Abstract:Abstract:Bergey's Manual of Systematic Bacteriology (hereinafter referred to as“Bergey's Manual”) is the collection of academic views accepted by taxonomists in many countries． It has scientificity，unitarity and practicality． “Bergey's Manual”(special issue of Actinomycetes) divided into two parts (part A and part B) was published in May，2012．Under the guidance and the organization of Michael Goodfellow et al.,the great work has been completed successfully in May 2012. “Bergey's Manual”made a great modification on the systematic of Actinomycetes and formally set up the phylum of Actinobacteria，which encompasses 6 classes，23 orders (include one order incertae sides)，53 families，222 genera and about 3000 species．The taxonomic catalogue is Bacteria，phylum of Actinobacteria，under the phylum there are class，order，family，genera and species．“Bergey's Manual”collected a great deal of new taxa，which were published in IJSEM (International Journal of Systematic and Evolutionary Microbiology) by Chinese scientists． We need to indicate that due to its too rigorous，conservative writing purpose and long publication periods，“Bergey's Manual”fails to collect new research results using the molecular approaches of multilocus sequence analysis“MLSA”，gene chip technology and genome technologies，which however will profoundly change the taxonomy of prokaryotes in the near future．

Abstract:Abstract:Tolerance of Saccharomyces cerevisiae to monoterpenes is important in both metabolic engineering of the yeast to produce these chemicals de novo and efficient use of biomass containing these chemicals．Understanding the mechanisms in the tolerance of S． cerevisiae to monoterpenes could facilitate the construction of yeast strains with enhanced monoterpenes resistance，and therefore improve related bioprocesses． Monoterpenes could disturb the redox balance in S．cerevisiae，therefore increase the accumulation of reactive oxygen species (ROS) and result in cell death．S．cerevisiae has to systematically improve its antioxidative ability to deal with the ROS induced damage． The current review summarized the recent developments in demonstration of the tolerance of S． cerevisiae to different typical monoterpenes mainly from the aspect of the antioxidative mechanisms．Based on the analysis of the previous works，further attempts to demonstrate the mechanisms were proposed．

Abstract:Abstract:It is difficult to stimulate efficient gut mucosal immune response to intestinal infection．This article critically reviews the research progressin Escherichia coli strain Nissle1917 (EcN) actingas a safe vehicle for the intestinal mucosal immunity，to restore gastrointestinal disorder and relieve ulcerative colitis． EcN is an orally administered probiotics，combining the excellent colonization and non-immunogenic character，and can be an ideal live vector candidate．This strain could be a tumor-targeted delivery of TAT-Apoptin fusion gene to colorectal cancer．In the treatment of ulcerative colitis and Crohn's disease，the recombinant strain of EcN can be used as a target therapeutics for defensins presenting．Genetically modified EcN could be an ideal carrier organism for gut-focused in situ synthesis and expression of specific localized antigen delivery into the intestine，and stimulate specific mucosal immune response． In vitro trial demonstrated that intestinal recombinant E． coli Nissle-HA110-120 has the potential to stimulate antigen specific response，but EcN itself does not induce mucosal immune response and influence peripheral tolerance to self-antigen． At the same time，there are evidences that EcN is safe．Recombinant E．coli Nissle-HA110-120 does not migrate，clonally expand and activate specific CD4 + T cells，neither in healthy mice nor in other animals with acute colitis，even when the intestinal epithelium suffer from inflammation and the barrier function of the epithelial layer being destroyed．

Abstract:Abstract:［Objective］The aim of this study was to further increase the yield of insulin precursor by Pichia pastoris．［Methods］For this，we transformed the expression vector pPICZα-IP into P． pastoris X-33 using electroporation and screened two mutant strains B4 and S6 on the YPD plate containing 100 μg/mL zeocin． Both could overexpress human insulin precursor． Taking B4 and S6 as start strains，we repeatedly transformed SacI linearizing pPICZα-IP into P．pastoris X-33 by electroporation，then screened a new mutant strain 2B4 (with 7 copies) on the 1000 μg/mL zeocin YPD plate．［Results］ After cultivation，the human insulin precursor yield of 2B4 strain was 2.7 fold higher than that of B7．Meanwhile，the cell growth was not inhibited．The target gene transcription level of 2B4 was 2 fold higher than that of B7 by real-time quantification PCR．［Conclusion］The strategy of combining resistance screening and repeated electroporation was efficient to increase the copy number of target gene，so as to facilitate higher transcription level and enhance objective recombinant protein yield．

Abstract:Abstract:［Objective］This study aimed to investigate the effects of Cu2 +，Cr2+ and Pb2 + stress on Nostoc flagelliforme cell．［Methods］ The response of Nostoc flagelliforme cell was analyzed under the stress．The modified BG11 culture medium containing different heavy metal ions of 0，0.1，1.0，10，100 mg/L was used to cultivate Nostoc flagelliforme cell at 25℃ and light intensity of 80 μmol/(m·s)．Electrolyte leakage，the activities of superoxide dismutase，the content of malondialdehyde，proline，soluble protein and trehalose were analyzed．［Results］ Under 1－100 mg/L Cu2 +，Cr2 + and Pb2 + stress，electrolyte leakage and malondialdehyde contents in Nostoc flagelliforme cell were higher than those in the control group during heavy metal ions stress． Meanwhile，superoxide dismutase activity increased slightly under 10 mg /L，but was lower afterwards．The contents of proline，soluble protein and trehalose increased under 10mg /L heavy metal ions stress，while declined under extreme heavy metal ions stress (100 mg/L)．［Conclusion］Nostoc flagelliforme cell has resistance to low heavy metal ions stress，but is damaged badly under extreme heavy metal ions stress．

Abstract:Abstract:［Objective］A gene encoding solvent-resistant glucose dehydrogenase was cloned from Bacillus sp．YX-1 and expressed in Escherichia coli．The recombinant enzyme was then characterized．［Methods］ The glucose dehydrogenase gene was amplified from Bacillus sp． YX-1 genome according to its conserved sequences in Bacillus sp．The recombinant enzyme was over-expressed in E．coli and purified by HisTrap HP affinity chromatography．The purified enzyme were characterized．［Results］The glucose dehydrogenase gene contains an open reading frame of 786 bp encoding 261 amino acids．The maximum activity was observed at 45℃ and pH 8.0．The recombinant enzyme was highly resistant to several organic solvents．More than 90% of the activity was maintained when the enzyme was incubated in 50% cyclohexane，octane，decane at home temperature for 1 h．In addition，the enzyme displayed broad substrate spectrum and has catalytic activity for several sugars to afford reduced coenzymes．It exhibits similar capability to regenerate either NADH or NADPH with specific activity of 8.37 U/mg and 8.62 U/mg for NAD + and NADP + ．［Conclusion］The organic solvent-tolerant glucose dehydrogenase was explored successfully on the basis of bioinformatics analysis． The work supplied a new biocatalyst for the cofactor-regeneration during the reaction in organic phases catalyzed by oxidoreductases．

Abstract:Abstract:［Objective］Crenarchaeota is a major archaeal lineage in terrestrial hot springs and important in biogeochemical cycles of life-essential elements．In this study，we investigated the diversity of Crenarchaeota in hot springs and the surrounding environments in Kamchatka，Russia．In addition，we compared crenarchaeotal community structures in Kamchatka，Russia and Yunnan province，China．［Methods］ Crenarchaeal 16S rRNA gene clone libraries were constructed and the sequences and abundances of representational clone were obtained． Phylogenetic analysis was then performed and the community structures in different samples were compared．［Results］ The high temperature spring Burlyashi Liza (BSL，89℃) comprised Thermoprotei．The moderate temperature spring TF Vent 2 (TFV，49℃) harbored unidentified Thermoprotei group，unidentified crenarchaeal group，HWCG-II (hot water crenarchaeotal group II)，and Group1.1b (one thaumarchaeotal subgroup)．Most of sequences that obtained from surrounding environments (＜15℃) are closely with the representational clone pJP from a Yellowstone hot spring．Jackknife cluster and Principal coordinates analysis (PCoA) showed that the samples have more similarity in crenarchaeal communities at similar temperatures． ［Conclusion］The diversities of Crenarchaeota in Kamchatka hot springs are somewhat different from those in Yunnan province． Terrestrial hot springs obviously affect the crenarchaeotal communities in surrounding environments．Temperature is the major factor controlling the community structure in terrestrial hot springs．

Abstract:Abstract:［Objective］ The diversity of Fe-hydrogenase based on Fe-hydrogenase gene of Clostridium was studied for exploring the biochemical mechanism of soil microbial hydrogen production and revealing the Fe-hydrogenase microbial community structure changes during the paddy soil flooding incubation ［Methods］ We used denatured gradient gel electrophoresis and real-time quantitative PCR to achieve the goal． ［Results］ The band number of Fe-hydrogenase denatured gradient gel electrophoresis fingerprints indicated Fe-hydrogenase microbes structure varied significantly during the completely flooding incubation． The Principal Component Analysis (PCA) showed the highly similar communities of Fe-hydrogenase microbial was divided into three groups: 1 d and 20 d，the 5 d，30 d and 40 d．With the growth of the flooding incubation time Fe-hydrogenase microbial community structure became relatively stable and convergence． α diversity index analysis found that the richness index (R)，Shannon-Weaver index (H')，Simpson index (DS) numerical values of 1 d and 10 d were lower compared with other points，indicated the two time points of low Fe-hydrogenase diversity，simple Fe-hydrogenase microbial community structure and successive variation of community structure during the whole flooding incubation． After sequencing 15 Fe-hydrogenase preponderant bands (labeled by G1－G15)，the phylogenetic tree of Fe-hydrogenase showed that the preponderant bands all had high similarity with the Clostridium Fehydrogenase in earlier flooding incubation stage and non-Clostridium Fe-hydrogenase in the later stage．Real-time quantitative PCR results demonstrated that the Fe-hydrogenase gene copy number was 106 level．［Conclusion］ In the research we found 4 kinds of Clostridium Fe-hydrogenases and three kinds of non-lostridium Fe-hydrogenases，the corresponding Fe-hydrogenase microbial community structure had successively significant variation in the early incubation stage and tend to be relatively stable and convergence in the late stage．

Abstract:Abstract:［Objective］Baculovirus is known as a safe vector candidate due to its non-replication in mammalian cells．The tropism to different cells and transduction efficiency can be improved by introducing cell-specific promoter，VSV-GED and different functional regulatory elements． The optimized pseudotyped recombinant baculovirus can express eGFP gene in primary chicken cells，which provides us a new approach to develop engineered poultry vaccines．［Method］ The pseudotyped recombinant baculoviruses were constructed with cytomegaoviyns (CMV) promotor，VSV-GED，woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and inverted terminal repeats (ITRs)．The recombinant baculoviruses contained eGFP reporter gene were transfected chicken primary cells，and the eGFP protein expression levels mediated by different baculoviruses were analyzed．［Results］The expression of eGFP was detected at 12 hours after infection．The transduction efficiency of the pseudotyped recombinant baculoviruses increased from 36% to 48.2% by inserting VSV-GED． The expression effect of eGFP in recombinant baculovirus carrying WPRE element was similar to that by adding 10mmol/L butyrate． However，the WPRE elements are nontoxic to cells． Within 72 hours，the expression intensity of eGFP in the recombinant baculovirus with ITRs increased gradually．［Conclusion］The VSV-GED element can improve the transduction efficiency and WPRE can increase the reporter gene eGFP expression levels mediated by baculovirus in chicken primary cells． The recombinant baculovirus with the ITRs elements can extend the expression time of eGFP.

Abstract:Abstract:［Objective］We developed a synthetic vaccine against foot-and-mouth disease type A．［Methods］We studied two peptide-based vaccines containing residues 131 to 159 of VP1，20 to 35 of VP4，21 to 35 of 3A and 29 to 42 of 3B of the AF/72 strain of foot-and-mouth disease virus ( FMDV) coupled with a CpG oligodeoxynucleotide (5'-TCGCGAACGTTCGCCCGATCGTCGGTA-3') in guinea pigs． We assayed the FMDV-specific IgG level， serum neutralizing antibody titer，splenic lymphocytes proliferative capacity and peripheral blood T lymphocyte CD4-CD8 subsets distribution． ［Results］The data show that high dose did not ensure a good immunity． In our study，8% (4/5) of peptide 364-2. 5-inoculated guinea pigs (2.5 μg of peptide 364 per animal) were protected against AF/72 strain challenge，while the protection ratio from other peptide-immunized groups was lower except the inactivated vaccineinoculated group which showed a full protection．Our results also indicated that the stimulatory ability of CD4 + T lymphocyte response played a key role in evaluating effective FMDV vaccine．The highest percentage of CD4 + T lymphocyte was 36.6% appeared in inactivated vaccine-immunized guinea pigs，the second was 33. 7% in peptide 364-2. 5-vaccinated group，whereas the remaining ranged from 18.1% to 27.7%．There was no obvious relation between CD8 + T cells and anti-FMDV infection; our data showed that the CD4/CD8 ratio was not always appropriate for assessing the immune system status．［Conclusion］In general，we not only designed an effective vaccine against FMDV type A，but also discovered some useful information of humoral and cellular responses induced by foot-and-mouth disease vaccines．

Abstract:Abstract:［Objective］Gene knock out technique is very important for gene function study．We developed a simple and efficient method to knock out chromosomal genes in Escherichia coli．［Methods］Using the Escherichia coli Keio singlemutant library，we combined Red homology recombination with P1 phage transduction and developed a method to knock out the genes in Escherichia coli MG1655．［Results］We obtained β-oxidation mutants △fadD，△fadE and △fadD-△fadE and fatty acid synthesis mutants △fabH，△fabF and△fabH-△fabF. There were no obvious growth changes between △fadD or △fadE mutant strains and MG1655． However，△fabH and △fabH-△fabF mutant strains grew much slower than the wild type strain． The fatty acid contents in △fadD，△fadE and △fadD-△fadE were 18.2 mg/L，20.0 mg/L and 19.2 mg/L respectively，higher than 17.5 mg/L in wild type．The fatty acid contents in △fabH，△fabF and △fabH-△fabF were 12.6 mg/L，15. 2 mg/L and 11.2mg/L respectively，lower than that in wild type．［Conclusion］Using Keio mutant library，P1 phage transduction and resistant gene elimination，we have established a simple and efficient method for gene knock-out in Escherichia coli．

Abstract:Abstract:［Objective］We compared alkaline tolerance between Bacillus thuringiensis(Bt) and B． subtilis(Bs) and to determine the effect on growth when the clpP gene encoding caseinolytic protease is disrupted after alkaline shock．［Methods］B．thuringiensis HD73 mutant with the deletion of clpP gene was constructed by homologous recombination．The effects of clpP deletion on the growth after alkaline shock，sporulation and germination were analyzed． ［Results］Bt can recover growth from alkaline shock when the medium pH was between 8.9 and 9.1 whereas that was between 8.2 and 8.4 for Bs．Bt tolerated alkaline more than Bs，leading Bt to adapt in alkaline environment of the midgut as a pathogen of insect．Deletion of clpP gene had no influence on sporulation and germination．The clpP mutant grew slower than Bt HD73 in the LB medium in addition to NaOH of 30 mmol/L．It indicates that the ClpP plays an important role in the alkaline tolerance of Bt strain．

Abstract:Abstract:Micoromonospora sp．40027，the producer of fortimicin A，harbors two plasmids pJTU101 and pJTU112．［Objective］Cloning and sequencing of replication region of pJTU112，analyzing replication region sequence of pJTU112．［Methods］Different fragments of pJTU112 were cloned and introduced by conjugation into Micromonospora sp． LXH20．The replication region of pJTU112 was identified．［Results］The replication region of pJTU112 was located in the 4.7 kb SacI-KpnI DNA fragment．DNA sequencing and analysis revealed that the 4.7 kb SacI-KpnI DNA fragment encoded five open reading frames．The pJTU112.1 and pJTU112.2 were related to plasmid conjugation，pJTU112.3，pJTU112.4 and pJTU112. 5 were related to plasmid conjugation．［Conclusion］ The replication region of pJTU112 was located in the 4.7kb SacI-KpnI DNA fragment．

Abstract:Abstract:［Objective］Bacterial strain F5-1 isolated from the Homarus americanus was characterized and its changes in membrane fatty acid composition in response to low temperature were also studied．［Methods］ The physiological and biochemical characteristics were carried out by using VITEK 2 compact automated microbiology system．The 16S rRNA gene was sequenced and subjected to phylogenetic analysis．Fatty acids were detected by gas chromatography-mass spectrometry (GC-MS)．［Results］Strain F5-1 was Gram-negative and susceptible to the vibriostatic agent O /129．Strain F5-1 was resistant to Penicillin．The isolated strain exhibited the highest levels of 99% probability to Vibrio metschnikovii based on the conventional physiological test． The sequence analysis of 16S rRNA gene of F5-1 isolation and comparison with that of other related vibrios showed that F5-1 was very close to V．metschnikovii (GenBank No．HQ658055)．The similarity was 99%．The major fatty acids were C12∶0，C14∶0，C16∶0 and C16∶1 (n-7)．Palmitoleic acid was the dominant unsaturated fatty acids．The major change in fatty acid composition occurred in response to low temperature，with an increase in palmitoleic acid from 34% to 40%．［Conclusion］Bacterial strain F5-1 isolated from Homarus americanus was identified as V．metschnikovii and was sensitive to multiple drugs．The fatty acid composition of F5-1 was different from V．metschnikovii isolated from a drinking water reservoir near Vladivostok City in the Russia Far East．Results of this study indicated that environmental conditions allowed modulation of the fatty acid composition of V．metschnikovii．