Paenibacillus sp. 1-49 is a nitrogen-fixing bacterium that has the potential as a fertilizer. However, it grows poorly (OD600≤1) in both mineral medium and rich medium. To achieve high-yield biomass, we optimized fermentation medium for Paenibacillus sp. 1-49. Plackett-Burman Design and Central Composite Design in response surface methodology were used to optimize medium composition. The optimal fermentation medium contained:(per liter) 36.22 g Sucrose, 5.31 g Tryptone, 10.92 g Yeast Extract, 0.51 g MgSO4, 3.5 g NaCl, 0.02 g Na2MoO4 and 0.02 g FeSO4. The maximum OD600 of 10.280±0.009 was obtained in shake flask fermentation, reached 94.6% of the predicted value. We succeeded in using a response surface methodology to optimize the fermentation medium for Paenibacillus sp. 1-49. Our results will be useful for largescale fermentation of this strain and other Paenibacillus spp.

To understand the molecular biology information of ibeB gene of meningitic Escherichia coli isolates in calves. The strain used was isolated from the brain and liver tissue of calves died from Meningitis. It was identified to be an O161-K99-STa pathogenic Escherichia coli strain and named as bovine-EN and bovine-EG. Based on the sequence of ibeB gene of meningitic Escherichia coli K1 RS218 strain in GenBank, a pair of primers was designed and the ibeB gene was cloned from isolates by PCR. Part molecular biology information of ibeB among different strains was compared. The sequence length of isolates ibeB gene was 1500 bp, containing a 1371 bp open reading frame (ORF) encoding 457 amino acids. Bioinformatics analysis showed that the nucleotide and amino acid homology of ibeB gene of bovine-EN strain shared 90.5% and 96.9% identity with Escherichia coli K1 RS218 ibeB gene, respectively, while bovine-EG strain shared 99.4% and 100.0% identity with Escherichia coli K12 respectively. The ibeB gene of bovine-E strains encoded water-soluble protein whose molecular weight was 50.26 kDa and isoelectric point was 6.05. This protein contained a signal peptide A but no transmembrane domain. Subcellular localization of ibeB belonged to the secreted protein, which secretory signal path site (SP) proportion was 0.939. The ibeB gene was cloned from meningitic E. coli isolates and had higher homology and similar biological characteristics with meningitis E. coli K1 RS218ibeB, which belongs to extraintestinal pathogenic Escherichia coli.

We improved the thermostability of LipA from Burkholderia cecapia ZYB002 by protein engineering technology to expand the application of lipase LipA. On the basis of B-factor value of lipase LipA, series of potential mutation hotspots were selected for iterative saturation mutagenesis and the corresponding small mutation gene libraries were then constructed to screen the hyperthermal variants. From the above mutation libraries, we obtained a series of mutants whose enzyme half-life at 55 degrees C increased by 1.7 to 2.2-fold. B-factor iterative test (B-FIT) is feasible to mutate thermostable strains.

The roles of CRIPR-Cas systems in bacterial host protection against mobile genetic elements ( MGEs) are now well known, but there is mounting evidence that these systems modulate other processes, such as the genetic regulation of group behavior and virulence, DNA repair and genome evolution. Here, we reviewed the structure, types and mechanism of interference of CRISPR-Cas system as well as the additional functions of CRISPR-Cas beyond adaptive immunity. Furthermore, we discussed the mechanisms for phages to overcome bacterial CRISPR-Cas system, and the prospective evolution of interactions between phage and host.

[ OBJECTIVE] Polyhydroxybutyrate-co-hydroxyvalerate (PHBV) was used as solid carbon source and biofilm carrier to remove nitrate from recirculating aquaculture system (RAS). Dynamics of microbial community structure in biofilm coating on carbon source packed into denitrification reactor were investigated. [METHODS] Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial community in biofilm from denitrifiation reactor. Bacteria degrading PHBV were isolated from the reactor using pure culture method. [RESULTS] Nitrate decreased remarkably in the RAS connected with dentrification reactor. In contrast, Nitrate increased continuously in the conventional RAS without dentrification reactor. According to the phylogenetic analysis, the microbes in the biofilm samples from denitrification reactor were divided into Proteobacteria ( p-proteobacteria, γ-proteobacteria and δ- proteobacteria) , Firmicutes and Bacteroidetes. The major advantageous populations were Acidovorax and Bacillus in the 40-day reactor. The advantageous populations in the 150-day reactor were in order of Clostridium, Desulfitobacterium, Dechloromonas, Pseudoxanthomonas and Flavobacterium. Pure cultures of bacteria degrading PHBV isolated from denitrification reactor were classified into Acidovorax, Methylibium, Pseudoxanthomonas and Dechloromonas. [CONCLUSION] Nitrate could be removed effectively from RAS using PHBV as carbon source. Advantageous bacteria and their dynamic changes were ascertained in biofilm from denitrification reactor packed with PHBV.

The effect of transgenic cotton on the rhizosphere bacteria can be important to the risk assessment for the genetically modified crops. We studied the rhizosphere microbial community with cultivating genetically modified cotton. The effects of transgenic Bt + CpTI Cotton (SGK321) and its receptor cotton (SY321) on rhizosphere total bacteria and ammonia oxidizing bacteria population size were studied by using droplet digital PCR. We collected rhizosphere soil before cotton planting and along with the cotton growth stage (squaring stage, flowering stage, belling stage and boll opening stage). There was no significant change on the total bacterial population between the transgenic cotton and the receptor cotton along with the growth stage. However, the abundance of ammonia oxidizing bacteria (AOB) in both type of cottons showed significant difference between different growth stages, and the variation tendency was different. In squaring stage, the numbers of AOB in rhizosphere of SY321 and SGK321 increased 4 and 2 times, respectively. In flowering stage, AOB number in rhizosphere of SY321 significantly decreased to be 5.96 x 10(5) copies/g dry soil, however, that of SGK321 increased to be 1.25 x 10(6) copies/g dry soil. In belling stage, AOB number of SY321 greatly increased to be 1.49 x 10(6) copies/g dry soil, but no significant change was observed for AOB number of SGK321. In boll opening stage, both AOB number of SY321 and SGK321 clearly decreased and they were significantly different from each other. Compared to the non-genetically modified cotton, the change in abundance of ammonia oxidizing bacteria was slightly smooth in the transgenic cotton. Not only the cotton growth stage but also the cotton type caused this difference. The transgenic cotton can slow down the speed of ammonia transformation through impacting the number of AOB, which is advantageous for plant growth.

To screen for compounds against influenza A virus by using the viral promoter reporter cell line HeLa-IAV-Luc and investigate their anti-viral mechanism. We screened the inhibitors of influenza A virus by infecting HeLa-IAV-Luc cells with influenza A virus WSN/H1N1 and treating it with the compounds isolated from microbial metabolites. The cell lysates were then subjected to the luciferase assay. We conducted the pesudovirus assay to analyze whether the compound affected the function of hemagglutinin (HA). We carried out the influenza viral promoter reporter assay to examine whether the compound could inhibitinfluenza viral RNA polymerase (vRNP) activity. The effect of anti-viral compoundon influenza viral RNA synthesis was measured by real-time fluorescence quantitative PCR. Colletodiol inhibited the replication of influenza A virus in HeLa-IAV-Luc cells by luciferase assay. It did not inhibit the function of HA protein based on the results of the time-of-addition experiment and pseudovirus assay. The influenza viral polymerase promoter reporter assay indicated that Colletodiol could inhibit the activity of vRNP dramatically. Due to the inhibition of vRNP, the influenza viral RNA synthesis decreased significantly. Compound Colletodiol was an inhibitor of influenza A virus. It blocks the replication of influenza A virus by reducing the activity of vRNP.

To investigate the composition of culturable bacteria from soil of Grove Mountains, East Antarctica and analyze the production of extracellular hydrolytic enzymes and antibacterial and antifugal activities of these culturable bacteria. We used spread plate method to obtain culturable bacteria. Phylogenetic relationship was analysed based on their 16S rRNA gene sequences. We used plate method and agar block method to detect the production of extracellular enzymes and antibacterial and antifungal activities of these bacteria, separately. We obtained a total of 39 isolates from all the soil samples. They belonged to 20 genera and grouped into Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, Deinococcus-Thermus lineages, of which each group occupied 48.7%, 25.6%, 20.5%, 2.6%, 2.6% of the total, respectively. Bacillus was dominant. We isolated different strains from soil stored at different temperatures, and this may be explained as viable bacteria were different at diverse temperatures. Of the 39 isolates 33 showed the activity of producing at least one extracellular enzyme. Amylase-producing strains were the most (64.1%), 6 strains were able to inhibit the growth of at least one tested bacterium or fungus. Strains with extracellular enzyme activity and antibacterial and antifungal activity would contribute to the exploration of cold-active enzymes and antibacterial (antifungal) material in Antarctica.

To construct a transformation system in Gliocladium sp. 6.102, a Cordyceps-colonizing fungus producing a variety of epipolythiodioxopiperazine (ETP) compounds with drug potentials. Agrobacterium tumefaciens-mediated transformation (ATMT) was used to transform Gliocladium sp. 6.102. The factors of bacterial cell concentration, co-cultivation time, pH and acetosyringone concentration were optimized. RESULTS; A total of 50 -100 transformants per 10(6) fungal conidia was obtained via the optimal ATMT method. The genes encoding hygromycin B phosphotase and enhanced green fluorescent protein (EGFP) were transferred into Gliocladium sp. by the optimal ATMT method. The marker genes were successfully expressed and stably maintained in the transgenic fungus. A transformation system was established for Gliocladium sp. 6.102 and this system may be useful to identify ETP biosynthetic genes in Gliocladium.

High-level expression system of heterologous protein mediated by internal ribosome entry site (IRES) in Saccharomyces cerevisiae was constructed, which could be used for other applications of S. cerevisiae in metabolic engineering. We constructed co-expression cassette (promoter-mCherry-TIF4631 IRES-URA3) containing promoters Pilv5, Padh2 and Ptdh3 and recombined the co-expression cassette into the genome of W303-1B-A. The URA3+ transformants were selected. By comparing the difference in the mean florescence value of mCherry in transformants, the effect of three promoters was detected in the co-expression cassette. The copy numbers of the interested genes in the genome were determined by Real-Time PCR. We analyzed genetic stability by continuous subculturing transformants in the absence of selection pressure. To verify the application of co-expression cassette, the ORF of mCherry was replaced by beta-galactosidase (LACZ) and xylose reductase (XYL1). The enzyme activities and production of beta-galactosidase and xylose reductase were detected. mCherry has been expressed in the highest-level in transformants with co-expression cassette containing Pilv5 promoter. The highest copy number of DNA fragment integrating in the genome was 47 in transformants containing Pilv5. The engineering strains showed good genetic stability. Xylose reductase was successfully expressed in the co-expression cassette containing Pilv5 promoter and TIF4631 IRES. The highest enzyme activity was 0. 209 U/mg crude protein in the transformants WIX-10. Beta-galactosidase was also expressed successfully. The transformants that had the highest enzyme activity was WIL-1 and the enzyme activity was 12.58 U/mg crude protein. The system mediated by Pilv5 promoter and TIF4631 IRES could express heterologous protein efficiently in S. cerevisiae. This study offered a new strategy for expression of heterologous protein in S. cerevisiae and provided sufficient experimental evidence for metabolic engineering application of this system in yeast.