Abstract:

Abstract:The ability of the foodborne pathogen Listeria monocytogenes to develop biofilm in food-processing environment is a major concern for the food safety, because formation of biofilm facilitates bacteria to survive in the adverse environment and resist desiccation, UV light and treatment with antimicrobial and sanitizing agents. However, the molecular mechanism of biofilm formation in L. monocytogenes has not been fully understood. PrfA is a key transcriptional activator that positively regulates most of the known listerial virulence genes expression. In order to explore the role of PrfA on Listeria biofilm development, we compared the abilities of biofilm formation in this study for L. monocytogenes wild type strains (EGD and EGDe) and their prfA deletion mutants (EGD??prfA and EGDe??prfA), nonpathogenic Listeria innocua, as well as the recombinant strains that can constitutively express PrfA in L. innocua (LI-pERL3-prfA*) and in EGDe??prfA (EGDe?prfA-pERL3-prfA*). Our results showed that the wild types of L. monocytogenes had strong abilities to develop “a network of knitted chains” biofilm structures on polyvinyl chloride microtiter plates, while unstructured biofilm was observed in L. innocua. Biofilm formation was reduced in L. monocytogenes mutants lacking PrfA and rescued in the strain with constitutive expression of PrfA. However, PrfA had no impact on L. innocua biofilm formation. Our results suggest that PrfA plays a significant role only in the L. monocytogenes biofilm formation but not in L. innocua. PrfA might indirectly regulate expression of certain genes involving in L. monocytogenes biofilm formation.

Abstract:

Abstract:Various substrates feeding strategies for cephalosporin C (CPC) efficient production by Cephalosporins acremonium were studied and conducted in a 7 L fermentor. A novel ammonium sulfate feeding strategy in couple with soybean oil addition was proposed. With this strategy, NH4+ concentration in fermentation broth could be stably maintained in a range of 3?6 g/L throughout fermentation period, simultaneously satisfying the demands on nitrogen and sulfur sources for cells growth and CPC synthesis, promoting Cephalosporins acremonium hyphal split, and creating the prerequisite for the subsequent CPC efficient production. Based on the novel ammonium sulfate feeding strategy, perform-ance of the subsequent CPC fermentations with different soybean oil feeding strategies, namely inter-mittent, constant rate and automatic DO-Stat feeding was compared. Feeding ammonium sulfate in couple with the soybean oil feeding in a manner of DO-Stat+oxygen-enriched air aeration at late fer-mentation phase could control carbon source concentration and DO at adequate levels, ensuring effi-cient CPC synthesis in a way of high CPC concentration and low by-product accumulation. With this combinational feeding strategy, final CPC concentration and CPC yield could reach 35.77 g/L and 13.3%, respectively, the major by-product, de-acetoxycephalosporin (DAOC) and DAOC/CPC were only 0.178 g/L and 0.5%.

Abstract:A bacterium, named CP1, capable of effectively degrading chlorpyrifos was isolated from activated sludge from a pesticide plant. Based on the phylogenetic analysis of 16S rRNA gene and Biolog test, the isolate CP1 was preliminarily identified as a strain of Genus Ochrobactrum. Both the orthogonal experimental design and Box-Behnken design were employed to optimized the main factors which influences the chlorpyrifos-degrading efficiency by strain CP1. The optimum conditions for chlorpyrifos biodegradation were as follows: the initial concentration of chlorpyrifos was 100 mg/L, the pH was 7.0, and the culturing temperature was 28.5 °C. Under the optimum condition, the biodegrada-tion efficiency of chlorpyrifos increased from 70.26% to 75.18%. Therefore, the optimization of chlor-pyrifos-degrading condition could improve the biodegradation efficiency of chlorpyrifos by Ochrobac-trum sp. strain CP1.

Abstract:Bacillus subtilis strain B47 is an endophytic bacterium of tomato and can produce substance to inhibit the growth of Bipolaris maydis which can cause southern corn leaf blight. The optimal nitrogen source, carbon source and salt for the production of antimicrobial substance by strain B47 were tested. The medium composition and fermentation conditions were optimized by orthogonal experiments. The results showed that the optimal nitrogen source, carbon source and salt were yeast extract, sucrose and MgSO4·7H2O, respectively. The best medium was YSB (Yeast extract-sucrose-beef extract). The composition of the medium was 2% (W/V) sucrose, 2% (W/V) yeast extract, 1.5% (W/V) beef extract, 0.06% (W/V) MgSO4?7H2O and 0.000 9% (W/V) FeSO4·7H2O. The optimal fermentation conditions were the combination of temperature 30 °C, initial pH 7.0, incubation time 6 d, 1% inoculum volume percentage and medium volume 40 mL/200 mL.

Abstract:267 endophytic bacteria of tobacco were isolated from roots, stems and leaves by the method of grinding separation. A preliminary analysis was made on the diversity of the isolates by phenotypic characteristics and molecular classification methods based on partial 16S rDNA sequences, the analyti-cal comparison was also made between them. 267 bacteria isolates were separated with 8 different phenotypic characteristics, and were grouped into 5 clusters and 56 subclusters at the level of 2.15 with numerical classified based on the unweighted pair group method with arithmetic averages algorithm, and they were clustered into 21 molecular groups based on partial 16S rDNA sequences. The results show that there are much more phenotypic types than molecular groups, phenotypic characteristics was not consis-tent with the results of molecular classification exactly. The result of sequence analysis of the 16S rDNA showed a 98%?99% homology with 21 species in the GenBank. The dominant bacterial groups of to-bacco endophytic bacteria belong to Firmicutes, Bacillus genera.

Abstract:pHsh is a novel high level expression vector of Escherichia coli, in which the regulatory promoters are recognized by the 32-kD sigma factor (σ32). In normal E. coli cells, the total time length of heat-shock response is about 12 min, however, in E. coli cells carrying recombinant high-copy pHsh vectors, the heat-shock response can sustain 4?10 h. In order to understand the mechanism of hign level expression of foreign gene in E. coli carrying pHsh vector, we employed xynIII gene encoding a xylanase as the representative of foreign genes. Firstly, the effect of copy-number of pHsh on the expression level of xynIII gene was tested, then the difference of the concentration of σ32 between in the E. coli cells harboring pHsh-xynIII and in the E. coli cells harboring pLac-xynIII was assayed by using western-blot under either inducing (30 °C→42 °C) or non-inducing conditions (30 °C). Finally, under different temperatures, the heat-shock level at steady state in recombinant E. coli cells harboring pHsh-xynIII was evaluated by the xylanase activity. the results showed that the high expression level of foreign genes in pHsh should attribute to the following three aspects: high-copy-number of pHsh enhanced the foreign gene dosage accessible for expression and leaded to a high productivity; Owing to the present of the pHsh, σ32 level in E. coli cells was significantly higher than that in E. coli cells without pHsh, thus the heat-shock level was significantly enhanced in E. coli cells harboring pHsh; The considerable heat-shock level at steady state in recombinant E. coli cells harboring pHsh was helpful for high level expression of foreign genes.

Abstract:To analysis the entophytic bacterial diversity of citrus and find the companion bacteria populations associated with Huanglongbing pathogen-infected and healthy citrus plant tissues for decipher the co-cultivation of HLB pathogen, we selected varied parts of citrus tissues collected from different loca-tions of citrus planted area. The facultative anaerobic entophytic bacteria were isolated and purified based on bacterial morphology, physiology, biochemistry characteristics and the molecular method of PCR-DGGE (Denaturing gradient gel electrophoresis) analysis based on the sequence of 16S rRNA V6-V8 fragment gene. By the directional isolation of the facultative anaerobic entophytic bacteria and 16S rDNA amplification, total 12 genera of bacteria were identified from 19 cultivable bacterial popula-tions. The dominant bacterial population in infected citrus plants were Curtobacterium sp. (IF: 29.07%), Bacillus sp. (IF: 23.12%), Microbacterium sp. (IF: 21.09%) while in healthy citrus tissues belonged to Bacillus sp. (IF: 21.03%), Planococcus sp. (IF: 20.69%), Pseudomonas sp. (IF: 17.44%). From 50 target bands obtainded by the DGGE approach, 9 genera of cultivable bacteria were recognized. The dominant bacterium population belonged to Serratia sp. (IF: 28%) and Pantoea sp. (IF: 14%) followed by it. Can-didatus Liberibacter asiaticus was only found in tangerine pith of deformed orange fruit, which sug-gested that the content (>1%) of Huanglongbing was more in diseased fruits and other tissues of citrus had low abundance percentage. The density and species of entophytic bacteria were also observed in re-markable difference between infected and healthy citrus plant from the PCR-DGGE profiles.

Abstract:Isolates from the stroma of Ophiocordyceps sinensis, produced generously ascocarps with reticuloperidium. Morphological characteristics and molecular identification indicated that the repre-sentative strain Pseu-F was Pseudogymnoascus roseus Raillo. The optimum temperatures for mycelial growing were 17.5 °C?20.0 °C. Submerged culture media were screened by means of orthogonal design experiments. Test factors included sucrose, glucose, peptone, yeast extract, potato, soybean, mineral salt and Vitamins. Based on the results of tests, the optimized combinations of culture medium are sucrose 20 g, glucose 10 g, peptone 10 g, yeast extract 5 g, soybean 50 g, potato 100 g, in 1 L liquid medium.

Abstract:A good Bacillus strain G1 antagonistic against pathogenic Aeromonas hydrophila of sturgeons was isolated and screened from the sediment of aquaculture ponds, which produced the inhibition zone of 18.50 mm in diameter against A. hydrophila strain S1. Strain G1 (GenBank accession number: HM245965.1) was identified as Bacillus amyloliquefaciens through APICH50 bacterial identification system and 16S rRNA sequence analysis. Its 16S rRNA sequence had homology of 99%?100% with those of Bacillus sp. strains submitted to GenBank, and showed the most close relative to Bacillus amyloliquefaciens strain Ba-74501 (GenBank accession number: DQ422953.1). The best growth pH and temperature of strain G1 were 7 and 30 °C, its growth curve in the condition of 30 °C and 200 r/min was as follows: the lag phase was 0?6 h, the log phase was 6?54 h, the stationary phase was 54?90 h, the decline phase was after 90 h. In addition, strain G1 also exhibited good antagonistic activity against other tested A. hydrophila strains. The experimental results were conductive to fill in the data gaps about the taxonomic position of antagonists against A. hydrophila, and provide scientific data to the biocontrol on sturgeon aeromonasis.

Abstract:Bacillus thuringiensis (Bt) strains, which can produce insecticidal crystal proteins, were widely used as biological pesticides. In this paper, we comprehensively analyzed the distributions, structures and putative biological functions of two-component transduction systems (TCS) from the genomes of Bt strains YBT-1520, CT-43 and BMB171, which have been sequenced by our laboratory. And more importantly, we constructed a preliminary TCS regulatory networks. This study should open a novel research direction in Bt for the growth, metabolism, regulator of toxic gene expression, as well as the formation mechanism of parasporal crystals.

Abstract:OmpT, located in Escherichia coli (E. coli) outer membrane, is a protease that demonstrates highly substrate specificity. In order to estalish the approaches for expression and refolding of membrane protein OmpT, and examine the demonstrated protease activity of OmpT, the ompT gene was first amplified by PCR and inserted into pET28a (pET-ompT) and introduced by Asp85Ala site-directed mutagenesis to generate mutant Asp85Ala (pET-ompT85). Then, the two recombinant plasmids were transformed into BL21 (DE3), OmpT and the mutant were expressed in the form of inclusion bodies, purified and refolded by N-Dodecyl-N,N-dimethyl-1-ammonio-3-propanesulphonate. The addition of lipopolysaccharide (LPS) to the recombinant OmpT was critical to refold its protease activity in vitro. Finally, the ability of the recombinant wide-type OmpT to hydrolyze protamine and rabbit muscle creatine kinase (RMCK) was confirmed by SDS-PAGE, bacteria agglutination and growth curve in contrast to the mutant. The results suggest that the desired recombinant OmpT was obtained, which showed the significant protease activity in the protection of E. coli against protamine in vitro.

Abstract:Pythium aphanidermatum was cultured in PD medium and the mycelia were extracted with ethyl acetate and methanol, and the methanol extracts were separated gradually by silica gel column using ethyl acetate mixture and petroleum ether (V/V=3:1 and V/V=2:1) as the developers. Eluents were collected and each 50 mL were considered as a fraction and bioassayed. The results revealed that fraction 21?24 showed strong activity against Digtaria sanguinealis with the inhibition level of 4. The combined fractions of 21?24 were eluted with the mixture of ethyl acetate and petroleum ether (V/V=2:1) as the developer and 20 fractions were collected. Bioassay results showed that fraction 3 had the stronger inhibitory activity against Digtaria sanguinealis. HPLC analysis suggested that this fraction mainly contained 3 components, the retention time of which was 12.7, 14.0 and 30.5 min respectively.

Abstract:Inosine 5'-monophosphate dehydrogenase (IMPDH) is one of plasminogen (Plg) receptors on the surface of Staphylococcus aureu (S. aureus). Plg through its lysine binding sites (LBS) binds to IMPDH. Apolipoprotein(a) [Apo(a)], which is one component of Lipoprotein(a) [Lp(a)], has a high homology with Plg and both of them contain LBS in their Kringle (K) domains, and a strong LBS is identified in KIV10 of Apo(a). Therefore, we previously reported that Lp(a) might bind to Plg receptor on the surface of S. aureus, subsequently competitively inhibiting the interaction of S. aureus with Plg. To further test our hypothesis, the IMPDH gene of S. aureus was cloned into pASK-IBA37 and the re-combinant IMPDH(rIMPDH) was expressed in E. coli BL21. The interaction between rIMPDH and Lp(a) was investigated by enzyme-linked immunosorbent assay (ELISA), and affinity chromatogra-phy-binding assay followed by Western blotting. The results indicated that rIMPDH could specifically bind to purified Lp(a) and rKIV10, and the lysine analog EACA inhibited the binding. Unexpectedly, Lp(a) and rKIV10 did not significantly inhibit the interaction between rIMPDH and Plg.

Abstract:To reveal the content and composition of polysaccharide in chlamydospore wall of Ustiloginoidea. virens, a study of the optimum method to extract polysaccharide of the chlamydospore wall was conducted. Five different methods were adopted to extract polysaccharide of its black chlamydospore wall, and the contents of polysaccharide were determined by phenol-sulfuric acid method. The results showed that the optimum extracting method was the complex enzyme-hot water extraction-sevag and the most appropriately extracting conditions were that the complex enzyme, pH 4 extracting tempera-ture extracting time and the material ratio were 4%, 70 °C for 120 min, 1:75 (V/V), respectively. Based on above the optimum method, the relatively crude polysaccharide and polysaccharides rates in the black and yellow chlamydospore wall were separately 21.2% and 72.3%, and 17.5% and 66.7%. Con-sequently, the complex enzyme-hot water extraction-sevag and its optimizing conditions among the five different methods were simple, efficient and particularly suitable to the determination polysaccharide in chlamydospores wall of U. virens.

Abstract:The family Nodaviridae contains two genera, Alphanodaviruses and Betanodaviruses, which predominantly infect insects and fish, respectively. The genome of nodaviruse consists of two sin-gle-strand positive-sense RNAs (RNA1 and RNA2). RNA1 encodes protein A, catalytic subunit of RNA-dependent RNA polymerase (RdRp), and RNA2 encodes coat precursor protein which undergoes an autocatalytic mature cleavage into two viral capsid proteins β and γ. During the course of RNA rep-lication, a sub-genome RNA3 is synthesized which is not packaged into the virion from the 3’ termini of RNA1. RNA1 can self-replicate automatically absence of RNA2 and produce the sub-genome RNA3 persistently. The mechanism of RNA3 synthesis is the mechanism of premature termination. The paper also reviewed the regulation of RNA replication, the functions of non-structural proteins and the local-ization of RNA replication of the nodaviruses.

Abstract:(p)ppGpp is a well known intracellular signal that mediates bacterial stringent response to environment stresses through the change of its concentration level thus controls many important cellular processes for bacterial survival. In this review, we outline the mechanism of (p)ppGpp action, the enzyme system involved in (p)ppGpp metabolism, and summarize the signal transmission, the regulation and the diversity of (p)ppGpp metabolism. Moreover, we give a brief introduction on our achieved results recently about (p)ppGpp metabolism in cyanobacteria, and predict that a (p)ppGpp new metabolic mechanism different from those known exists in cyanobacteria.

Abstract:To improve the quality of education of Environmental Engineering Microbiology, it is necessary to innovate the curriculum in many aspects. It is concluded the author’s exploration and practice in textbook construction, classroom teaching, experiment, multimedia application and so on, which had some effectiveness.

Abstract:Purple nonsulfur bacteria are one branch of purple phototrophic bacteria, which are ecologically important and have valuable applications. In this study, we established paraffin wax overlay plate method for the isolation and the enumeration of purple nonsulfur bacteria. Compared with several tra-ditional methods, the new one was found to be more rapid and convenient. We applied this method to-gether with molecular approaches to investigate purple nonsulfur bacteria in paddy soil, and found that it can well reveal the diversity of purple nonsulfur bacteria at culturable level, when formate acts as carbon source. We believe the new method would be of great help to the investigation of purple non-sulfur bacteria in environment.

Abstract:In this study, the effect of extracellular metabolites produced by Saccharomyces cerevisiae on intracellular protein expression of Non-Saccharomyces cerevisiae and on the quality of wine was investigated by dialysis tube fermentation method. By using dialysis tubes with molecular weight cut-off value of 10.0 kD and 3.5 kD respectively in the mixed fermentation, the survival time of non- S. cerevisiae was extended to 18 days and 22 days correspondingly. Consequently the survival time of non-S. cerevisiae can be changed by limiting the exchange of metabolites between strains. 65 proteins, namely 13% of the total protein, were found differently expressed, with mass spectrometry results in-dicating their close relation to the biosynthesis of steroids, lysine, organic acids and ATP. Compared with 10.0 kD dialysis tube fermentation, in 3.5 kD dialysis tube, the concentration of tartaric acid in-creased by 5.1% while acetic acid decreased by 44.3%, which indicates that by allowing communication of metabolism between cells with limited molecular weight, we can adjust the yield of wine’s titratable acidity and volatile acidity.