB and its intermediates in the reaction system were determined periodically by GC-MS. Moreover, the morphology and growth of JS100 were observed. [Results] The NZVI-JS100 reaction system could degrade about 55.4% of PeCB at 36 h. Compared with the single system of NZVI or JS100, it showed better degradation efficiency The reaction process accorded with pseudo-first-order reaction kinetics, and the rate constant was 0.020 48 h–1. Based on the results from GC-MS of the reaction intermediates, the mechanism of PeCB degradation could be rationally hypothesized. At aerobic conditions, hydroxyl radicals were generated from NZVI, and then attacked PeCB, leading to the production of lower chlorinated benzenes. While JS100 used them as nutrient for growth successively. At the same time, it provided multiple attachment sites and reduced the aggregation of NZVI, and thus, improving the efficiency of the NZVI-JS100 system. [Conclusion] The NZVI-JS100 system synthesized in this study exhibited a high degradation efficiency for PeCB. Moreover, NZVI-JS100 system provided an important reference for the remediation of organic pollutants such as higher chlorinated benzenes in complex environments.

Abstract:[Background] Anaerobic ammonium oxidation (Anammox) is one of the important processes of nitrogen cycling. Previous studies have confirmed that anaerobic ammonium-oxidizing bacteria existed in various habitats, but there is no comprehensive understanding about their ecological distribution. [Objective] In this paper, we analyzed the diversity distribution of anammox bacteria in multiple habitats. This study will demonstrate the characteristics of community structure of anammox bacteria in different habitats and will illuminate the relationship between diversity distribution and environmental factors. [Methods] Based on the established anammox bacterial 16S rRNA gene sequence database, high throughput sequencing technology was used to analyze the diversity distribution of anammox bacteria in different habitat. [Results] The diversity of anammox bacteria in mangrove, bay and estuary habitats were relatively higher than that of sludge and red soil. Anammox bacteria in these habitats were dominated by Candidatus Brocadia, Ca. Scalindua and unclassified genes based on the phylogenetic analysis. From river to mangrove, the dominant species of Anammoxers shifted from Ca. Brocadia to Ca. Scalindua with the increase of salinity, and redundancy analysis showed that salinity was an important factor in shaping the community of anammox bacteria. [Conclusion] The community structure of anammox bacteria is different in different habitat, and environmental differences directly affected the population distribution and the evolution of anammox bacteria.

Abstract:[Background] Xishuangbanna Nature Reserve is rich in biodiversity, but few reports about plant growth promoting rhizobacteria especially actinomycetes in this reserve are found. [Objective] Screen the plant growth promoting rhizobacteria in Xishuangbanna Nature Reserve and test their growth-promoting ability. [Methods] Plant growth-promoting rhizobacteria was selected by five types of medium and followed by taxonomic identification based on 16S rDNA sequence analysis. The ability of obtained strains to produce IAA, siderophores, dissolved phosphorus, cellulase, and amylase activities were determined by Salkowski method, CAS method, molybdenum anti-chromogenic method, CMC-Na method and improved Young method, respectively. [Results] Fourteen strains of growth-promoting rhizobacteria, were isolated and purified from soil samples and identified as Streptomyces, Nocardia, Bacillus, Ensifer, Mesorhizobium, Azospirillum and Stenotrophomonas. Among the 14 strains isolated, strain B433 had the maximum capacity of indole acetic acid (9.23 mg/L) at 12 d. The strains of B351, B453 and B546 had strong iron siderophore production capacity, where the Su value were all >80%, and the maximum was 86.67%, with a strength of +++++. Strain B541 showed the strongest phosphorus solubility, and the concentration of dissolved phosphate reached 9.79 mg/L. The comprehensive cellulase production capacity of strain B442 was 31.86 U/mL. The amylase activity of strain B412 was 16.07 U/mL. [Conclusion] The rhizosphere soil in Xishuangbanna Nature Reserve contained a variety of growth-promoting rhizobacteria with strong and broad spectrum growth-promoting ability, which is valuable for research and exploitation. This study provides reliable basis of bacterial strain resource for the development of microorganism resource in Xishuangbanna Nature Reserve.

Abstract:[Background] Anaerobic digestion is the main method for treating restaurant food waste in China. Microorganism plays a crucial role in the anaerobic fermentation of restaurant food waste, however, there is limited study on the microbial structure and biodiversity in this field. [Objective] This work is aiming at providing scientific evidences for improving restaurant food waste treatment technologies and bio-resource recycling efficiency, by analyzing the microbial diversity and community structure for each processing unit. [Methods] Three waste liquid samples from typical units of oil-water separation, anaerobic fermentation, and residue dewatering were collected in a typical restaurant food waste treatment plant. The high-throughput sequencing technology of 16S rRNA gene was applied to analyze the composition, abundance, dominant microorganism, and their impacting factors. [Results] The microbial communities in anaerobic fermentation and residue dewatering samples showed higher microbial diversity than those in oil-water separation samples. At the phylum level, Firmicutes accounted for the highest proportion of 81.1% in all samples, followed by Bacteroidetes and Chloroflexi, accounting for 15.81% and 4.59% respectively, and at the genus level, Lactobacillus, Syntrophomonas, etc. had higher relative abundance. Several generic microbes had both environmental and resource properties, e.g., Pseudomonas, which accounts for 32.67% relative abundance in residue dewatering process, had the function of producing polyhydroxyalkanoates (PHA) but contained few pathogenic or opportunistic pathogenic bacteria. The most significant impacting factor of microbial diversity was pH, followed by the ammonia nitrogen content. [Conclusion] Characteristics of microbial community structure and diversity in a typical restaurant food waste treatment plant were found in this work, in addition, several recommendations for optimizing the treatment process and promoting bio-resource recycling were put forward.

Abstract:[Background] Kalidium foliatum are typical salt-tolerant plants widely distributed in saline-alkali desert areas. Salt accumulation constructed a salt island in the rhizosphere. There was rare investigation about how the microorganism succeed and adapted in the salt accumulation process. However, it was important to reveal the salt tolerance of Kalidium foliatum and formation of the salt island. [Objective] We analyzed the distribution characteristics of microorganism in soils of the rhizosphere, the root zone and the environment, and determined the effect of different salt concentration treatments on metabolic diversity of rhizosphere bacteria using Biolog Eco microplate. [Methods] Soils of the rhizosphere, the root zone and the environmental were sampled from Heshuo County, Xinjiang. The Biolog Eco microplate technique was used to analyze the utilization of microbial carbon sources. The rhizosphere soil samples were diluted by different NaCl concentrations, and then the dilution was taken to add into Biolog Eco microplates. The effect of the salt concentration on rhizosphere microbial activity and diversity was analyzed. [Results] Microbial metabolic activity of the samples showed a trend of the rhizosphere > the root zone > the environment. With the increase of distance from the root, the proportion of microorganisms using amino acids and amines as carbon sources decreased significantly, while the proportion of microorganisms using polymeric compound carbon sources increased significantly such as Tween, Glycogen and so on. Analysis of diversity showed that the Shannon index (H) decreased with significant difference. The significant effect of dilution solutions in different NaCl concentrations was observed on the metabolic activity and distribution of microbial community. And microbial activity of the rhizosphere was the highest at 5% NaCl solution as dilution solution. [Conclusion] The composition and distribution of microorganisms around the roots of Kalidium foliatum showed obvious evolution regularity. And it is necessary to optimize the cultivation condition using Biolog Eco microplate.

Abstract:[Background] Bayinbuluke grassland in Xinjiang is the second largest alpine grassland in China. There is a kind of white mushroom (Tricholoma mongolicum) that grows on the grassland belongs to Tricholoma, which can form a typical mushroom ring. [Objective] In order to understand the species composition and community structure of fungi around the mushroom ring, and provide a theoretical basis for study the formation and growth of mushroom circle and its fruiting body. [Methods] We utilized high-throughput sequencing technology through Illumina HiSeq system to analyze the species richness and microbial structure diversity of the mushroom fungus. [Results] The valid sequences were annotated into 809 OTUs, and they were divided into 5 phyla, 26 classes, 79 orders, 166 families, and 232 genera. The species richness, community structure diversity and specific species on the mushroom ring are lower than those outside the circle and within the circle. There are some special species on the fairy ring, such as Xylodon nothofagi, Agaricales, Phaeococcomyces, Ochrocladosporium adansoniae, Coniochaeta ligniaria, Tomentella amyloapiculata, Dothideomycetes, Incertea_sedis_Helotiales. [Conclusion] This study showed that the formation of mushroom rings affected the distribution of soil fungi, and the dominant fungal group on the circle had inhibitory effects on other fungal groups. The outcrop growth of mushroom rings may be related to the imbalance of microbial community structure.

Abstract:[Background] High-efficiency composting agents, especially fungi, have become a research hotspot for improving the efficiency of green waste compost. Due to the disadvantaged of fungi application such as its sensitivity to oxygen and substrate, the role of bacteria in composting process has gradually been studied. Bacillus subtilis (B. subtilis) BL03 was screened from the composting process of green waste using CMC-Na as substrate in our laboratory. The BL03 strain with good cellulose decomposition ability could improve the speed of cellulose degradation and humus synthesis in the green waste composting. [Objective] To further improve the cellulase activity of BL03. [Methods] The atmospheric pressure room temperature plasma (ARTP) was used to mutate the bacteria. Three rounds of screening were carried out via measuring the diameter of the hydrolyzed transparent circle on CMC-Congo red solid medium and detecting the activity of cellulase after liquid fermentation. The genetic stability of the mutant was measured after 10-generations culturing. The optimum growth temperature and initial pH of medium for mutant fermentation were determined by setting gradient temperature and pH culture. Orthogonal design was adopted to determine the industrial raw materials for the fermentation of the mutants. [Results] Two mutant strains, of which the enzyme activity increased by 69% and 72% respectively, were obtained. Also, the cellulase activity of the two mutants were stable after 10 generations culturing. The optimum culture temperature for the highest cellulase activity mutant, BLA3890, was 37 °C, and the initial pH of the medium was 5.0?6.5. An economical fermentation medium was obtained from this study. [Conclusion] BLA1973 and BLA3890, which obtained from Bacillus subtilis BL03 mutated by ARTP, are valuable in composting process of green waste or other cellulose degradation applications and need further research.

Abstract:[Background] Due to the extensive use of antibiotics, antimicrobial resistance has become a big problem. Searching for new antibacterial drugs has become a research hotspot. [Objective] Cloning and expression of quorum quenching enzyme and investigate its effect on the pathogenicity of Pseudomonas aeruginosa. [Methods] Quorum quenching enzyme gene aiiA gene from quorum quenching bacterium Bacillus sp. QSI-1 was amplified by PCR methods. The aiiA gene was cloned into the expression vector pET30a and transformed into E. coli BL21(DE3). The expression AiiA protein was purified with a HiTrap Q Sepharose column. P. aeruginosa PAO1 was cultured in medium containing different concentration of quorum-quenching enzyme. The supernatant was used to detect the level of pyocyanin, rhamnolipid and total protease, and biofilm also been detected. The quorum-quenching enzyme was applied to Caenorhabditis elegans infected with P. aeruginosa, the survival rate of the nematode was calculated. [Results] We successfully cloned an N-acylhomoserine lactonase gene from Bacillus sp. QSI-1. The purified quorumquenching enzyme significantly inhibited the production of virulence factors and biofilm formation in P. aeruginosa and reduced the mortality of nematodes infected by P. aeruginosa. [Conclusion] As a substance that can effectively inhibit pathogenic bacteria, quorumquenching enzyme may become a new drug for clinical treatment of bacterial infections.

Abstract:[Background] The cyanobacterial tricarboxylic acid cycle, which produces succinic acid, is different from that of other species. The existences of α-ketoglutarate decarboxylase and succinic semialdehyde dehydrogenase in cyanobacterium make its tricarboxylic acid cycle complete. Succinic semialdehyde dehydrogenases that catalyze the conversion of succinic semialdehyde to succinic acid, exist in cyanobacteria extensively. [Objective] Clone, express and purify the protein encoded by the cce4228 gene of Cyanothece sp. ATCC51142, and characterize its biochemical properties in vitro. [Methods] cce4228 gene was cloned into pET-28a vector, overexpression of recombinant cce4228 protein was induced by isopropyl β-D-thiogalactoside (IPTG) in E. coli BL21(DE3) and then the protein was purified by Ni-NTA affinity chromatography. The biochemical properties of cce4228 protein were characterized by spectrophotometric and bioinformatics methods. [Results] An expression plasmid pET-28a-cce4228 was constructed and cce4228 protein was overexpressed in soluble form in E. coli BL21(DE3). Recombinant cce4228 protein with purity higher than 90% was obtained. Steady-state kinetic and bioinformatic studies demonstrated that cce4228 protein was a NADP+-dependent succinic semialdehyde dehydrogenase. [Conclusion] The cce4228 gene in Cyanothece sp. ATCC51142 encodes a succinic semialdehyde dehydrogenase, which prefers to use NADP+ rather than NAD+ as its cofactor. These results will provide basis to understand the structure-function relationship and catalytic mechanism of cce4228 protein further.

Abstract:[Background] PilZ domain is the earliest discovered cyclic diguanylate (c-di-GMP) receptor signaling molecule, regulates the activity of the target gene or protein after binding to c-di-GMP, and plays a vital role in the growth of bacteria. However, the study of PilZ domain in Brevibacillus breves is a relative deficiency. [Objective] To mine the gene of PilZ domain in Brevibacillus breves GZDF3 strain and construct the recombinant expression system for further research. [Methods] The PilZ domain model was downloaded from the Pfam database. HMMScan software was adopted to scan the genome of GZDF3 strain. Protein conserved domains were analyzed in Conserved Domain Database (CDD). The product of protein was predicted by protein BLAST. The physical and chemical properties of the protein were determined by online bioinformatics analysis softwares in ExPASy website. The recombinant expression vector of the gene was constructed to obtain recombinant protein. [Results] Five genes coding protein containing PilZ domain were discovered in GZDF3 genome. The gene named as Gene4836 encoded a glycosyltransferase by homology analysis. The Gene1423 was a protein-coding gene of YcgR superfamily and Gene1723 encoded hyaluronan synthase belonged to glycosyltransferase superfamily 2. The other two genes Gene2571 and Gene2956 encoded hypothetical proteins. The result of bioinformatics analysis suggested that the molecular weight of the product of Gene4836 was 24.08 kD, and the isoelectric point was 6.39. It was an acidic hydrophilic protein. A conserved PilZ domain was found in the C end of the protein of Gene4836. The optimal expression condition was 0.5 mmol/L lactose for 20 h at 30 °C. A recombinant protein about 25 kD was detected by SDS-PAGE electrophoresis, consistent with the result of bioinformatics analysis. [Conclusion] It is the first report for recombinant expression protein with PilZ domain in Brevibacillus breves. It would provide a foundation for further study of the Pilz domain function.

Abstract:[Background] Xanthomonas campestris pv. campestris (Xcc) is a causal agent of black rot in cruciferous plants. The diffusible signal factor (DSF)-dependent quorum sensing (QS) system and RpfB-dependent QS exit mechanism are closely associated with Xcc pathogenicity. [Objective] We investigated the effects of 18 amino acids on the biosynthesis of DSF-family QS signals, to provide a clue for developing new strategy to control plant diseases. [Methods] The Xcc mutant strain ΔrpfC was grown in the XYS medium supplemented with 18 amino acids, respectively. The crude DSF extracts after 24 h to 36 h inoculation were then analyzed by high performance liquid chromatography (HPLC). [Results] 1) Among the 18 amino acids tested, methionine, tryptophan and cystine were found to significantly decrease DSF and BDSF levels in the ΔrpfC culture. The inhibitory effect was closely associated with amino acid concentration; 2) Additive inhibitory effects between three amino acids were observed; 3) Addition of methionine, tryptophan or cystine had no significant effect on the levels of DSF and BDSF in the ΔrpfCΔrpfB culture. [Conclusion] Methionine, tryptophan and cystine probably induce Xcc to exit from quorum sensing stage via the RpfB-dependent pathway.

Abstract:[Background] The arid zone in central Ningxia is characterized by perennial water shortage, sparse vegetation, serious desertification and fragile ecological environment. Land desertification leads to the decline of land productivity and restricts the development of agriculture in this area. Therefore, it is of great significance to improve the farmland ecological environment in the arid zone of central Ningxia. [Objective] In order to provides basic data and theoretical basis for the rational utilization and development of land resources and the further study of soil fungal diversity in arid areas. [Methods] This research used Illumina MiSeq high-throughput sequencing technology to study the species and diversity of soil fungi, and analyze the fungal diversity index and community structure. [Results] Among the five treatments, fungi species were abundant and richness index had no difference. The diversity index of fungi was highest in Zhangzagu No. 5 millet and lowest in quinoa, and there were significant differences. Ascomycota was the most dominant phylum in the rhizosphere soil of different crops, and the relative abundance ranged from 73.00% to 89.14%, which was much larger than the subdominant phylum——Basidiomycota (3.9%?16.5%) and showed a very obvious advantage; Apart from that Acremonium and Schizothecium were common dominant genera. Through DCA correspondence analysis and correlation analysis, it was found that soil available phosphorus and soil alkali-hydrolyzed nitrogen affected the structure and functional diversity of soil fungi community. The change of soil microbial community structure and functional diversity was the result of interaction between soil physical and chemical properties and microorganisms. [Conclusion] The soil nutrients of recreational and crop-growing farmland can be increased in different degrees, the soil pH decreases, and the fungal community structure and diversity change. It shows that rational farmland use is conducive to enriching the structure and diversity of soil microbial community, improving soil characteristics, and then promoting the stability of soil ecosystem in the region, and improving the rational utilization of farmland land resources.

Abstract:[Background] Lycium barbarum is a traditional edible herb, and dark septate endophytes (DSE) is an important component of endophytic fungi in L. barbarum. [Objective] DSE strains were isolated from the roots of cultivated and wild varieties of L. barbarum in Ningxia. In order to analyze the diversity and the community constitution of DSE associated with L. barbarum and understand the colonization and distribution of DSE in L. barbarum. [Methods] The roots of Ningqi-1, Ningqi-3, Ningqi-5, Ningqi-6, Ningqi-7, Ningqi-8, L. barbarum var. auranticarpum and L. ruthenicummurr were collected from Ningxia wolfberry cultivation garden. Strains were identified by morphological characteristic and rDNA-ITS sequence, and DSE of roots from L. barbarum were validated by Koch’s Rule. [Results] DSE live as mircosclerotia in the roots of L. barbarum. In this paper, 279 DSE were isolated from roots of eight kinds of L. barbarum, belonging to 18 genera, which are rich in species diversity. Fusarium were a common and dominant genus in various breeds. The relative frequency was up to 85%. Monosporascus, Talaromyces and Earliella were firstly reported in L. barbarum. There were significant differences in biodiversity index, evenness index and Simpson’s index of DSE community among different varieties of L. barbarum. [Conclusion] DSE of L. barbarum were rich in resources of cultivated and wild varieties of L. barbarum, DSE could form a symbiotic relationship with L. barbarum and enhance the adaptability of wolfberry to the ecological environment.

Abstract:[Background] Citrus Huanglongbing (HLB), one of the most devastating diseases in citrus production, is mainly caused by “Candidatus Liberibacter asiaticus” (CLas). With the available of CLas whole genome sequence, the gene expression study and functional verification of CLas genes become possible. [Objective] To screen the reference genes of CLas and assess the expression of reference genes in different infection stage and different plant hosts. [Methods] Twenty-three reference genes of CLas were screened according to the categories of gene function and analyzed by real-time quantitative PCR (16S rRNA gene as the control). Combined analysis of the standard deviation of Ct values, geNorm, NormFinder and RefFinder were used to evaluate the expression stability of the reference genes. [Results] Fourteen pairs of primers showed superior specificity and stability in plant sample collected from different infection stage of CLas and different citrus cultivars. The stability of reference gene were ranked as: ftsZ>gyrA>rpoB1>ftsA>secA>gap>zapE>gmk2>rpoD>secY>rpoO>ftsW>gmk1>recA. Based on the geNorm pairing variation value Vn/n+1, ftsZ and gyrA were selected as reference genes for further evaluation. By using ftsZ+gyrA and 16S rRNA gene as reference genes, a CLas pathogenic gene (LasΔ5313) showed similar expression patterns among above reference genes. [Conclusion] The expression of housekeeping genes associated with DNA replication and cell division function of CLas were relatively stable. ftsZ and gyrA can be used as reference genes for the expression analysis of CLas genes. This study provides foundation for the analysis of CLas genes by using real-time PCR and the pathogenic mechanism of CLas.

Abstract:[Background] Salmonella typhimurium is an important zoonotic pathogen that causes economically devastating to livestock breeding and potential threats to human health. Regulatory proteins are involved in the regulation of the survival and infection of pathogens. [Objective] To analyze the effect of regulatory protein RstB on the biological characteristics and pathogenicity, the rtsB gene mutant and complemented strain of S. typhimurium were constructed and characterized. [Methods] We constructed the rtsB gene mutant strain and complemented strain of S. typhimurium SAT52 using the Red recombination system and complementary plasmid. Then we analyzed the growth characteristics, motility, biofilm formation, cells adhesion and invasion ability, and pathogenicity. [Results] The deletion of rtsB gene did not affect the growth rate of SAT52. However, the mutant strain showed significantly enhanced motility and decreased biofilm formation ability. Compared with the wild-type strain, the capacities of adherent to Hela cells and survival in RAW264.7 cells were significantly decreased for the mutant strain. The results of animal infection experiments showed that the virulence of mutant strain was attenuated. [Conclusion] The rtsB gene plays an important role in the pathogenesis of S. typhimurium infection, providing a certain basis for further understanding of the pathogenic mechanism of S. typhimurium. These data indicated that regulator RtsB plays an important role in the process of S. typhimurium infection, which would help us to comprehensive understand the pathogenic mechanism of S. typhimurium.

Abstract:[Background] The multi-drug resistance of Salmonella is becoming more and more serious. It is particularly urgent to study the mechanism of antibiotic resistance in Salmonella. The two-component system (TCS) is closely related to bacterial antibiotic resistance. [Objective] The baeSR gene deletion and complementary strains of Salmonella typhimurium were carried out to study the effect of BaeSR on antibiotic resistance in S. typhimurium. [Methods] S. typhimurium selected in vitro to obtain cipro?oxacin-resistant strain CR, and take it as the study object, deleted mutants CRΔbaeSR were obtained by homologous recombination mediated by suicide plasmid pLP12. And we used chloramphenicol and arabinose-induced vmt toxicity gene for selecting. In addition, the complemental strains of CR C△baeSR were generated through transformation of a recombinant expression plasmid of pBAD-baeSR into CR△baeSR. And minimum inhibitory concentration (MIC) of 11 antibiotics against CR, CR△baeSR and CR C△baeSR strains was determined by broth microdilution method. Growth curve, migration ability and biofilm formation ability of three strains were also measured. [Results] The results showed that the MICs of CIP, ENR, SAR, CEF, GEN, AMK, APR were lower than those of wild strains; The growth rate of CR△baeSR was slightly slower than CR, and the final concentration was relatively low, but there was no significant difference (P>0.05). The migration ability (P<0.05) and biofilm formation ability (P<0.01) of CR△baeSR decreased significantly. [Conclusion] The deletion of baeSR gene in S. typhimurium can affect the ability of migration and biofilm-forming to affect the sensitivity to antibiotics.

Abstract:[Background] Escherichia fergusonii is a pathogen highly homologous to Escherichia coli, but its antimicrobial resistance has rarely been reported. [Objective] In this study, two E. fergusonii strains, EFCF053 and EFCF056, which were isolated from chicken feces in Zhejiang province, were tested and analyzed for antimicrobial resistance. [Methods] MICs were determined by microbroth dilution method. The whole genome sequences were obtained by using the next-generation sequencing, and acquired antimicrobial resistance genes were predicted via ResFinder database. Plasmids and resistance genes were identified by S1-PFGE and Southern blotting hybridization. [Results] It was found that both strains were resistant to ampicillin, gentamicin, florfenicol, sulfamidoxazole, trimethoprim/sulfamethoxazole and tetracycline. The strain EFCF056 was also resistant to colistin, ceftiofur, spectinomycin, enrofloxacin and ofloxacin. It was predicted that resistance genes existing in the strains were beta-lactam blaTEM-1A, blaCTX-M-65, blaOXA-1, blaTEM-1B, blaCTX-M-55; aminoglycoside aac(3)-IId, aph(3')-Ia, aph(3'')-Ib, aph(6)-Id, rmtB, aac(6')-Ib-cr, aadA2; colistin mcr-1; quinolone qnrS2, aac(6')-Ib-cr, oqxA, oqxB; fosfomycin fosA; macrolide mph(A); phenicol floR, catA1, catB3; rifamycin ARR-3; sulfonamides sul1, sul2, sul3, dfrA12, dfrA14; tetracycline tet(A). In addition, the data indicated that the plasmid harboring mcr-1 gene had the ability of conjugative transfer. [Conclusion] The results showed that E. fergusonii might be a reservoir of antimicrobial resistant genes. It is necessary to distinguish it from E. coli in the epidemiology of antimicrobial resistance. Further studies are needed to investigate the MIC frequency distribution, important drug resistance genes mcr-1 and ESBL for ensuring the accuracy of clinical detection.

Abstract:[Background] Bdellovibrio-and-like organisms (BALOs) can lyse pathogenic bacteria in aquaculture, so BALOs have enormous potential value in application. However, in practical applications, the inconsistency between BALOs’ growth conditions and the environment results in poor or even no effect. Therefore, it is critical to obtain BALOs with a broad range of adaptation. [Objective] The aim of this study was to screen out a euryhaline BALO for extensive application and increase its bdelloplast densities for better preservation. [Methods] A euryhaline BALO was isolate from a shallow bay with the host bacterium Bacillus subtilis. Molecular biological identification were carried out, and the biological characteristics as well as lysis properties of BDN-1 were studied. In addition, the factors affecting its bdelloplast densities such as monosodium glutamate, Ca2+ and Mg2+, indole were researched. [Results] A strain of euryhaline BALOs (named BDN-1) was isolated and identified. The optimal range of temperature, salinity and pH for BDN-1 were 20?30 °C, 0.5%?3.0%, 6.0?8.5. Lysis experiments on 30 tested strains showed that the lytic rate of BDN-1 was 86.7%, and for the 16 tested vibrios, the lytic rate was 87.5%. The monosodium glutamate, indole, Ca2+ and Mg2+ have effects on the bdelloplast densities of BDN-1. [Conclusion] This study provided a strain of BALOs with strong lysis ability which can be applied to sea and fresh water. Besides, the biological characteristics and factors of bdelloplast densities were identified, which laid the foundation for the efficient application of BALOs.

Abstract:[Background] Phage can specifically kill host bacteria especially the strains resistant to antibiotics. Phage can be a new antimicrobial agents alternative to conventional drugs. [Objective] No Enterobacter ludwigii phage has been reported until now. The purpose of this work is to isolate and characterize E. ludwigii phage. [Methods] The double-layer plate method was used for isolation, purification, and quantification of phage; SDS-PAGE was used for viral structural protein analysis; transmission electron microscopy (TEM) was used for phage morphology analysis; and the Crystal Violet staining and Congo Red agar methods were used for biofilm analysis. [Results] The phage GM20 was isolated from the environmental samples with the strain E. ludwigii GM20 as indicator. The plaques were transparent with the average diameter about 0.47 mm. TEM showed that GM20 has a contractile tail, and it was classified as a member of the Myoviridae family. The titer of phage is 7.65′109 PFU/mL, suggesting that it is a virulent phage. The one-step growth curve showed that GM20 had a latent period of 15 min and a burst size of 164.3 PFU/infection center. Further experiments showed that GM20 could inhibit bacteria growth effectively, and the average mutation frequency of phage-resistant mutants was about 1.06′10?5. [Conclusion] The virulent phage GM20 can effectively kill the host strain E. ludwigii X20 and may be used in the prevention and control of E. ludwigii infection.

Abstract:[Background] Recent studies have found that microgravity conditions can affect the proliferation and toxicity of some pathogenic microorganisms. Candida albicans is a typical conditional pathogenic fungus, which is ubiquitous in space environment and human body. It is of great significance to study the proliferation and toxicity of C. albicans under microgravity conditions. [Objective] Rotating cell culture system (RCCS) was used to simulate the microgravity environment for continuous subculture of C. albicans. The changes of proliferation, toxicity and gene expression of C. albicans in simulated microgravity environment were detected. [Methods] C. albicans were inoculated in high aspect rotating vessel (HARV) and cultured in RCCS for 14 days. After the culture completed, the proliferation rates of C. albicans were measured. The proliferation abilities were detected under different pH conditions, and the cytotoxicity and animal toxicity were determined. The differentially expressed genes were identified by RNA-seq. [Results] In the simulated microgravity group, the logarithmic phase of C. albicans was earlier and the proliferation rate was faster, the proliferation ability was generally improved under suitable pH conditions, but its relative biolfilm formation and toxicity to LoVo cells and mice was weakened. RNA-seq revealed that 280 genes expressed differentially more than 1.5 times (P<0.05), of which 248 were up-regulated and 32 were down-regulated in simulated microgravity environment. The differential genes were enriched by GO and KEGG, and the gene expressions of cell membrane formation and cell division were up-regulated in simulated microgravity environment , while the gene expressions of biofilm, cell adhesion and symbiotic adhesion host were down-regulated. [Conclusion] Simulated microgravity environment can change the proliferation and toxicity of C. albicans, and the related changes can provide reference for studying on the influence of microgravity environment on microorganisms.

Abstract:[Background] H5N1 influenza virus can cause severe respiratory infection in human with high fatality. [Objective] To analyze the virus A/Nanjing/1/2015, a human infected highly pathogenic avian influenza virus H5N1 confirmed in our laboratory, and to investigate its possible origins and genomic molecular characterization. [Methods] Whole genome sequencing was performed on the samples of patient?s sputum. CLC Genomics Workbench 9.0 was used for sequence assembly. Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 5.22 software. [Results] The virus belonged to H5 subtype clade 2.3.2.1c. The 8 segments were highly homologous with those viruses isolated from domestic poultry in Jiangsu and Zhejiang area and no re-assortment was observed. Molecular characterization analysis revealed that the amino acid sequences of hemagglutinin (HA) protein cleavage site were PQRERRRR/G. The Receptor Binding Sites (RBS) was in avian type, however, D94N, S133A and T188I substitutions enhanced the affinity to human receptors. A 20 amino acid deletion at position 49?68 in neuraminidase (NA) stalk, and a deletion at position 80?84 together with a P42S mutation in the non-structural protein 1 (NS1) were observed. Several amino acid mutations enhancing virulence and affinity to human cells in other proteins were also found in the present strain. Analysis on drug resistance sites revealed the presence of oseltamivir resistant mutation H274Y, and the virus remained sensitive to amantadine. [Conclusion] The human infected highly pathogenic avian influenza A (H5N1) virus A/Nanjing/1/2015 belonged to clade 2.3.2.1c and was avian in origin. The key sites in the present strain were relatively conservative; however, there were still several amino acid evolution and mutations to make it more favorable for infecting human. H5N1 avian influenza virus is evolving actively and continued surveillance is warranted.

Abstract:[Background] Streptococcus suis type 2 is an important pathogen that harms the pig industry and causes significant losses to the pig industry in China. So far, there is still a lack of model of encephalitis caused by Streptococcus suis type 2 infection, so we establish appropriate models for clinical drug development and mechanism research to lay the foundation for prevention and control of the disease. [Objective] Construction of Streptococcus suis type 2 infection in piglets induced encephalitis model, the clinical changes observed in piglets infected, pathological changes, Isolation and PCR analysis confirmed successful model building. [Methods] Six healthy ternary crossbred piglets were randomly divided into two groups, A and B. Group A piglets were inoculated with Streptococcus suis, and group B was inoculated with normal saline as the control. The clinical symptoms, necropsy changes and histopathological changes of group A piglets were observed. [Results] Group A piglets had elevated body temperature, depression, lethargy, and severe neurological symptoms, or even death. Severe lesions occur in the organs, such as swelling, necrosis, and dissolution of neurons in the spinal cord and brain. Focal necrosis and post-necrotic space are seen in the purkinje cell layer and granular layer of the cerebellar gray matter. [Conclusion] A model of encephalitis in piglets infected with S. suis type 2 was successfully constructed.

Abstract:[Background] Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. Type V secretion system (T5SS) plays an important role during the infection of APEC. [Objective] This study was carried out to analyze the distribution of T5SS in APEC and its correlation with E. coli phylogenetic group and other virulence factors. [Methods] The 15 E. coli T5SS were selected for primers designing, and the distribution of these T5SS in APEC clinical isolates were detected by PCR. The phylogenetic group and virulence factor of APEC strains were determined. The correlations between T5SS distribution and phylogenetic group and other virulence factors of APEC were analyzed. [Results] T5SS was widely distributed in APEC isolates. T5SS ydeK and pplfP was present in 98.55% and 92.03% of the APEC isolates, respectively. However, the distribution rates of T5SS upaC and pic in APEC were less than 10.00%. Most APEC isolates belonged to A, B1 and D phylogenetic groups, following by B2. The combinations analysis showed that T5SS ehaA, ehaB, pic and vat were predominated present in phylogenetic group D APEC isolates. While, the phylogenetic groups A and B1 were the predominated groups that harbor the ehaG, ag43/flu and apaC. However, no significant correlation between the distribution of T5SS and other virulence genes in APEC were found. [Conclusion] T5SS is widely distributed in APEC, and some T5SS was correlated with the phylogenetic group of APEC.

Abstract:Peri-implantitis diseases are characterized by the presence of an inflammatory process that affects the peri-implant tissues under loading, caused by microorganisms and might lead to the implant failure. Investigations on the microbiota of peri-implant biofilm would be important to define effective preventive and therapeutic strategies for peri-implantitis. With the development of sequencing technology, sequencing and analysis technology of 16S rRNA gene have recently been used to evaluate the microbiota associated with osseointegrated implants. These results expanded the knowledge on the diversity of the microbial communities associated with the disease; they also suggested that the microbial structure of peri-implantitis might be different with periodontitis. Here we review the recent progress about the microbiology of peri-implantitis biofilm based on 16S rRNA gene sequence analysis technology.

Abstract:The gut microbiota plays various important roles in the health of the host. Bees are highly socialized insects. The gut microbiotas of bees are distinctly different from most insects. They are composed of facultative anaerobic and micro-aerobic bacteria. They maintain a highly conservative and specific core microbiome. Recent studies show that gut microbiota of bees plays vital roles in metabolism, immune function, growth and development, and protection from pathogens. Gut microbiota plays an important role in bee health and disease. The destruction of gut microbiota will have adverse effects on bee health. In this paper, we review the characteristics and advances in research of colonization of microbiota in the honeybee. We address the effects of age, colony and season on the gut microbiota of honeybee, and the effects of host function and metabolism on the gut microbiota.

Abstract:Many fungi are closely related to human health, industrial and agricultural production and material circulation in the ecosystem. Fungi synthesize a variety of sex hormones, plant hormones and animal hormones. These endogenous hormones and exogenous hormones produced by plants and animals can be perceived by fungi and affect fungal growth and development, fruit body formation, metabolism, pathogenesis and symbiosis. However, little is known about the synthesis and signaling pathways, origin and evolution of the fungal hormones. The establishment of fungal hormonology will greatly promote the research and application of fungal hormones.

Abstract:Antibiotics are widely used in the treatment of human and animal diseases. Unreasonable use and abuse of antibiotics lead to the production and spread of resistant bacteria and resistance genes. Metagenomics can analyze the diversity of antibiotic resistance genes in different environments, and improve the existing or construct new metagenomic libraries, which will provide powerful references for future gene comparison. This article will review the application of metagenomics in the detection of microbial antibiotic resistance genes in humans, animals and the environment, and provide technical support for future assessment of resistance gene risks and addressing antibiotic resistance.

Abstract:Since the concept of a type 3 secretion system was proposed, the study of related molecular mechanisms has furthered our understanding of it. In contrast to the signal peptide in other secretory machineries, there is no conserved signal sequence when the protein is secreted or transported through the bacterial type 3 secretion system. A variety of secretory signals that guide the secretion of type 3 secreted protein have been discovered in the recent study. This article introduces the categories of bacterial type 3 secretion system, the types of secreted proteins in the secretory system and focuses on molecular characters of secretion signals, providing new ideas for the development of new antibacterial drugs.

Abstract:Escherichia coli Nissle1917 (EcN), as a probiotic, is mainly used to treat a variety of gastrointestinal disorder including inflammatory bowel disease, chronic constipation. EcN prevents human intestinal epithelial cells from adherent and invasion of evasive pathogen, and protects intestinal mucosal barrier and inhabits pathological inflammation. EcN also enhances intestinal immune function and regulates cytokine secretion. Recently, EcN has shown anti-cancer efficacy via tumor targeting ability. Furthermore, EcN enhances the tumor targeting effect when combined with chemotherapeutic drugs, providing a promising application of EcN in precise treatment of cancer. EcN provides a great potential in developing novel approach of cancer treatment.

Abstract:In 1975, the sequencing of the first cDNA genome, the phage фX174 genome, marked the beginning of the sequencing era. Since 1996, the launch of the Human Genome Project has greatly promoted the application and development of sequencing technology. The second generation sequencing technology is also known as high-throughput sequencing technology. Single-molecule DNA sequencing technology is a new generation of sequencing technology called third-generation sequencing technology, which developed in the last 10 years. It includes single-molecule real-time sequencing, true single-molecule sequencing and single-molecule nanopore sequencing. Because SMRT sequencing technology has the characteristics of long reading length, short sequencing period, without requirement for template amplification and direct detection of apparent modification sites, it provides researchers with new options. This paper reviews the basic principle and performance of SMRT sequencing technology and its application in microbial 16S rRNA gene, microbial whole genome sequencing and microbial macrogenome sequencing, and analyzes the advantages and problems of SMRT sequencing technology in environmental microorganisms.

Abstract:Nitrite reductase (NiR, EC1.7.2.1) is a kind of enzyme that catalyzes the reduction of nitrite (NIT). NiR is a key enzyme in the natural nitrogen cycle, which can degrade nitrite into NO or NH3. The classification, structural characteristics, catalytic mechanism and current application fields of nitrite reductase are reviewed in this report, which provides reference for further study of nitrite reductase.

Abstract:Hybrid teaching model of Microbiology courses based on “Large Lecture Class Combined with Small Tutorial Class” is a valuable model to guarantee the teaching effect when the software and hardware conditions are insufficient, and the main purpose is to implement the “student-centered teaching philosophy”. Teachers explain the contents of the syllabus to students in the “Large Lecture Class”, while provide students with review, case discussion, classroom test and supervise their learning process in the “Small Tutorial class”. This kind of hybrid teaching model changed the students’ role from passive listeners to active participants.

Abstract:Experimental teaching plays a key role in training applied talents in local university. Flipped classroom is a type of teaching strategy that students complete self-study before class and engage in discussions with mentor in class. Here, in order to apply the flipped classroom to experimental teaching, one experiment named ‘sausage making’ in the course of Food Fermentation Technology was performed, and an online course platform was used to deliver learning contents. This teaching practice including several parts, such as completing tasks before class, group discussion and experimental design in class, and making video after class. This flipped classroom model not only motivated the students to study more efficiently by themselves, but also improved their critical thinking and innovative skills. In the meanwhile, it also provided reference for the reform of experimental teaching.