Haihua Yang , Jianjun Qiao , Wenbo Sun , Mingzhang Wen
2012, 52(1):1-5.
Abstract:摘要:本文详细介绍了2011 年度国家自然科学基金委员会生命科学部微生物学学科的项目申请、受理和资助的情况,着重介绍了项目申请中应当引起申请人普遍关注的问题,对获资助项目情况的特点进行了概括和分析,希望为科研人员今后申请基金项目提供有用的参考。
Dongchang Sun , Yanmei Zhang , Yuefeng Shi
2012, 52(1):6-11.
Abstract:Abstract:Naturally transformable bacteria are able to take up DNA to acquire new genetic traits in the environment.To be naturally transformed,bacteria need to establish a physiological state,called natural competence,in which DNA uptake and processing genes are expressed. DNA uptake proteins assemble a complex to pull exogenous DNA into the cytoplasm where it can recombine with the genome DNA or establish as a plasmid. In general,DNA uptake of bacteria could be divided into two stages: DNA is transported from the milieu to the periplasm at the first stage (for Gram-negative bacteria) and is translocated across the inner membrane at the second stage. Our work and other studies revealed new plasmid DNA transformation modes in Escherichia coli. Here,we first reviewed recent advances in the molecular mechanism of natural transformation and then described the distinctive plasmid transformation mode in E.coli.
2012, 52(1):12-21.
Abstract:Abstract:Gene Transfer Agent (GTA) particles are released by bacteria and resemble small,tailed bacteriophages.GTA particles contain small,random pieces of host DNA rather than GTA structural genes or a phage genome.Gene transfer mediated by GTA is efficient and species specific based on knowledge of currently best studied GTAs produced by 4 anaerobes.Genome sequencing projects have revealed a remarkable distribution of GTA gene clusters in the genomes of marine bacterioplankton,implying GTA may be an important mechanism for horizontal gene transfer in ocean.On basis of characterization of the 4 best studied GTAs,this review described GTAs released by numerically dominant marine bacteria,discussed their properties that were important for horizontal gene transfer in ocean,and gave future perspectives to advance GTA research.
Chongde Wu , Juan Zhang , Liming Liu
2012, 52(1):22-29.
Abstract:Abstract:As cell factories,lactic acid bacteria are widely used in food,agriculture,medicine and other industries,and play a great role in industrial processes. However,lactic acid bacteria encounter various environmental stresses both in industrial processes and in the gastrointestinal tract,which impair their physiological functions and food manufacture efficiency. Recently,the development of metabolic engineering and system biology brings unprecedented opportunity for the physiological modification of lactic acid bacteria. In this review,we addresses the progress of lactic acid bacterium system biology,and based on this,the metabolic engineering strategies for manipulating and optimizing lactic acid bacteria physiological function were summarized.
Sheng Huang , Na Li , Jun Zhou , Jing He
2012, 52(1):30-37.
Abstract:Abstract:[Objective]Many natural product biosynthetic gene clusters are too large to be entirely cloned into one cosmid for heterologous expression. Because bacterial artificial chromosome (BAC) vectors are well known for their capacity of cloning large DNA fragments,we constructed a new BAC vector for cloning and heterologous expression of natural product biosynthesis gene clusters in Streptomyces.[Methods]The chloramphenicol resistance gene on the original BAC vector pCUGIBAC1 was substituted with a streptomycin resistance gene via λ RED-mediated PCR-targeting technique.The streptomycin resistance gene was then excised by digestion with NheI and the left gap was filled with the origin of transfer (oriT),the φC31 integrase gene,the integrating attP site,and an apramycin resistance gene.[Results]We achieved the final BAC vector pMSBBACs. To test the newly established vector,pMSBBACs was used to build up a genomic BAC library of Streptomyces U27. The average size of inserts in the library is about 100kb. A 140 kb BAC plasmid as a representative was successfully introduced into heterologous hosts,S.lividans and S.albus,by either conjugation or protoplast transformation. It demonstrated that the BAC plasmids constructed by pMSBBACs could be integrated into chromosomes via site-specific recombination for heterologous expression.[Conclusion]The newly constructed pMSBBACs was verified to be a good BAC vector for cloning of large DNA fragments and heterologous expression in Streptomyces.
Jing Shen , Zhenyu Tang , Ciying Xiao , Meijin Guo
2012, 52(1):38-43.
Abstract:Abstract:[Objective]The aim is to explore the self-regulation mechanism of the rex in Streptomyces rimosus M4018.[Methods]We cloned the rex of S. rimosus M4018 (Sr-rex) based on its homologoussequence in Streptomyces coelicolor A3(2) and its upstream rex operator (ROP) fragment using PCR and genome walking. An electrophoretic mobility shift assay (EMSA) was applied to analyze the regulation of rex to ROP in vitro.[Results]Sr-rex is 846 bp in length and has a 84% identity with the one in S.coelicolor A3 (2) in amino acid sequence. It was deposited in Genbank under the accession number GQ849479. The expressed Sr-Rex by E.coli was mainly composed of alpha-helixes and beta-sheets,which was in compliance with the prediction. An Electrophoretic Mobility Shift Assay (EMSA) confirmed the specific binding activity of Sr-Rex with ROP. Meanwhile,we synthesized a 22 bp DNA fragment (ROP1) based on the minimal binding site of ROP. The maximal binding ratio of this fragment to Sr-Rex was 5∶1(molar).NADH negatively affected the binding activity,however,NAD + had no impact on it.[Conclusion]In S. rimosus M4018,the Rex regulated the gene expression of ROP via sensing the intracellular level of NAD (H) .
Zhongming Li , Jiao Pan , Xudong Zhu
2012, 52(1):44-51.
Abstract:Abstract:[Objective]To identify genes in glucose repression of laccase in the human pathogen Cryptococcus neoformans.[Methods]We created a random insertional mutagenesis library containing over 200000 transformants by Agrobacterium tumefaciens-mediated transformation (ATMT).We screened the glucose derepression mutants under high-glucose condition and obtained the genes for glucose repression of laccase via inverse polymerase chains reaction ( PCR ) .[Results]Totally,we isolted 30 glucose derepression mutants from the library. We found that that 83% of the mutants contain a single T-DNA via Southern blot. We preliminarily identified 10 genes,which fall into a broad range of biological processes including: carbohydrate metabolism,sterol biosynthesis,chitin biosynthesis and glycosylphatidylinositol (GPI) anchor biosynthesis. Additionally,we found that three glucose derepression mutants have a single T-DNA insertion in the promoter region of LAC1,which encodes cryptococcal laccase.[Conclusion]As an effective way,ATMT can be utilized for identifying genes in glucose repression of laccase,which sheds lights on the roles of laccase in virulence and provides information for laccase production in industry.
Jizheng Chen , Qian Wang , Song Xu
2012, 52(1):52-59.
Abstract:Abstract:[Objective]We analyzed the effect of the stable expressed Hepatitis C virus core protein on PCK1 mRNA expression level and the molecular mechanisms involved in Huh7-lunet cells.[Methods]A retroviral vector mediated mammalian cell expression cell line of the HCV core protein was constructed. The mRNA and protein levels of PCK1,FOXO1 and PGC-1α were analyzed by Real-time PCR and luciferase assay in Huh7-lunet-core cells. [Results] HCV CORE upregulated the mRNA levels of PCK1 significantly. Both the mRNA and protein levels of FOXO1 were not affected in Huh7-lunet-core cells,whereas a decreased phosphorylation status of FOXO1 was exhibited. Moreover,activation of FOXO1 by HCV CORE was detected. Further,the mRNA level of PGC-1α was found to be significantly elevated in Huh7-lunet-core cells.[Conclusion]Our results revealed for the first time that HCV core protein expression-mediated FOXO1 activation and the increased PGC-1α leaded to the elevation of PCK1 at the mRNA level,which suggesting the immoderate gluconeogenesis in HCV-infected hepatocytes. Our findings contributed to the understanding of the molecular mechanisms of HCV-related insulin resistance and provided potential new clues for the prevention and therapy of diabetes.
Rui Sun , Ming Liu , Caixia Gong , Wei Wang , Aibing Zeng , Aiying Li
2012, 52(1):60-68.
Abstract:Abstract:Medermycin,an aromatic polyketide antibiotic produced by streptomyces,possesses a stereochemical-pyran-ring lactone critical for its strong anticancer activity. The med-ORF12 located in the biosynthetic gene cluster of medermycin encodes a stereochemical ketoreductase. Based on many indirect data,we proposed it to be involved in the enantioselective reduction at C-3 in the formation of the pyran ring of medermycin. The direct genetic evidence about the function of med-ORF12 in the medermycin-producing strain has yet to be obtained. Enzymatic features,expression and regulation pattern of Med-ORF12 in the medermycin-producer still remain obscure.[Objective and Methods]The present study aimed to investigate the expression profiles of med-ORF12 and relationship between Med-ORF12 and medermycin accumulation in medermycin-producers using prokaryotic expression,protein purification,polyclonal antiserum preparation,western blot.[Results]First,we established a prokaryotic expression system of med-ORF12 using a pET vector and optimized the induction conditions to accumulate the soluble Med-ORF12. Subsequently,we acquired the polyclonal antiserum against Med-ORF12 by immunizing the New Zealand rabbit with the purified protein. Finally,we detected the expression pattern of med-ORF12 in the medermycin producers with the obtained polyclonal antiserum,and found that med-ORF12 could express with a fairly high amount during the late stationary phase of the medermycin-producers,consistent with the accumulation of medermycin as a secondary metabolite.[Conclusion]These data indicated that Med-ORF12 expressing efficiently in the secondary metabolism could be involved in the biosynthesis of medermycin in the medermycin-producers.
Ee Li , Qi Chang , Xuena Guo , Xiuping He , Borun Zhang
2012, 52(1):69-76.
Abstract:Abstract:[Objective]There are a large numbers of tandem repeats in FLO1,which are highly dynamic components in genome leading to the unstable flocculation profiles in Saccharomyces cerevisiae. The effects of repeated unite B or D deletion on the function of flocculation protein was studied to provide theory basis for constructing genetically stable flocculation gene with minimal size.[Methods]We cloned the intact flocculation gene FLO1 from S.cerevisiae YS59 by PCR,and constructed the derived genes FLO1b and FLO1d with repeated unite B or D deletion respectively by fusion PCR. We analyzed the physiological characteristics of flocculation in yeast strains YSF1,YSF1b and YSF1d containing FLO1,FLO1b and FLO1d respectively.[Results]YSF1b and YSF1d displayed almost the same level of Flo1-type flocculation as YSF1. However,flocculation of YSF1b and YSF1d,especially YSF1d was more tolerant to pH change and mannose concentration than strain YSF1.[Conclusion]Tandem repeats regulate the function of flocculation protein. Deletion of repeated unite B or D,especially D increases the stability of flocculation protein.
Linxian Ding , Pinghua Zhang , Huachang Hong , Hongjun Lin , Akira Yokota
2012, 52(1):77-82.
Abstract:Abstract:[Objective]The purpose of the present study was to produce the Rpf (resuscitation promoting factor) protein by cloning and expressing the rpf gene,secreted by Micrococcus luteus IAM 14879,in Escherichia coli and to evaluate its role in the recovery of the VBNC (viable but non-culturable) state in high-GC Gram-positive bacteria.[Methods]Genomic DNA was extracted from Micrococcus luteus IAM 14879 and the rpf gene was amplified by PCR using specific primers. The PCR products was purified,cloned into a pET15b expression vector,and transformed into Escherichia coli BL21(DE3).Then the pET15b plasmid expression vector was used to confirm the purification of the recombinant proteins via SDS-PAGE. The VBNC state cells from the high-GC Gram-positive bacteria,Rhodococcus sp. DS471,were used to confirm the promotion and recovery of growth capacity. Rhodococcus sp. DS471 were isolated from soil and closely related to Micrococcus luteus IAM 14879. [Results]The gene sequences confirmed that the rpf gene from Micrococcus luteus IAM 14879 that was expressed in Escherichia coli,was 672 bp. SDS-PAGE analysis showed that the recombinant Rpf protein was obtained successfully,and further studies showed it capable of promoting the recovery of the VBNC state by about 100-fold relative to the control.[Conclusion]Rpf of Micrococus luteus IAM 14879 can be successfully cloned and expressed in Escherichia coli and shows a strong ability to promote the recovery of the VBNC state of cells of Rhodococcus sp. DS471.
Qinghe Zhu , Honghua Jia , Yan Li , Lisha Jia , Yingying Ma , Ping Wei
2012, 52(1):83-89.
Abstract:Abstract:[Objective]We characterized alcohol dehydrogenase from Rhodococcus erytropolis to catalyze ketoesters or ketones.[Methods] We cloned alcohol dehydrogenase gene (adh) of 1047 bp from Rhodococcus erythropolis ATCC 4277,inserted the open reading frame of adh into vector pET-22b(+) and expressed in auto-inducing media for 24 h at 15℃. The enzyme activity was determined at 30℃ using acetophenone as substrate.[Results]Under the above conditions,the specific enzyme activity of crude extract was 2.6U/mg. The optimal pH was between 6.0 and 6.5 and the enzyme can survived up to 60℃. After incubation at 60℃ for 5 h,80% enzyme activity remained.The optimal substrate among β-ketoesters examined was ethyl acetoaetate. Ethyl 4-chloroacetoacetate was catalyzed by whole cell in aqueous phase. After chiral liquid chromatography,the product showed (R) -enantioselective. [Conclusion]The study shows that the enzyme might have potential in β-ketoesters transformation on industrial scale.
Xiaoping Fu , Wenyan Wang , Guangya Zhang
2012, 52(1):90-95.
Abstract:Abstract:[Objective]This paper reports the purification of xylanase using the shortest,sensitive ELP [KV8 F-20][Methods]We designed a thermophilic xylanase gene,and recombined it with the ELP via a random coil sequence to generate the vector pET-22b-SoxB-M2-S-ELP. The expressed xylanase was purified by inverse transition cycling through high-speed centrifugation,and then we characterized the purified xylanase.[Results]The phase transition temperature of the ELPs dropped to 22℃ with 0.5 mol/L sodium carbonate (pH=7).Under this condition,SoxB-M2-S-ELP was purified by 3.16 folds after centrifugation. The recovery rate was 21.2%,and purity of the xylanase was 64.3%.[Conclusion]Elastinlike polypeptide as a purification tag to purify recombinant proteins is simple,fast,gentle and cheaper.The expression vector we constructed here might be a very useful and reliable tool to purify many other target proteins.
Guangyun Wang , Juan Wu , Wei Xie , Yucheng Li , Rong Jia
2012, 52(1):96-103.
Abstract:Abstract:[Objective]To provide effective microorganisms for the treatment of water polluted by microcystins (MCs),the strains capable of biodegrading microcystin LR(MC-LR) were isolated from the sediment of Chaohu Lake. The degradation of microcystin-LR by the intracellular extracts of the strains were studied. [Methods]The enrichment culture using MCLR as the sole carbon source was utilized to isolate the microcystin-degrading strains. The isolated strain was identified according to the observation of morphology,the physiological and chemical tests and the analysis of 16S rRNA gene sequences. The degradation of MC-LR by the intracellular extracts was studied. [Results]Strain M6 effectively degrading MC-LR was isolated and the strain M6 could grow utilizing MC-LR as the sole carbon source. Phylogenetic analysis based on 16S rRNA gene showed the similarity of 99% between strain M6 and Bacillus cereus. The experimental results suggest that the active substances for degrading MC-LR were the intracellular extracts which were the tissue enzymes of cells rather than induced enzymes. The degradation of MC-LR may be due to the catalytic effects of three enzymes. The degradation rate of 98.7% could be reached under the following conditions: pH 8.0,404.9mg/L of intracellular extracts,and 10 mg/L of the initial concentration of MC-LR.[Conclusion]Strain M6 which could biodegrade MC-LR efficiently was identified as Bacillus cereus. The influences of pH,the concentration of intracellular extracts and the initial concentration of MC-LR on the enzymatic reaction were obvious.
Lihua Hui , Ji Zhao , Linhui Wu , Yuqin Shao , Jingyu Li , Bing Zhu
2012, 52(1):104-113.
Abstract:Abstract:[Objective]To investigate the structure of ammonia-oxidation microbial communities in the wetlands to dry-up process at 99 degraded lakes of the Huitengxile grassland in the Inner Mongolia Plateau.[Methods]The microbial quantity of ammonia-oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB) were examined by most probable numberpolymerase chain reaction (MPN-PCR).The clone libraries of amoA were constructed and phylogenetics were analyzed.With analysis of the soil properties,we evaluated the effects of wetlands degradation on ammonia-oxidation microbes communities.[Results]In 75% of the samples,the quantity of AOB communities was higher than that of AOA; moreover,quantity of bacterial were up to 18. 1-fold more abundant than Archaea's. The AOB microbial quantity was strongly correlated with NH4 + -N content in the soil. Phylogenetic analyses of the amoA gene fragments showed that most AOB sequences from degraded wetlands were affiliated with Nitrosomonas-like species and a few close to Nitrosospira. All AOA sequences belonged to the kingdom Crenarchaeote.[Conclusion] Experimental results showed that quantity of ammonia-oxidation microbes increased but community diversity declined during wetlands degradation,and oxidation conditions and ammonium concentration in the soil might play important roles in the community structure of both the AOA and AOB.
Pinghua Li , Xingwen Bai , Zengjun Lu , Pu Sun , Guocai Qi , Chenghao Han , Zaixin Liu
2012, 52(1):114-119.
Abstract:Abstract:[Objective]A series of type O foot-and-mouth disease (FMD) outbreaks occurred in China seriously affect development of Chinese husbandry. Its causative agent—type O foot-and-mouth disease virus (FMDV) has been evolved three topotypes: Cathay,Middle East-South Asia and Southeast Asia,specifically,the viruses of Cathay topotype are highly adapted to pig,representing the biggest threat on Chinese pig industry. The available FMD vaccine in China provides insufficient protection against some arising viruses of Chathay topotype,which exert important obstacle to control porcinophilic FMD epidemics in China. To develop vaccine candidate with characteristics of good immunogenicity and broad spectrotype,a full-length infectious cDNA clone of inter-genotypic chimeric FMDV was constructed,which replaced part VP3 and VP1 gene of O/HN /93 strain with the corresponding to the variants of Cathay topotype (mainly replaced the B-C and G-H loop of structure protein VP1).[Methods] Linearized recombinant plasmid and plasmid expressing T7 RNA polymerase were cotransfected into BHK-21 cells to rescue the chimeric virus in vivo.[Result]The transfected cells showed apparent cytopathic effects (CPE) after 36 h post-transfection. The rescued virus was checked by RT-PCR,indirect immunofluorescence,electron microscope. Results show that chimeric FMDV was successfully rescued in vivo.The study of sulk mice pathogenicity show chimeric FMDV has attenuated pathogenicity for suckling mice compared with its parental virus.[Conclusion] The construction of inter-genotypic chimeric FMDV will lay the basis for developing novel vaccine against FMD.
Tingting Zuo , Yanwei Li , Xuelian Han , Jun He , Qing Duan
2012, 52(1):120-123.
Abstract:Abstract:[Objective]To define the sequence type (ST) isolates of Bacillus anthracis by multilocus sequence typing (MLST).[Methods]Fragments of seven housekeeping genes (glpF,gmk,ilvD,pta,pur,pycA,and tpi) were amplified by PCR using the standard primers as described on the website for MLST of Bacillus and the sequences were compared with existing allele sequences on the MLST website.[Results]Two novel allele combinations of the seven loci were found in two isolates 17003-14 and 17003-32.[Conclusion]Two novel ST isolates of B.anthracis were identified by this study and confirmed by the MLST website,and the pubMLST ids were id-1053 and id-1054.
2012, 52(1):124-129.
Abstract:Abstract:[Objective]To explore the cholesterol-degrading in mice by Lactobacillus plantarum LpT1 and LpT2 in vivo.[Methods]The hypercholesterolemia mice were randomly classified into 4 groups: A,B,C and D and fed with strain LpT1,LpT2,lovastatin and distilled water,respectively,and then TC,TG,HDL-C,LDL-C and AI were determined.The tissue slice of liver was made and observed by transmission electron microscope.[Results]The hypercholesterolemia mice model was successfully constructed after feeding the hypercholesterol forage for 7 days. Lactobacillus plantarum LpT1 and the positive control lovastatin could significantly degrade the total cholesterol content (p<0.01) after 28 days,higher than Lactobacillus plantarum LpT2 (p<0.05). However,the negative control water could not degrade it. The transmission electron microscope result showed that the strains could effectively regulate the lipid metabolism to develop towards the regular trend after they were absorbed into the intestines.[Conclusion]The results further laid foundation for studying the cholesterol- degrading mechanism by Lactobacillus plantarum in vivo.
Chunyan Qin , Xu Zhang , Gu Chen
2012, 52(1):130-135.
Abstract:Abstract:[Objective]It is a conserved mechanism in bacteria that metalloprotease site-2 protease (S2P) cleaves transmembrane anti-sigma factor to release sequestered sigma factor in response to extracytoplasmic stress. However,the function of site-2 protease homologs in cyanobacteria remains elusive,so we investigated the metalloprotease activity of Slr0643 and Sll0862,the site-2 protease homologs from Synechocystis sp. PCC6803. [Methods]Recombinant Slr0643 and Sll0862 were constructed and overexpressed in Escherichia coli BL21 (CE3).Their protease activities were tested against β-casein and then resolved on SDS-PAGE.[Results]Results from caseinolytic assay indicated that Slr0643 and Sll0862 have proteolytic activity which is blocked by o-phenanthroline,a metalloprotease inhibitor. These metalloprotease activity of Slr0643 and Sll0862 in vitro provide the foundation for futher analysis of their substrates in vivo.[Conclusion]The site-2 protease homologs in Synechocystis sp. PCC6803 have metalloprotease activity.
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