Abstract:Abstract:Growing attention has been focused on probiotic lactic acid bacteria because of their important health-promoting effects． Nowadays，probiotic-based products have become fashionable nutraceuticals of choice．Before a newly developed probiotic-based product is to be introduced into the industry，it is important to ensure not only the desirable properties of the probiotic strain but also a good genetic stability． This article firstly introduces the research methods for investigating genetic stability，followed by summarizing the latest research progress in China and overseas．

Abstract:Abstract:As successful enteric bacteria，Salmonella spp．has to overcome the extreme acid condition in the stomach before invading into host intestinal epithelial cells．Salmonella spp． has evolved an adaptation to its replicative niche in the acidic environment． This review summarizes acid resistant characteristics of Salmonella，and introduces several mechanisms to acid resistance，including keeping internal pH homeostatic，synthesizing acid shock protein through several regulatory pathways and altering membrane character．The achievements will be significant for understanding and controlling Salmonella infections in the future．

Abstract:Abstract:Abatract: For most viruses in Paramyxoviridae，cell fusion requires both attachment protein and fusion protein．The attachment protein is responsible for the binding to its cognate receptors，while the interaction between fusion protein and attachment protein triggers the fusion protein which is responsible for the fusion．However，the Metapneumovirus fusion in Pneumovirinae subfamily displayed different mechanism where the attachment protein is not required．The cell fusion is accomplished by fusion protein alone without the help of the attachment protein． Recent studies indicate that low pH is required for cell fusion promoted by some hMPV strains．The fusion protein of aMPV type A is highly fusogenic，whereas that of type B is low．The original fusion models for Paramyxovirus cannot explain the phenomenon above．The mechanism to regulate the cell fusion of Metapneumovirus is poorly understood．It is becoming a hot spot for the study of cell fusion triggered by Paramyxovirus where it enlarged the traditional scope of Paramyxovirus fusion．In this review，we discuss the new achievements and advances in the understanding of cell fusion triggered by Metapneumovirus．

Abstract:Abstract:［Objective］We evaluated the difference of three standard curves in quantifying viable Vibrio parahaemolyticus in samples by real-time reverse-transcriptase PCR (Real-time RT-PCR).［Methods］ The standard curve A was established by 10-fold diluted cDNA. The cDNA was reverse transcripted after RNA synthesized in vitro．The standard curve B and C were established by 10-fold diluted cDNA．The cDNA was synthesized after RNA isolated from Vibrio parahaemolyticus in pure cultures (108 CFU/mL) and shrimp samples (106 CFU/g)(Standard curve A and C were proposed for the first time). Three standard curves were performed to quantitatively detect V．parahaemolyticus in six samples，respectively (Two pure cultured V．parahaemolyticus samples，two artificially contaminated cooked Litopenaeus vannamei samples and two artificially contaminated Litopenaeus vannamei samples)．Then we evaluated the quantitative results of standard curve and the plate counting results and then analysed the differences.［Results］The three standard curves all show a strong linear relationship between the fractional cycle number and V．parahaemolyticus concentration (R2＞0.99);The quantitative results of Real-time PCR were significantly (p＜0.05) lower than the results of plate counting．The relative errors compared with the results of plate counting ranked standard curve A (30.0%)＞standard curve C (18.8%)＞ standard curve B (6.9%); The average differences between standard curve A and standard curve B and C were－2.25 Lg CFU/mL and－0.75 Lg CFU/mL，respectively，and the mean relative errors were 48.2% and 15.9%，respectively; The average difference between standard curve B and C was among ( 1.47－1.53) Lg CFU/mL and the average relative errors were among 19.0%－23.8%.［Conclusion］Standard curve B could be applied to Realtime RT-PCR when quantify the number of viable microorganisms in samples.

Abstract:Abstract:［Objective］The study was aimed at understanding the roles of polygalacturonases in the pathogenicity and the interaction between Rhizoctonia solani and rice．［Methods］According to the sequences of Rspg1 of R．solani deposited in GenBank，a pair of specific primers was designed．The gene Rspg1 was cloned and expressed using prokaryotic expression tool to elucidate its biological characteristics．The structures of the protein RsPG1 were predicted using bioinformatics tools．［Results］A 1395-bp fragment including an open reading frame (OFR) of Rspg1 was amplified from the genomic DNA of the pathogen．Compared with RT-PCR results，it was found that this sequence fragment contains five introns (positions 278-334，545-601，657-715，1090-1155 and 1244-1304) and one 1095 bp ORF．The ORF was predicted to encode 364 amino acids． Bioinformatics analysis showed that RsPG1 contains an 18-amino acid signal peptide and 4 conserved sequence segments (180NTD，202DD，223GHG and 255RIK) characteristic of all the polygalacturonases．The main structural elements of the secondary structure are α-helix，β-sheet and random coil．Six cysteines form three disulfide bonds (Cys24 -Cys40，Cys204 -Cys220 and Cys329 -Cys333)．Transmembrane prediction analysis suggested that RsPG1 could be secreted outside the cell．Tertiary structure is a right-handed helix which consisted of ten repeated β-sheet，forming an opening activity cleft．［Conclusion］ RsPG1 is tentatively a 40 kDa protein with polygalacturonase enzyme activity at 277.78 U/mg．It is probably a secreted protein and has characteristics of all the polygalacturonases．The results can help to further understand the roles that R． solani polygalacturonases play during the pathogenicity and how the pathogen interacts with the host．

Abstract:Abstract:［Objective］We regulated the carbon flux distribution of Torulopsis glabrata CCTCC M202019，an efficient pyruvate-producing microorganism，for improved 2，3-butandione production.［Methods］ We overexpressed the acetolactate synthase (ALS) from Bacillus subtilis and then used the genome-scale metabolic model (GSMM) for T．glabrata (named iNX804) to evaluate the importance of deleting the ILV5 gene． In addition，the BDH gene was deleted to restrict the degradation of 2，3-butanedione.［Results］Overexpression of the ALS resulted in a 4. 6-fold increase in ALS activity and increased the extracellular concentration of 2，3-butanedione to 0.57 g/L from 0.01 g/L. The deletion of the ILV5 gene was found to increase the 2，3-butanedione accumulation level by 28.1% ，attributed to the disruption of Lvaline and L-leucine biosynthetic pathway． With the deletion of the BDH gene，the enzyme activity levels of butanedione reductase and butanediol dehydrogenase were decreased by 74.4% and 76.1%，respectively．And the accumulations of 3-hydroxybutanone and 2，3-butanediol were decreased by 52.2% and 71.4%，respectively．The final 2，3-butanedione concentration was 0.95 g/L，which was 30.1% higher than that of the control strain.［Conclusion］The GSMM based system metabolic engineering can be a functional strategy to redistribute the carbon flux from pyruvate node to 2，3-butanedione and achieve efficient accumulation of 2，3-butanedione.

Abstract:Abstract:［Objective］ To explore the regulation of iron on siderophore production，cell growth and photosynthetic pigments biosynthesis by siderophore-producing anoxygenic phototrophic bacteria.［Methods］Siderophore production was determined using Chrome Azurol S (CAS) assay. The siderophore types were determined by Arnow method，Csaky test and Shenker test. The compositions and contents of photosynthetic pigments were determined by spectrophotometry and HPLC analysis.［Results］Rhodopseudomonas palustris (Rps. palustris) CQV97 was capable of producing hydroxamatetype of siderophore．Siderophore production reached the highest yield in the absence of ferric chloride．With increasing ferric chloride concentrations，the lag phase of cell growth was shortened，and the cell growth rate，final biomass and the total amounts of carotenoid and bacteriochlorophyll a were increased significantly．The characteristic absorption maxima of carotenoids from pigment extracts were blueshifted. Iron concentration had little effect on the compositions and relative contents of bacteriochlorophylls a，whereas predominately affected carotenoid compositions，rhodopin was present as major carotenoid component instead of spirillxanthin. Culture tends to accumulate the Cars having shorter conjugated double bonds at the expense of longer conjugated double bonds as the ferric chloride concentration increased． The changes in carotenoid composition were consistent with those of the blue shift of absorption spectra of pigment extracts.［Conclusion］ Rps. palustris CQV97 can produce siderophore and the changes in microbial growth，siderophore production and photosynthetic pigments accumulation of anoxygenic phototrophic bacteria are related to the iron concentration in the medium.

Abstract:Abstract:［Objective］ To study biochemical functions of the Legionella pneumophila eukaryotic-like effector protein LegK3，the budding yeast Saccharomyces cerevisiae was used as an alternative host in which growth defect induced by the ectopic expression of LegK3 was assessed． ［Methods］ Using genomic DNA of the L. pneumophila strain Lp02 as template，we respectively amplified and inserted the ORF sequences of legK3，ralF or lidA into the plasmid pESC-HK to yield the ectopic-expression plasmids． Then，the recombination plasmids were transformed into the yeast strain W301-1A．With 2% -galactose induction，growth defect and carboxypeptidase Y (CPY) delay were determined simultaneously. In parallel，total yeast proteins before or after induction were extracted and subjected to Immunoblot assay. For detecting the expression of effector proteins or determining CPY delay，anti-c-myc or anti-PGK/anti-CPY antibodies were utilized respectively．［Results］The expression of LegK3 resulted in visible growth defect in yeast cells，together with obvious retard in CPY processing．［Conclusion］L．pneumophila eukaryotic-like effector LegK3 might target and interfere with the vesicle-trafficking pathways，thereby to inhibit the growth and division of host cells．

Abstract:Abstract:［Objective］We investigated biological activities，physiological and biochemical properties of two endophytic bacteria isolated from fresh leaves of tea plants．［Method］ We did morphological observation，biological activity test，physiological and biochemical assays，16S rDNA analysis，and compared their genotype and phenotype with those of 13 Herbaspirillum species．［Results］Their colonies were round，opaque，central uplift and regular edge with a milky white color． Their cells were Gram-negative，rod-shaped with the size of (0.5－0.7) mm×(1.4－1.8) mm and flagellers，but without spore．Both isolates produced indole-3-acetic acid (IAA) (18.7 mg/L for WT00C and 24.9 mg/L for WT00F)，ammonia and siderophores，but no nitrogen-fixing activity．The 16S rDNA had sequences similarities of 99.7% each other and 99% with 13 Herbaspirillum species. Two isolates used carbon source as described in the genus Herbaspirillum，except for propionate salt．The neighbor-joining tree built using the 16S rDNA showed that two isolates formed an independent group，which kept certain genetic distance from the 13 Herbaspirillum species. Their physiological and biochemical characteristics and genotypes were different from those for 13 Herbaspirillum species.［Conclusion］Two isolates WT00C and WT00F were classified as novel members in the genus Herbaspirillum.

Abstract:Abstract:［Objective］ To compare five selective media to isolate human Bifidobacterium［Methods］ Feces from six healthy human volunteers were diluted and cultivated on five Bifidobacterium selective media． After anaerobic cultivation，bacterial colonies were counted，selected and identified． Meanwhile，bacterial genomic DNA was extracted from the feces samples，and the Denaturing Gradient Gel Electrophoresis (DGGE) and Quantitative Polymerase Chain Reaction (q-PCR) were applied to reveal the diversity of Bifidobacterium.［Results］The amount of Bifidobacterium grown on BSM and BLM media was similar to the result detected by q-PCR and was significantly higher than that on three other media．Bifidobacterium isolated from BLM medium was similar to the identified result of DGGE profile.［Conclusion］ BLM medium is the best selective medium for Bifidobacterium isolation from human gastrointestinal tract.

Abstract:Abstract:［Objective］Sturgeons were the important economic species in Beijing．In July 2012，continuous mortality of cultured hybrid sturgeons was occurred on a farm in Huairou．［Methods］We isolated three pathogens from liver，kidney and spleen of the dying sturgeons with clinical symptoms，marked HRS12718L， HRS12718K and HRS12718S respectively．Then we analyzed theirs morphological，physiological and biochemical characteristics，and drug sensitivity．We also cloned and partially sequenced the 16S rDNA of strain HRS12718K． Moreover，we identified the pathogenic characteristic of strain HRS12718K by artificial infection．［Results］The morphological，physiological and biochemical characteristics of three strains were consistent with that of Streptococcus iniae isolated from other fishes in China，and 16S rDNA sequence of the strain HRS12718K was more than 99.1% homology with that of Streptococcus iniae．The LD50 of the pathogen to hybrid sturgeon was 4.42×105 CFU/mL，and challenged sturgeons presented the similar signs as the natural infected sturgeons． In addition，the bacterium was sensitive to norfloxacin，enrofloxacin，neomycin sulfate，doxycyline hyclate and tetracycline hydrochloride，and was highly sensitive to enrofloxacin． ［Conclusion］Streptococcus iniae was the pathogen to cultured hybrid sturgeons in Beijing area，and enrofloxacin can be used against the disease．

Abstract:Abstract:［Objective］ We explored the cause of cell growth inhibition by antisense RNA mediated nonessential gene silencing of rpsF gene in Escherichia coli．［Methods］The 41－230 bp fragment around 5' end of gene rpsF was reversely cloned into antisense expression vector pHN678，which is flanked with a paired-termini．The recombinant plasmid was named pHNF．Then it was transformed into E.coli to produce antisense RNA strain E.coli/pHNF．Antisense RNA expression was induced by isopropyl-β-D-thiogalactopyranoside (IPTG)，the difference of liquid growth phenotype was identified between E.coli/pHNF and the control strain E.coli/pHN678; and gene transcriptional level was measured by Real time RT-PCR.［Results］We obtained one antisense RNA strain targeted rpsF．We found that the reduced growth rate of this strain was positively related to the IPTG concentration． When IPTG was 100 μmol/L，the cell growth was not inhibited whereas the mRNA amount of rpsF had decreased by 36%，and mRNA of essential gene rpsR in the same operon did not decayed．However，when IPTG reached 200 μmol/L，the cell growth was obviously inhibited and rpsR mRNA was reduced by 12%.［Conclusion］ The essential gene transcription level of rpsR decreases with the nonessential gene silencing of rpsF in the same operon，and leads to the growth inhibition of E.coli/pHNF.

Abstract:Abstract:［Objective］ To construct the recombinant baculovirus with mammaliancell-specific promoter and woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)，to highly express Newcastle disease virus(NDV) F gene in the primary chicken embryo cells． ［Method］We extracted total RNAs from NDV La Sota strain． Then the F gene was amplified by reverse transcription polymerase chain reaction． We constructed the baculoviral vector (pCMV-WPRE-F) with F gene fused with the WPRE near its 3＇ end，which expressed under the control of the CMV promoter．The F gene recombinant bacmid was obtained by Bac-to-Bac system and transfected into sf9 insect cells to acquire F gene recombinant baculovirus．After amplification of recombinant baculovirus，the recombinant virus was transfected into chicken primary cells with 50 multiplicity of infection，and the proteins were harvested at 72 h after infection．The F protein expression levels mediated by WPRE regulatory element were analyzed．［Results］ Western blot results show that the F gene was successfully expressed in chicken primary cells． The product was a 56kDa protein and could be recognized by anti-NDV serum．The WPRE fusion significantly improved the F gene expression as 10mmol/L butyrate did，but different to butyrate，the WPRE regulatory element was nontoxic to cells．［Conclusion］The optimized recombinant baculovirus could efficiently deliver NDV F gene into chicken primary cells and express the F antigen protein．In addition，the WPRE regulatory element could increase the expression levels of exogenous gene mediated by baculovirus in chicken primary cells．The research provides us a potential basis for the gene engineered vaccines of NDV and other avian infectious disease based on baculovirus vector.