Abstract:摘要：历史上，微生物学的发展曾经历了两个辉煌的黄金时代，也经历了其发展的低谷时期。近20年来，随着基因组学、结构生物学、生物信息学、PCR技术、高分率荧光显微镜及其它物理化学理论和技术等的应用，使微生物学的研究取得了一系列突破性进展，微生物学己走出其低谷，开始进入它的第三个黄金时代。本文就下列几个方面谈谈自已对当今微生学发展的机遇、挑战和趋势的一些认识，供读者参考：（1）历史上微生物学发展的两个黄金时代；（2）微生物学复兴的机遇和挑战；（3）当前微生物学发展的特点和趋势；（4）整合微生物学——第三个黄金时代的重要特征。

Abstract:Abstract: The purpose of this review was to offer reference for the study of community analysis in which 16S rRNA was taken as target molecule. We summarized the information of 16S rRNA including its function in cells, transcription organization, the position of variable regions and conserved regions, copies numbers of 16S rRNA gene, secondary structure, some artifacts generated during PCR amplification and the solution to avoid the artifacts.

Abstract:Abstract: Tuberculosis remains a major global health threat. Nearly one-third of the world population infected with Mycobacterium tuberculosis, the etiologic agent of tuberculosis. M. tuberculosis is a typical and most successful intracellular pathogen. The pathogen can evade and manipulate the host immune response. Insights into the interplays between the pathogen and the host was pivotal to develop more sophisticated diagnosis methods and control measures to tuberculosis. No single model can address the full spectrum of this extraordinarily successful pathogen. Multiple models are urgently needed to explore diverse facets of this human being scourge. Zebrafish- M.marinum model was increasingly recognized as an ideal system for preliminary studies. Some key findings emerging from this model were summarized in this paper, such as the interactions between host and M. marinum when the bacterium invades and the contribution of the virulence determinants of M.marinum such as Erp, Esx-1, pmiA, Mel and KasB. Discoveries from different models will be complementary and conducive to find clues to eradicate Mycobacterium tuberculosis.

Abstract:Abstract: [Objective] We screened and isolated coronatine-producing stains from various samples. [Methods] The strains were isolated and selected from samples by the methods of streak plate and serially dilution. The samples were sick leaves/branches and soil in which plants got sick according to the symptoms of leaf blight disease and tuber enlargement. Coronatine production was determined by high performance liquid chromatography (HPLC). The strain was characterized by the physiological and biochemical analysis, the determination of (G+C) mol% contents and 16S rDNA sequencing. The molecule structure of the fermentation product was identified based on the data of ultraviolet spectrum, infrared spectrum and mass spectrum. [Results] Strain BCC933 was gram-negative, polar flagella, short rod and non-spore-forming bacterium and accumulated poly-β- hydroxybutyrate (PHB). It producted catalase, but not arginine dihydrolase nor oxidase, couldn’t grow at temperature 41℃. It hadn’t the abilities to hydrolyze starch and gelatine. No nitrate reduction and denitrification activity was detected. The (G+C) mol% content was 67.2%. We analyzed 16S rDNA nucleotide sequence, and ascertained the phylogenetic position of the strain. [Conclusion] Strain BCC933 with coronatine biosynthesis ability was identified as Burkholderia cepacia, which hasn’t been reported up to date.

Abstract:Abstract: [Objective] To construct a plasmid for analyzing gene functions and expressions and to study the MXAN1334 gene in Myxococcus xanthus with the plasmid. [Methods] We constructed the plasmid vector pZCY11, amplified MXAN1334 gene fragment from M. xanthus DK1622 by PCR, and inserted the fragment into a site upstream of lacZ, resulting in the recombinant plasmid pZCY13. The plasmid pZCY13 was transformed by electroporation to DK1622, producing a mutant ZC16-18 (?MXAN1334). [Results] The plasmid pZCY11 carried the resistance gene aph as the selectable marker, the replication origin of OriR6K and promoterless reporter gene lacZ. We examined the swarm expansions of ZC16-18 on CTT hard and soft agar, and the result indicated that MXAN1334 gene was probably involved in gliding motility in M. xanthus. In addition, β-galactosidase activity of ZC16-18 was detected by X-gal assay and the blue color developed was used to mark the colony growth. Time of colour showed that MXAN1334 gene was expressed in the early stage in M. xanthus. [Conclusion] The plasmid vector pZCY11 made it more convenient for the study on functions and the expressions of target gene in M. xanthus.

Abstract:Abstract: NtcA is a master transcriptional regulator in global nitrogen control in Anabaena. Heterocyst differentiation depends on the NtcA protein. A large number of genes regulated by NtcA have a consensus sequence, GTA-N8-TAC, for NtcA binding. Bioinformatics analysis of alr1390 in the chromosome of Anabaena sp. PCC 7120 indicates that there is a conservative NtcA binding box, GTA-TAGTTTTC-TAC, upstream the putative tsp position of alr1390. [Objective] To understand the relationship between alr1390 and NtcA, [Methods] Real-time RT-PCR and EMSA were used. [Results] Real-time RT-PCR results showed that the transcriptional level of alr1390 remained constant before and after combined nitrogen step-down in wild type Anabaena sp. PCC 7120. However, in the ntcA mutant, there was an increase in the transcriptional level of alr1390 after 12h of combined nitrogen step-down, which indicated that alr1390 should be subject to the regulation of NtcA. Additionally although no conspicuous NtcA-alr1390 complex was detected by EMSA in vitro, a smear appeared in gel electrophoresis. [Conclusion] These results suggested that alr1390 might be regulated by NtcA.

Abstract:Abstract: A Streptomyces strain X3-3, growing up to 50 ℃, was isolated from the composts prepared from swine manure and straw. This strain harbored a -7-kb plasmid pTSC2. [Objective] Cloning, sequencing and analyzing pTSC2, and identifying its replication mode as well. [Methods] The complete nucleotide sequence of pTSC2 was obtained by sub-cloning and primer-walking; the replication gene (rep), double-strand origin (dso) and single-strand origin (sso) were identified by multiple sequence alignments; and the replication intermediate was detected by Southern hybridization after neutral transfer. [Results] The complete nucleotide sequence of pTSC2 consisted of 7516-bp in length, encoded eight proteins, four of which resembled the replication and conjugation proteins of Streptomyces plasmid pIJ101 which replicates in a rolling-circle mode. The replication elements, rep, dso and sso, were also similar with pIJ101. The rep and dso was proved as essential components for plasmid replication by transformation of both S. lividans ZX7 and thermophilic Streptomyces spp. 2C, while sso was dispensable for replication. Southern hybridization detected a large amount of single-stranded DNA accumulated during replication. [Conclusion] We identified that the thermophilic Streptomyces plasimd pTSC2 replicated in a rolling-circle mode. It was first reported that the plasmid from thermophilic Streptomyces was cloned and sequenced, as well as the replication of which was characterized

Abstract:Abstract：Plasminogen activation and antimicrobial peptide hydrolysis contribute to pathogens invasion and survival in vivo. [Objective] To demonstrate the expression of outer membrane protease T in E. coli K1 pathogenic strain E44, its activity of plasminogen activator and protamine hydrolysis. [Methods] After Benzamidine Sepharose Fast Flow and SOURCE 30Q chromatography, we got E44 outer membrane mixed fraction, and examined its activity of plasminogen activation with chromogenic substrate S-2251 method. An ompT deletion mutant of E44 was constructed by using the suicide vector pCVD442, termed as E44ompT. We examined 0.1 mg/mL cationic antimicrobial peptide protamine susceptibility of E44, ompT mutant strain E44ompT and E44ompT harboring pUCT, which was constructed by inserting complete ompT open reading frame into pUC13. [Results] We got about 37 kDa E44 membrane extract, which could activate plasminogen, and activation was membrane extract dose dependent. This confirmed the expression of outer membrane protease T in the outer membrane of E44. E44ompT was more susceptible to 0.1 mg/mL protamine than E44, and E44ompT was partially complemented by pUCT. [Conclusion] Outer membrane protease T is expressed in E. coli K1 pathogenic strain E44, and can activate plasminogen and hydrolyze protamine.

Abstract:Abstract: [Objective] To isolate and characterize alkaliphilic bacteria with protease activity. [Methods] Protease-producing alkaliphiles were isolated by skim milk agar method. The morphological, biochemical and physiological characteristics, 16S rRNA gene sequence and DNA–DNA hybridization were determined to identify the taxonomic position of strain ZL223. Meanwhile, the enzyme characterization of the protease including optimal temperature and pH range, stability and oxidation resistance were tested by using casein hydrolysis test. [Results] A new strain named ZL223 was isolated from Bange Lake in Tibet, China. Cells were Gram-positive, spore-forming, motile rods. Growth pH range was from 7.0 to 10.0, optimum at pH 9.0. Growth temperature range was from 15 oC to 45 oC, optimum at 37 oC. The G+C content of DNA was 40.4 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain belonged to the genus Bacillus and most closely related to Bacillus pseudofirmus OF4 with 98.6 % sequence similarity. DNA–DNA hybridization analysis confirmed the close relationship of strain ZL223 with Bacillus pseudofirmu OF4 (86% relatedness). Strain ZL223 produced extracellular alkaline protease and its maximal enzyme activity was observed at 40 oC and pH 12.0. [Conclusion] Base on polyphasic taxonomy, strain ZL223 was a new member of Bacillus pseudofirmus. It produced alkaline protease which was worth further research.

Abstract:Abstract：[Objective] A novel (S)-specific carbonyl reductase gene (scrⅡ) was cloned from the genome of Candida parapsilosis CCTCC M203011, and its catalytic function for the biotransformation of chiral alcohol was verified. [Methods] The possible carbonyl reductase gene scrⅡ was amplified by PCR method from the C. parapsilosis genome. Using the recombinant Escherichia coli BL21/pET28a-scrⅡ as the catalyst and 2-hydroxyacetophenone as the substrate, the biotransformation was carried out. The optical purity and yield of the final product were investigated by HPLC analysis. The optimal pH and temperature of the reaction were also determined. [Results] The gene scrⅡ coded 279 amino acids with an open reading frame of 837 bp. It shared 85% identity with the reported gene scr. By analysis, SCRⅡ contained two typical motifs of the short-chain carbonyl reductase including a Rossmann-fold Thr40-Gly41-(X)3-Gly45-X-Gly47 and a conserved catalytic triad Ser172-(X)n-Tyr187-(X)3-Lys191. SDS-PAGE results showed that SCRⅡ was overexpressed at 30℃ after the induction of 0.1 mmol/L IPTG. When the concentration of 2-hydroxyacetophenone was 6 g/L, 10% (w/v) wet recombinant E. coli cells showed excellent performance to give (S)-1-phenyl-1,2-ethanediol with high optical purity of 99.1% enantiomeric excess in a yield of 89.6% under the optimal conditions of pH5.5 and 35℃. SCRⅡ catalyzed the tansformation of (S)-1-phenyl-1,2-ethanediol more efficiently than SCR. When compared with SCR, its substrate concentration was increased by two-fold, and the optical purity and yield of (S)-1-phenyl-1,2-ethanediol were improved by 10% and 28%, respectively. [Conclusion] The gene coding for novel carbonyl reductase SCRⅡ was isolated using the molecular cloning technique and its discovery supplied a solid research foundation for chiral alcohol preparation efficiently.

Abstract:Abstract：[Objective] To analyze the impact of tillage and fertilization on bacterial community structure under different depth in black soil, [Methods] We used denaturing gradient gel electrophoresis (DGGE) profile analysis of bacterial community based on the 16S rRNA gene combining soil physico-chemical characteristics. [Results] DGGE profiles showed that tillage and fertilization influenced more on bacterial community structrue in the surface soil than the subsurface and deep soil, and the divergence distance between the tillage soil and the control was about 4%. Subsurface and deep soil affected less compared with the control, and the divergence distance was 2%. The vertical similarity of bacterial community structure between subsurface and the deep soil was higher than that of suface soil. [Conclusion] Tillage and fertilization affected the surface bacterial community (0?30 cm), whereas little on the deeper soil (>100 cm) bacterial community.

Abstract:Abstract: [ Objective ] PCR method was used to detect Penicillium griseofulvum, a dominant species in marine contaminated sediments and thereby to deduce the contamination degree. [ Methods ] According to differences in internal transcribed space (ITS) sequences of Penicillium genus and specific IAO sequence, we designed species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum and established the corresponding PCR systems. By using PCR and nested-PCR，the detection sensitivity was compared. [ Results ] The primers could exclusively amplify destined DNA fragment from environment. Using AS1/RS4 as primers, the detection sensitivity could be 10 fg/μL and 10 spores. The detection sensitivity for the sediments was 102 spores/0.25g sediments. While the detection was unsensitive when using IAO1/IAO2 as primers. [ Conclusion ] It is feasible that the species-specific primers be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, because the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.

Abstract:Abstract: [ Objective ] Pathogens of the first influenza pandemic this century belong to influenza A H1N1 viruses, which are different from human seasonal H1N1 viruses in antigenic and genetic characterization. To better understand the genetic characteristics and evolution, timely detect variant strains with epidemiological importance, we analyzed in detail the molecular characterization of the early influenza A H1N1 (2009) virus. [ Method ] genomic sequences of reference influenza viruses were obtained from Influenza Resource Center of GenBank. Sequences were analyzed using the EditSeq and Megalign program with the Lasergene sequence analysis software package (DNAStar, Madison, WI, USA). A/California/07/2009 (H1N1) was selected as a representative strains of the novel influenza A H1N1 (2009) virus, and its molecular characteristics was determined. [ Results ] A/California/07/2009 do not contained the molecular characteristics of highly pathogenic influenza virus, and its 11 proteins retained most of the molecular characteristics of swine influenza virus, but also had some characteristics of avian and human influenza viruses. With a classical swine H1N1 and human H1N1 dual character, PB1-F2 protein of A/California/07/2009 terminates after 11aa, 57aa and 87aa，which is a unique molecular characteristics of influenza H1N1 (2009) virus. [ Conclusion ] This is the first report for detailed analysis of Molecular characteristics of the novel influenza A H1N1 (2009)virus. As the virus further adapt and persist in human populations, its molecular characteristics will change accordingly. So we should pay special attention to the effect on virus transmission and pathogenesis.

Abstract:Abstract: [Objective] This study was aimed to construct and characterize Salmonella vaccine strain C500 expressing the recombinant Pasteurella multocida toxin (PMT) antigen by the Asd+ balanced-lethal host-vector system. [Methods and results] The DNA fragment encoding C terminal of PMT was cloned downstream from the beta-lactamase signal sequence in the multicopy Asd+ pYA3493 vector to create pYA-PmtC. Fermentation patterns, serotype, and mean generation time of the vaccine strain C500 harboring pYA-PmtC (named with C501(pYA-PmtC)) were identical to those of the parent strain C500. The recombinant pYA-PmtC plasmid was very stable in C501(pYA-F1P2), which expressed secretorily a large amount of the recombinant PMT antigen (named with rPmtC). The virulence of C501(pYA-PmtC) with LD50 of 8.5×106 CFU was a little lower than C500 with LD50 of 4.4×106 CFU based on the method of Reed and Muench. All piglets inoculated with C501(pYA-PmtC) or C500 survived, and had no signs of disease during the entire experimental period. No significant differences were found between these two groups. [Conclusion] The recombinant vaccine strain C501(pYA-PmtC) had a series of biological characteristics silimar to the parent vaccine strain C500. It is likely that C501(pYA-PmtC) could be adapted to develop multivalent recombinant Salmonella vaccine against both infections with S. enterica serovar Choleraesuis and toxigenic P. multocida.

Abstract:Abstract: [Objective] To establish an experimental culture system of herpes simplex virus typeⅡ(HSV-2) latent infection and reactivation in SH-SY5Y cells. [Methods] Changes of biological character were observed after 20, 40, 60, 80,100, 120 and 140 μmoL/L ACV were added into cell cultures, and also the morphological observation was detected with phase-contrast microscopy after HSV-2 was inoculated into SH-SY5Y cells using MOI of 0.1, 1, 10 and 100. The optimum condition of time and temperature was approached using temperature of 41℃, 42℃, 43℃, 44℃ and 45℃, and time of 0.5 h, 1.0 h, 1.5 h, 2.0 h and 2.5 h to induce HSV-2 reactivation. The optimum concentration of Forskolin was also decided using 25, 50, 75, 100 and 125 μmoL/L to activate the virus from latency. PCR was used to authenticate HSV-2 latent infection and reactivation. The morphological changes were observed when the virus was reactivated from latency. [Results] The optimum concentration of ACV was 60 μmoL/L to establish latency in SH-SY5Y cells. Suitable infective dose of HSV-2 was 1-10 MOI to construct latency and reactivation in SH-SY5Y cells. The time of virus latency in SH-SY5Y cells could reach up to 14 d. Heat stress of 43℃, 1.5 h and 75 μmoL/L Forskolin were the optimum condition to induce virus reactivation. The morphologic changes in SH-SY5Y cells recurred by HSV-2: Cytopathic effects were more and more obvious with time lasting from 24 h to 72 h after reactivation from latency. PCR and results of electrophoresis proved the cell model of latent infection and reactivation was set up successfully. [Conclusions]The cell model system of HSV-2 latent infection and reactivation in SH-SY5Y cells was established. The research provided usefulness for study on HSV-2 of latency, reactivation and pathogenic mechanism.

Abstract:Abstract: [Objective] To purify and identify avian influenza viruses causing multi-subtype mixed infection. [Methods] By using chicken embryo end-point dilution or combining with specific antiserum neutralizing tests, the mix-infection samples of 2 or 3 subtyping avian influenza viruses were purified, and the results of purification were identified by RT-PCR and hemagglutination inhibition tests. These methods were used to purify and identify 214 positive samples of avian influenza viruses. [Results] The method of chicken embryo end-point dilution was able to purify the viruses from samples by dilution and subculturing six or seven passages. However, combining this method with the specific antiserum neutralizing tests, the samples could be purified after four or five passages. The accuracy of identification could be obviously enhanced by using RT-PCR and hemagglutination inhibition tests simultaneously. Based on these methods, 233 of avian influenza viruses, which constituted 13 subtypes, were purified from 214 samples. [Conclusion] Both chicken embryo end-point dilution method and its combination with specific antiserum neutralizing tests could be used to purify avian influenza viruses of mix-infection, however, the latter may be more focused and valid. In addition, the results of purification should also be evaluated by multiple means.

Abstract:Abstract: [Objective] Electronic tongue is a modern analysis instrument and can detect very well the comprehensive information of liquid samples. Based on this, we employed the electronic tongue to detect the bacteria liquid culture, with the purpose of developing a new rapid method for the detection and identification of food-borne pathogens.[Methods] A novel voltammetric electronic tongue, smart tongue, was used to detect and differentiate 11 species of pathogenic Vibrio. Principal component analysis (PCA) was used to extract the relevant information obtained by the smart tongue. From the data evaluation, the score plots of PCA were obtained. [Results] According to the plots, we chose the most feasible working electrodes at the most favorable frequency segments were determined for our purpose. The results showed that the best electrode arrays and frequency segments to differentiate pathogenic Vibrio were titanium electrode in 100 Hz, silver electrode in 100 Hz and tungsten electrode in 1 Hz and 10 Hz, respectively. We differentiated 11 species of pathogenic Vibrio independently in the score plot of tungsten electrode in 1 Hz. We also distinguished all the Vibrio with the combination of two of other three electrodes and frequencies. [Conclusion] Smartongue could differentiate well 11 species of pathogenic Vibrio from the results analysed by PCA. It has a promising future as a novel modern rapid analytical technology for detecting and distinguishing the pathogenic Vibrio. The method and results could be a good stencil-plate to the research of detecting the other food-borne pathogens with smartongue, and good reference information for the further study.

Abstract:Abstract: [Objective] To analyze the diversity and succession of microbes during traditional solid-state fermentation. Taking Zhenjiang vinegar for example, we compared 11 methods for extracting genomic DNA from microbes in solid-culture of Zhenjiang vinegar. [Methods] The yield and purity of the DNA were measured by ultraviolet spectrophotometer and real-time PCR. The bacterial and fungal diversity during solid-state fermentation was analyzed by denaturing gradient gel electrophoresis (DGGE). [Results] The highest extraction yield of total DNA was 93.2±1.5 μg/g dried culture. The quantity of bacteria and fungi reached to the maximum of 1.73×1013 copies?(gram dried culture)-1 and 6.49×1012 copies?(gram dried culture)-1, respectively. The community patterns revealed by DGGE were noticeably influenced by the DNA extraction methods. Hereinto, six methods of SDS-based DNA extraction resulted in the larger number of DNA bands. [Conclusion] The results showed that the most effective method was SDS high salt extraction combined with grinding in liquid nitrogen and lysozyme lysis.

Abstract:[Objective] MTM1 gene is essential for superoxide dismutase 2 activity and normal mitochondrial functions. MTM1 deletion results in decreased superoxide dismutase 2 activity, impaired mitochondrial functions and growth defect on nonfermentable carbon source. To promote understanding of MTM1 gene, we started a genetic screen for transposon insertions which are able to rescue the growth defect resulting from MTM1 deletion. [Methods] Routine screening strategy didn’t work because of the irreversible damage caused by MTM1 deletion. So we adopted the following screening strategy: we transformed a plasmid overexpressing MTM1 into wild type before deleting MTM1 in chromosome and got the resulting strain, designated YES2MTM1. Then we transformed mTn-lacZ/LEU2 transposon library into the YES2MTM1 strain. Transformants lost the plasmid overexpressing MTM1 after 5-Fluoroorotic acid treatment. We picked up the yeast strains able to grow on nonfermentable carbon source with MTM1 deletion and some transposon insertion and identified the insertion sites. [Results] We found transposon insertions in two genes were able to rescue the growth defect resulting from MTM1 deletion on nonfermentable carbon source. [Conclusion] Our study will provide reference for thorough understanding of MTM1 gene function.

Abstract:Abstract: [Objective] Investigation of ammonia-oxidizing microorganisms (AOMs) in hot springs is of significant importance to understand global nitrogen cycling. However, we still know little about the abundance of AOMs in hot springs. In this research, the abundance of AOMs in thirteen hot springs located in Yunnan Province, China, and the effects of environmental variables (e.g. temperature, pH and ammonia concentration, and certain ions) on the AOM abundance were studied. [Methods] Microbial abundance in collected hot spring samples was determined by using an integrated approach including reverse-transcription quantitative polymerase chain reaction and catalyzed reporter deposition-fluorescence in situ hybridisation. [Results] Total biomass in the collected hot spring samples was 108-109 cells/g, among which ammonia-oxidizing archaea (AOA) occupied 0.02-1.32%, whereas no ammonia-oxidizing bacteria were detected. Statistical analysis indicated that AOA abundance was significantly（p<0.05）correlated with concentrations of NH3, NO2-, NO3-, pH and temperature, but not related (p>0.05) to salinity and concentrations of Fe2+ and salinity. [Conclusion] AOA were the major component of AOM in the studied hot springs, and play an important role in ammonia oxidation in hot springs. Multiple environmental variables (e.g. NH3, NO2-, NO3-, pH and temperature) were together controlling the AOA distribution among hot springs of different conditions, and some environmental variables, such as Fe2+ and salinity may not be the key factors for AOA in hot springs.