Abstract:

Abstract:Lipopolysaccharide is the main component of outer membranes, which serve as a permeability barrier. In this study, we investigated the role of the structure of lipopolysaccharide on the permeability of bacterial cell membranes. Nine Escherichia coli strains which make different structures of LPS were selected or constructed. Lipopolysaccharide and lipid A were extracted from these strains, and their structures were analyzed by using thin-layer chromatography and electrospray ionization mass spectrometry. The membrane permeability of these strains was analyzed by using N-phenyl-1-naphthylamine fluorescent probe. Wild type E. coli showed the least permeability, while mutants in which LPS structures were changed by the deletion or expression of related genes showed higher permeability. The number of phosphate groups and the acyl chains, and the length of the polysaccharide of lipopolysaccharide all affect the permeability of E. coli. The chain length of the polysaccharide has the largest effect on the permeability, followed by the number of the acyl chains. The results indicate that the cellular membrane permeability is related to the structure of lipopolysaccharide.

Abstract:

Abstract:Xylose-utilizing and thermophilic Geobacillus sp. Y565-5 was isolated from surface soil of an oilfield in Yumen Town, Gansu Province, China. A xylose isomerase (XylA) gene was cloned from the strain by PCR. The open reading frame of xylA (1 182 bp) encoded a protein of 394 amino acids, which showed high sequence homology (99% identity) with that of Geobacillus sp. Y412MC52. The intact coding region was subcloned into pET28a(+) vector and expressed in Escherichia coli BL21(DE3). The molecular weight of the recombinant protein was 45 kD based on SDS-PAGE and its xylose isomerase activity was detected through cysteine welts thiazole method after the induction of isopropyl β-D-1-thiogalactopyranoside? (IPTG). The optimum temperature and pH for the partially purified recombinant XylA activity were 90 °C and pH 8.0, respectively.

Abstract:In order to enhance the yield of dextran dextrinase (DDase) produced by Gluconobacter oxydans DSM 2003, the effects of pH from 3.5 to 6 on cell growth and DDase activity were investigated in a 3 L fermentor. Based on time courses of cell growth and DDase activity, we developed a two-stage pH control strategy, in which pH was controlled at 5.0 for the first 6 h and then 4.0 for remaining time. Under the optimized strategy, the production of DDase had a significant improvement. Moreover, the maximal DDase activity reached 4.03 U/mL, 38.5% and 1 147% more than that from the strategy without pH control and in the 250 mL shake flasks, respectively. Meanwhile, the fermentation time of DDase in the fermentor was also shortened from 47 h to 15 h compared to that in 250 mL shake flasks.

Abstract:The β-dehalogenase gene from Bacillus sp. was amplified by PCR with primers designed according to the sequence of the β-dehalogenase gene (named bhd) in GenBank. Then the bhd was overexpressed in Escherichia coli BL21(DE3)-CondonPlus with plasmid pET-30a(+). The recombinant β-dehalogenase (rBhd) was purified with HisTrapTMFF affinity chromatography and the molecular mass of the protein was about 23.1 kD. Further study on the enzymatic characteristics showed that, the hydrolysis reaction of 3-chloroproionic acid to 3-hydroxypropionic acid catalyzed by the purified rBhd should be carried out in 100 mmol/L sodium phosphate buffer at 30 °C. Under the optimum conditions, the specific activity, Km and Vmax of the enzyme were 16.2 U/mg, 3.26 μmol/L and 17.86 mmol/(min·g protein), respectively. When 10 mmol/L of 3-chloropropanoic acid was used as substrate, the conversion ratio reached 93% after reaction for 36 h.

Abstract:As a protein resource, only a little of cottonseed meal was used in feed industry due to the presence of toxin, gossypol. To obtain strains for gossypol detoxification, 16 soil samples were collected from China and 144 strains were isolated. Among them, a strain (Y-2) possess gossypol detoxification markedly. The strain Y-2 was identified as Pichia guilliermondii by traditional and molecular genetic identification. This strain was non-pathogenic yeast, and was first reported used on degradation of gossypol. Mutant YUV-51 with the highest detoxification was obtained by UV mutation. The detoxification rate of gossypol was up to 58% under optimized culture conditions: inoculation of 0.025g wet cell/g cottonseed meal, 30 °C, initial moisture content of solid substrate 50%, 48 h. Moreover, to avoid degradation of a large part of free gossypol before the fermentation, a great lot of energy could be saved with no heat-moisture treatment.

Abstract:Seventeen endophtye strains were isolated from soybean seeds by enrichment culture, and three endophyte strains producing β-mannanase were screened using Congo red dye method. By the shaking-flask culture, a strain of bacterium with the highest enzyme activity of 54.59 U/mL was obtained, and it was identified as Bacillus subtilis by the analysis of morphological, physiological and biochemical characteristics and 16S rDNA sequences. Enzymic properties of the β-mannanase revealed that the optimal temprature and pH were 30 °C?50 °C and 7.0, respectively. The enzyme activity still remained 68% when the enzyme was treated at 50 °C for 2 h; and more than 64% enzyme activity was remained after 1 h treatment at pH 5.0?9.0. In addition, the enzyme can be activated by Zn2+, Co2+, Ba2+, K+, and Ca2+ which has the most significant effect on the enzyme activity to improve 31% of activity. But, Mn2+ and EDTA could inhibite the enzyme activity.

Abstract:A bacterial strain R-2, capable of degrading fipronil, was isolated from a long-term fipronil-polluted activated sludge in a fipronil manufacture factory. Strain R-2 was preliminarily identified as Paracoccus sp. based on its physiological and biochemical properties, as well as the 16S rRNA gene sequence analysis. Strain R-2 could utilize fipronil as the sole carbon source for growth, and degrade 85% of 50 mg/L fipronil within 3 d in mineral salts medium. The optimal pH value and temperature for fipronil degradation by strain R-2 were 6.0?7.0 and 30 °C, respectively. The degrading rate showed a negative correlation with the initial concentration of fipronil. The addition of 0.1 mmol/L Zn2+ or Fe3+ to the culture medium could significantly increase the degradation of fipronil. In both sterile and non-sterile soils, strain could remove 63.4%?71.2% of 100 μg/g fipronil within 10 d.

Abstract:Metabolic engineering for aromatic amino acid biosynthesis pathway in Escherichia coli to acquire high-level biosynthesis of shikimic acid was reported. Knockout of aroL, ydiB genes and knock-in of T7-RNA-Polymerase gene which expression was controlled by L-arabinose based on the initial strain DH5α△ptsHIcrr (DHP), resulted in a series of shikimic acid-producing host strains. A series of tandem genes consisting of aroE, aroB, tktA, glk or aroFfbr were controlled by T7 promoter on plasmids were transformed into these host strains. According to the concentration of shikimic acid in shake-flask culture, all the engineered strains displayed high-potentiality compared to the control strain DHP, and the strain DHPYA-T7/pAOC-TGEFB synthesized the highest yield of 392 mg/L of shikimic acid. This study layed a strong foundation for constructing a high-level shikimic acid-producing engineered strain.

Abstract:To study the diversity of cultured symbiotic fungi isolated from Galaxea fascicularis L. in the South China Sea. By dilution plate technique and ITS-rDNA sequence analysis to research the diversity of the cultured symbiotic fungi with the coral, determined sequences data were submitted to GenBank and compared with those known sequences. Phylogenetic tree were built up with MEGA 4.0 program. 19 different morphological strains were isolated from surface and inner of the coral Galaxea fascicularis. ITS-rDNA sequences analysis showed that, symbiosis cultured fungi mainly included Aspergillus sp., Cladosporium sp., Xylariales sp., Penicillium sp., Stachybotrys sp., Gibberella moniliformis, Fusarium sp. etc. The similarity of strain 4-13 sequence to the most closely related sequences in GenBank was only 89%. It is concluded that there are diverse fungal symbionts with coral Galaxea fascicularis, which have potential new microorganism resources, deserved for further study.

Abstract:Microbial biocontrol agents SL19 has strong antimicrobial activity to many plant pathogens. By morphological, physiological and biochemical experiments and 16S rDNA sequence homology analysis, SL19 was identified as Bacillus velezensis. Antifungal spectrum was confirmed by confrontation tests. The results showed that SL19 has significant inhibitory effect against Verticillium dahilae, Fusarium oxysporum, Botrytis cinerea, Rhizoctonia solani, Sreptomyces scabies, etc. Active substances were separated and purified using ammonium sulfate method, and physical and chemical properties were studied. Antifungal substances were heated at 60 °C, 80 °C for 20 min, respectively, and antifungal activity has no difference; After 100 °C treatment for 20 min, antifungal activity decreased to 75.3% of the control; after 120 °C treatment for 20 min, antifungal activity was completely lost. Antifungal substances were not sensitive to trypsin, pepsin, proteinase K, UV radiation and chloroform. SDS-PAGE analysis revealed that the antifungal substances contained a kind of about 50 kD protein. It can be preliminarily concluded that the antifungal substance secreted by this strain was mainly protein. Tests suggested that the antifungal protein could inhibit the growth of hyphae and spore germination of Verticillium dahilae. The study on this strain would provide theoretical basis for biological control.

Abstract:The beta-cypermethrin degradation conditions including inoculum amount, temperature, pH, liquid volume, and initial concentration by Streptomyces HP-S-01 were systematically investigated. The results showed that Streptomyces HP-S-01 degraded beta-cypermethrin rapidly with a degradation rate up to 96% within 3 d, under the conditions of inoculum amount 0.6 g/L, 28 °C, pH 7.5, and liquid volume 50 mL/250 mL. The strain also could effectively degrade beta-cyfluthrin, beta-cyhalothrin, d-phenothrin and tetramethrin. Furthermore, the degradation reaction followed first-order kinetics and half lives (T1/2) were 0.78, 0.88, 1.08 and 1.24 d, respectively. The Andrews model was used to describe the beta-cypermethrin degradation process. The degradation kinetic data collected fitted the model well. The calculated parameters of qmax, Ks and Ki for the model were 1.826 3 d?1, 58.951 3 mg/L and 359.378 2 mg/L, respectively. And the optimal concentration of initial beta-cypermethrin was 145.553 5 mg/L.

Abstract:The minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of enrofloxacin against 12 strains of Vibrio alginolyticus, V. mimicus, V. harveyi, V. vulnificus were determined by two-fold broth macrodilution method and the results showed that the MIC and MBC were 0.1?0.8 mg/L and 0.4?3.2 mg/L respectively. On the basis of these, 4 sensitive strains of 12 vibrio srtains were measured with the killing-curves and the PAE in different concentrations of enrofloxacin. The results showed that every concentration of enrofloxacin against V. alginolyticus X040625-R had a strong antibactericidal effect, but that against V. harveyi M071202-H, V. vulnificus Q050723-C, V. mimicus H010911-Z showed that the three bacteria was inhibited in the lower concentration and gradually killed with the increasing concentration. The PAE of enrofloxacin had a direct relationship with the concentration and the mixing time of the drug. The PAE of enrofloxacin against V. alginolyticus X040625-R was the longest while that against V. harveyi M071202-H was the shortest.

Abstract:The activities and components of extracellular metabolites of a mutant Bacillus SC27 which was isolated from mangrove soil and was mutated via combined nitrosoguanidine (NTG) and UV were analyzed. The results showed that the Bacillus SC27 produced lactic acid of 5.04 g/L. The activities of protease, amylase, cellulase in the fermentation fluid were 1 316.59 U/mL, 176.2 U/mL and 513.3 U/mL, respectively, while no lipase activity was detected. The extracellular metabolic products showed higher anti-bacteria activities against Gram-positive bacteria than those against Gram-negative bacteria, and the anti-bacterial products of Bacillus SC27 could withstand treatment of high temperature, enzymes of papain, proteinase K and trypsin. Main chemical compositions of fermentation fluid extracted by dichloromethane were hydroxytoluene of 10.28%, dimethoxydimethylsilane of 7.87%, 2,4-bis(1,1-dimethylethyl)-phenol of 2.92% and 2 unidentified compounds of 4.47% and 2.36%.

Abstract:Lactic acid bacteria is an important probiotics in body. Because of its function and safety benefits, it has been widely used in the food industry and health care. In addition, Lactic acid bacteria, as an oral vaccine carrier and drug delivery carrier, has become a hotspot of research. However, the survival capability of Lactic acid bacteria is limited by the harsh environment of gastrointestinal tract. In this article, we used chemical method to prepare calcium alginate microcapsules of Lactococcus lactis, which can express Gfp stablely. Then we used Gfp as a viable marker to test the protective effect of calcium alginate microcapsules on Lactococcus lactis. In vitro experiments, it is showed that after acid treatment 30 min, 60 min, 90 min, 120 min, the survival rates of calcium alginate microencapsulated Lactococcus lactis were increased by 1 370, 525, 235 and 105 times, respectively. In vivo experiments, after 2 h stomach gavage, the number of alive Lactococcus lactis in calcium alginate microencapsules was increased by about 90 times in the intestine. The results suggested that microcapsulation of Lactococcus lactis with calcium alginate had significant protective effect on Lactococcus lactis in the gastrointestinal tract environment and provided an important frame for Lactococcus lactis in the future research of oral agents.

Abstract:In this paper, the influence to bacterial surface characteristics and interaction mode with liposomes of antimicrobial peptides BF2-A and BF2-B, two analogues of Buforin Ⅱ, had been researched. The both peptides could enhance the electronegativity and hydrophobicity of cell surface of Gram-positive bacteria and Gram-negative bacteria, which determined by Zeta potential electrometer and hexadecane extraction, respectively. BF2-A/B could cause calcein release from large unilamellar liposomes consisted of the anionic lipid phosphatidylglycerol and the zwitteronic lipid phosphatidylcholine, which reflected the compositions of bacterial cytoplasmic membrane. BF2-A displayed much weaker leakage than that of BF2-B, suggesting that BF2-B might cause perturbation of the phospholipid bilayer of the plasma membrane. However, BF2-A/B didn’t collapse the membrane of liposomes. Then the peptides were labeled with FITC. The blue shift of fluorescence spectra and the augmentation of quantum yield of FITC-peptides were discovered after the addition of liposomes. And the fluorescence quenching of FITC-peptides by acrylamide was prevented under the protection of liposomes, which implied that the N-terminal of BF2-A/B inserted into the phospholipid bilayer of membrane.

Abstract:Salmonella is a facultative intracellular pathogen that is associated with gastroenteritis, septicemia, and typhoid fever. It survives and replicates in macrophages during the course of infection and can be exposed to a number of stressful environments during its life cycle. Among the major antimicrobial products of macrophages are reactive intermediates of the oxidation of nitrogen (RNI) and the reduction of oxygen (ROI) which belong to oxygen-dependent antimicrobial systems of phagocytic cells. Resistance to ROI and RNI is a key feature of Salmonella virulence in vivo. Bacterial small non-coding RNA (sRNA) plays diverse physiological roles in stress responses, regulation of metabolism, control of bacterial envelope composition and bacterial virulence. In this study, we studied regulatory function of salmonella sRNA istR associated with resistance of S. enterica serovar Enteritidis to RNI and ROI. We constructed an istR-deletion mutant of SE2472 (SE2472△istR) using the red recombination system. The △istR mutant of SE2472 was complemented by cloned the wild-type allele of istR into plasmid pHDB3. To study the contribution of istR to the resistance of Salmonella enteritidis to RNI and ROI, we studied the differences of the survival rate of wild type SE2472, SE2472△istR mutant and complement strain to NaNO2 (pH 5.0, 20 mmol/L) and H2O2. The SE2472△istR was more sensitive to the bactericidal activity of NaNO2 (pH 5.0, 20 mmol/L) than wild type SE2472, but there is no difference in survival rate in the present of H2O2. To confirm the results, we constructed the complement plasmid and transferred it into SE2472△istR to rescue the survival defect. Results showed that the survival defect of SE2472△istR was rescued well when complement strain grown with NaNO2. The study of survival rate of the wild type SE2472 and istR deletion mutant in the presence of NaNO2 (pH 5.0, 20 mmol/L) and H2O2 demonstrated that sRNA istR acts as an important regulator of the resistance of SE oxygen-dependent antimicrobial systems.

Abstract:In order to express and characterize the biological function of protein Reg3A, the partial coding sequence without signal peptide of Reg3A gene were sub-cloned into vector of pET-32a to construct recombinant prokaryotic expression vector pET-32a-Reg3A. After induction with IPTG, the strain of E. coli BL21-Codonplus was transformed successfully with recombinant constructs, which was found to be expressing the recombinant protein in high yield and existed in the form of inclusion bodies. The inclusion body dissolved in urea was refolded into natural conformation after dialysis. The expressed protein was purified by Ni-NTA column. The purified protein, about 95% purity, was confirmed by Western blot. It was further demonstrated that the recombinant protein could effectively inhibited growth of Gram positive bacteria, which indicated that our recombinant Reg3A retained antibacterial activity. The cloning and expression of the Reg3A proteins provide basis for further characterization of the Reg3A biological function.

Abstract:The aim of this study was to investigate the effect of partial solar eclipse on the carbon metabolic diversity of airborne microorganisms. We used Biolog metabolic fingerprinting method to analyze the variation of the carbon metabolic diversity of airborne microbial community in Urumqi before and after partial solar eclipse. The results suggested that the ability of carbon sources utilization for microbes on the day was higher than other days. The analysis of variance of the microbial community diversity indices showed that Shannon-Wiener diversity was the highest on the day; and principal component analysis demonstrated that carboxylic acids was the mainly carbon sources to the differentiation of the airborne microorganisms. Hence partial solar eclipse may affect the functional diversity of air microbial community in Urumqi.

Abstract:Magnetotactic bacteria can absorb abundant ferrum from surroundings and synthesize nano- magnetic particle termed magnetosome in cells. Genome features of several kinds of magnetotactic bacteria are compared in this review and current research progress on synthesis mechanism of magnetosome are summarized in aspects of magnetosome island and functions of magnetosome-associated genes.

Abstract:The appressorium is the key structure for the rice blast fungus to infect the host. The initiation and development of appressorium are regulated by cAMP, mitogen-activated protein kinase and Ca2+ signal pathways. And they are also regulated by the protein for host surface recognition, melanin and glycerol generation, autophagy, and the SNARE protein. This paper has reviewed the appressorium initiation and development from the above aspects.

Abstract:In order to help students master the basic operating techniques and cultivate the practical abilities and student’s initiative, we made some improvements for microbiology experiment teaching in the following aspects including the teaching methods and content arrangements for sections of basic experiment and comprehensive experiment, respectively, as well as evaluation methods for the experiments of students. It was proven that these improvements were effective.

Abstract:For the purpose of improving microbiology teaching effect, old concepts and methods should be abolished, good teaching habits and ethos should be established. In this paper, our practice in microbiology teaching under the complete credit system in four aspects including teaching and educating, circumstance designing, creative thinking and practice strengthening were introduced sequentially. It was indicated that our efforts in teaching reform had significant effect on microbiology study, and also could meet with the requirement for training high quality talents in biological science and technology in the new era.

Abstract:LAMP (Loop-mediated isothermal amplification, LAMP) rapid detection method for Clostridium perfringens in food was established, with the specific primers designed on the CPa gene. The results of the study indicated that the method had perfect specificity, as 5 strains of Clostridium perfringens were able to amplify specific fragments, while 13 strains of non-Clostridium perfringens were not, and no false positive or false negative results occurred. The test could be done within 1 hour with the sensitivity low to 10 fg/μL. The established method provides a better choice for rapid detection of Clostridium perfringens.

Abstract:Prunus necrotic ringspot virus (PNRSV) is considered a quarantine pathogen of fruit trees disease in some parts of the world, and also a plant quarantine virus issued by Chinese government. A pair of primer and a TaqMan probe based on the conserved nucleotide sequence of coat protein gene of different PNRSV strains were designed and synthesized. Then through optimizing the concentration of primers, probe, Mg2+ and dNTPs, a real-time fluorescent RT-PCR was developed for detection of PNRSV in fruit trees. when optimal concentrations of primers, probe, Mg2+ and dNTPs was 400 nmol/L, 333 nmol/L, 5 mmol/L and 0.43 mmol/L, the assay for specific detection was highly sensitive, which could detect the template concentration as low as 23 copies, and could detect PNRSV in leaves tissues of cherry trees successfully. This reliable, sensitive, quick and easy-handling method is suitable for detection and identification of Prunus necrotic ringspot virus.

Abstract:Lactobacillus is used as probiotics and there has been much interest. The detection and identification of Lactobacillus from pickle need a rapid method. We designed a pair Lactobacillus-specific primer by analyzing 16S rDNA of 14 Lactobacillus species whose complete genomes had been sequenced. The results of colony PCR showed that an 800 bp band was generated when Lactobacillus and Leuconostoc strains used as template but no band appeared when Staphylococcus epidermidis, Bacillus subtilis, or Lactococcus lactis used as template. Lactobacillus of Sichuan pickle was rapidly detected by a procedure including selection of MRS culture, Gram stain and colony PCR. By sequencing the 800 bp fragment, the detected strain was identified at the species level. 15 Lactobacillus strains were detected from 16 samples of Sichuan pickle. 14 Lactobacillus strains were identified as L. plantarum and a Lactobacillus strain need more identification. The method would detect new Lactobacillus species.