Abstract:Abstract:Cyclic diadenosine monophosphate (c-di-AMP)，a new second messenger found recently in bacteria，regulates various aspects of bacterial physiology，including cell growth，cell wall homeostasis and virulence．In addition to its functions in bacterial physiology，c-di-AMP represents a putative bacterial secondary signaling molecule sensed by eukaryotic host cells and triggers innate immunity．The level of c-di-AMP in bacteria is regulated by the activities of diadenylate cyclase (DAC) and phosphodiesterases (PDE)，the former harbors a DisA_N domain，and the latter a DHH or DHH/DHHA1 domain．This review gives an overview on metabolic pathway，regulatory mechanism，receptor proteins and biological function of c-di-AMP in bacteria，as well as its application and trends of development.

Abstract:Abstract:The human intestinal microbiota is essential in nutrients utilization，organ development，host metabolism，pathogen resistance，and regulation of immune responses．The establishment of early gut microflora in newborn infants experiences periods from less to more，simple to complex and unstable to stable and can be influenced by various factors．This article summarizes the effects of delivery mode，delivery period，rearing methods as well as antibiotics on the development of intestinal bacterial community．

Abstract:Abstract:［Objective］The aim of this study was to screen and identify carrageenase-producing strain from mangrove soil leaf and to characterize produced carrageenase.［Methods］The culture medium withκ-carrageenan as sole carbon source was used to isolate the strain exhibiting carrageenase activity．The isolated strain was identified by morphology observation and 16S rDNA sequencing．κ-carrageenase produced by Pseudoalteromonas sp．ASY5 was purified and characterized by DNS method.［Results］A bacterial strain ASY5 with high carrageenase activity was isolated from mangrove soil humus，and was identified as Pseudoalteromonas sp．The molecular mass of the purifiedenzyme was estimated to be 30 kDa．The optimal temperature and pH of the enzyme were 60℃ and 7. 5，respectively．The enzyme was stabileat 50℃，and more stable between pH 7.0 and 9.0．The enzyme could convert κ-carrageenan．The Km and Vmax values of the enzyme for κ-carrageenan was 2.28 mg/mL and 147.06μmol/(min·mg)，respectively．The enzyme was significantly stimulated by Na+，K+，Ca2+ ，Mg2+ and Al3+．The enzyme was inhibited strongly by Ag+、Zn2+、Cd2+ and SDS.［Conclusion］κ-carrageenase produced by Pseudoalteromonas sp．ASY5 was stable at high temperature and alkaline pH，with potential applicationin carrageenan oligosaccharides production.

Abstract:Abstract:［Objective］Phytoene desaturase PDS is a eukaryotic nuclear membrane binding protein，we used different expression methods to search for the soluble expression strategy of membrane binding protein in Escherichia coli.［Methods］We cloned the full-length cDNA sequence of PDS from Dunaliella salina through RACE．First，we utilized prokaryotic expression vector pET-28a to construct pET-28a-PDS vector．Then，we substituted PLtac promoter for T7 promoter in pET-28a to construct pET-PLtac-PDS vector．Last，we constructed pET-Mistic-PDS fusion vector by integrating Mistic sequence into pET-28a． All were transformed into BL21 (DE3) for protein expression.［Results］The 2237-bp full-length cDNA sequence of PDS was cloned，including a 1749-bp open reading frame，encoding 582 amino acids (NCBI accession: GQ923693.1)．The expression of PDS protein was low via pET-28a-PDS and pET-PLtac-PDS vector，and proteins were mostly expressed in inclusion body．The expression of PDS protein was significantly increased via pET-Mistic-PDS vector，in addition most were expressed as soluble protein which possessed dehydrogenase activity.［Conclusion］Mistic as the solubilization label was able to promote proper folding of membrane proteins and improve solubility． Protease activity assay proved that Mistic could maintain the enzyme activity.

Abstract:Abstract:［Objective］In order to identify Sigma factor associated with biofilm formation of Salmonella pullorum，we determined the gene expression in the wild type strain and biological characteristics of deletion mutants.［Methods］Biofilm forming ability of S． pullorum strain was detected by crystal violet assay．The rpoS gene-dependent or -independent strain for biofilm formation was determined by catalase test．Real-time PCR was established to compare the expression of six different Sigma factors during biofilm formation．Deletion mutants were constructed using the Red recombination system，and their resistance to environmental stress was determined.［Results］ S. pullorum strain S6702 had strong ability of biofilm formation．The result of catalase test was negative，indicating that S6702 was an rpoS-independent strain for biofilm formation．The expression of rpoE gene was the highest during 4 h and 24 h post-incubation．Compared to wildtype strains，ΔrpoS kept the biofilm-forming ability，whereas ΔrpoE mutant could not produce biofilm．Both mutants with deletion of the rpoS and rpoE genes had reduced resistance to environmental stress.［Conclusion］The rpoE gene was identified as one of biofilm formation associated genes in a S． pullorum rpoS-independent strain．The finding may help to elucidate the regulatory mechanism of Salmonella biofilm formation.

Abstract:Abstract:［Objective］ To further improve the efficiency of xylose fermentation by modifying the pentose phosphate pathway (PPP) and the aldehyde reductase gene h16_A3186 in Ralstonia eutropha W50-EAB．［Methods］The transketolase (tktA，cbbT2) and transaldolase (tal) gene were cloned from R. eutropha chromosome by PCR and inserted into expressing vector pBBR1MCS-3．The resulting recombinant plasmids were transformed into W50-EAB to generate W50-KAB，W50-CAB and W50-TAB，respectively．The aldehyde reductase gene h16_A3186 was shortened from 834 bp to 135 bp by in-frame deletion from strain W50-E in which the xylE gene coding for xylose transporter was chromosomally integrated to construct recombinant strain W50'-E．Then the xylAB gene coding for xylose isomerase and xylulokinase from Escherichia coli were expressed in W50'-E to generate recombinant strain W50'-EAB．Recombinant plasmid pWL1-TAL was transformed into W50'-EAB to construct the strain W50'-TAB．The fermentation characteristics of the engineered strains were investigated． ［Results］The expression of tktA，cbbT2 and tal genes in R.eutropha W50-EAB was confirmed by enzyme assay．The deletion of h16 _A3186 gene was confirmed by PCR analysis and enzyme assay．Amplification of transketolase activity in R.eutropha W50-EAB showed negative effect on cell growth and D-xylose consumption．The recombinant strain W50-TAB and W50'-EAB exhibited a faster growth than W50-EAB with the maximum specific growth rate of 0.039 h-1 and 0.040 h－1，respectively，when cultivated on 0.1 mol/L D-xylose．And the PHB accumulation of W50-TAB and W50'-EAB reached 16.2±1.01% and 19.8±1.05% on the basis of cell dry weight，respectively．Furthermore，recombinant strain W50'-TAB exhibited better fermentation performance with the maximum specific growth rate of 0.042 h－1 and PHB content of 27.9±0.47%，respectively．Meanwhile，the recombinant strains W50-TAB，W50'-EAB and W50'-TAB showed higher biomass and more PHB accumulation when using glucose (0.01 mol/L) and Dxylose (0.09mol/L) mixed sugars as fermentative substrate.［Conclusion］Overexpression of the tal gene resulted in incressed D-xylose consumption．Deficiency of the aldehyde reductase relieved inhibition to D-xylose metabolism．Combination of the two strategies contributed to a higher efficiency of D-xylose utilisation and more PHB accumulation of the engineered R.eutropha strain.

Abstract:Abstract:［Objective］ To identify and characterize an unknown microorganism causing contamination in several mammalian cell cultures.［Methods］This bacterium was identified by 16S rRNA sequencing and studied by DAPI and DiOC6 (3) staining，Gram staining，acid-fast staining，and electron microscopy． The isolated bacterium was also used to infect host cells to observe antibiotic effectiveness and its relationship with host cells.［Results］The 16S rRNA sequence analysis shows that this rod-shaped microorganism belongs to the family Caulobacteraceae，class Alphaproteobacteria，and was most closely related to Phenylobacterium zucineum HLK1T strain．The bacterium collected in the“swimming”stage was Gram staining negative，but Gram staining positive in the “sessile”stage．Under the electron microscope both flagellated and non-flagellated types were found． So far，no antibiotics were effective to inhibit this microorganism．The contamination with this bacterium frequently led to failed resuscitation of thawed cells． We found that the cells resuscitated with the used culture supernatants were increased in number by 3－4 folds as compared to those resuscitated with freshly prepared media． ［Conclusion］ Phenylobacterium may have a dimorphic life cycle including a swimming stage and a sessile stalked stage．

Abstract:Abstract: ［Objective］The function of Mms6 related to biomineralization on the magnetosome formation in Magnetospirillum magneticum AMB-1 was studied． ［Methods］ The transcript of mms6 was analyzed under static and aerobic conditions with Real-time RT-PCR．We observed the cell growth and magnetism of the mutation in which mms6 was mutated.［Results］The transcript of mms6 increased with the formation of magnetosomes．Mutation of mms6 caused about 50% decrease of magnetism in AMB-1 under static conditions，however，the cell growth of mutant was similar as to that of the wild type.［Conclusion］Gene mms6 is involved in the magnetosome formation of AMB-1.

Abstract:Abstract:［Objective］The aim of the present study was to compare the binding activity of Lactococcus lactis peptidoglycan protein anchor (PA) with different number of motifs.［Methods］L．lactis PA gene sequences with 2 and 3 lysin motifs (LysM) were obtained by PCR amplification．Then，the recombinant plasmid of pET-32a(+) containing PA gene was constructed and transformed into E．coli BL21 (DE3)，and induced to express the fusion protein PA2 and PA3．The purified and refolded PA2 and PA3 were incubated with gram positive enhancer matrix (GEM)．The binding activities of PA2 and PA3 were identified and compared by Western blot，TEM and SDS-PAGE.［Results］The PA2 and PA3 could bind to GEM．The anchoring activity of PA3 was obviously superior to the PA2.［Conclusion］The data indicated that PA with 3 LysMs had a better binding capacity compared with 2 LysMs．The results provided foundations to further improve the design of GEM-PA display system．

Abstract:Abstract:［Objective］We used 50-amino acid-long peptides from the N-terminus of 4 different non-classically secreted proteins to study the secretion efficiency of Bacillus subtilis LipaseA via non-classical secretion pathway.［Methods］We amplified the coding sequences (CDs) of LipaseA and N-terminus of non-classically secreted proteins，constructed 8 fusion protein expression vectors containing both LipaseA CD and different secretion signal peptide and transformed them into B．subtilis WB800．Secretion efficiency of these fusion proteins was analyzed by enzyme activity，SDS-PAGE and Western-Blot． ［Results］Recombinant LipaseA containing coding sequences of PdhA or N-terminus of SodA and Eno as secretion signals was efficiently secreted．［Conclusion］ Parts of non-classically secreted proteins or N-terminus (50amino acids) could guide LipaseA protein secretion．

Abstract:Abstract:［Objective］We studied the correlation between the microbial diversity in Panax notoginseng root soil and its root rot diseases，to find biological control approaches in Panax notoginseng soil borne diseases.［Methods］We isolated bacterial strains from the rhizosphere soils of healthy and root rot Panax notoginseng plants that are cultured continuously for 6 years in Wenshan Region．After separation and purification，we obtained DNA．On the basis of 16S rRNA’s general primer we carried out PCR amplification，conducted blast gene similarity and analyzed phylogenetic information． ［Results］The isolated bacterial strains distributed to 4 phyla，40 genera of bacteria，179 isolates from the samples of healthy Panax notoginseng rhizosphere soil belong to 30 genera and Burkholderia，Arthrobacter，Streptomyces and Bacillus are the dominant microflora．Additionally，117 isolates from the samples of root rot Panax notoginseng rhizosphere soil belong to 29 genera and Ralstonia，Sphingomonas，Stenotrophomonas as the dominant microflora．Among them，Flavobacterium and Enterobacter were only isolated from the samples of root rot Panax notoginseng rhizosphere soil．At least 5 isolates are novel species; the ions concentration and electrical conductivity value show distinct discrepancy between the two groups (P＜0.05);the microbial amount of dominant species in the healthy soil samples present negative correlation with electrical conductivity value，the concentration of NO3－，SO42－，CO32－，K+ and total salt (P＜0.01)．［Conclusion］In addition to pathogen infection，the physical and chemical characteristics，microbial community structure and the proportion of dominant species are also closely related to notoginseng continuous cropping soil borne disease．Especially the beneficial microorganisms (Burkholderia，Bacillus，Streptomyces，etc.) abundance is significant to evaluate the soil healthy condition and accurately disease control ＆ forecast for Panax notoginseng cultivation.

Abstract:Abstract:［Objective］The diversity of mycorrhizal fungi isolated from Vaccinium uliginosum L in the northern region of Daxing’anling mountains was examined for the first time.［Methods］Morphology and ITS sequence analysis were used to identify the fungal communities.［Results］Six groups of fungi were isolated from Vaccinium uliginosum root samples: one belongs to Hymenoscyphus; one to Phialocephala; one to Lachnum; one to Cadophora; one to Marasmius and one to Mycena．Among them，87.10% belong to ascomycetes and 12. 90% belong to Basidiomycotina.［Conclusion］ The diversity of fungi associated with Vaccinium uliginosum is abundant and the fungi are from heterogenous group.

Abstract:Abstract:［Objective］The aim of this study was to express Mycobacterium tuberculosis MPT83 protein and to evaluate its immunogenicity in murine model as well as the serological diagnosis potential value for bovine tuberculosis.［Methods］The fragment of mpt83 gene was amplified and constructed into pET30a(+) -mpt83 recombinant plasmid．MPT83 fusion protein was purified with His affinity chromatography column from strain of BL21(DE3) -pET30a(+)-mpt83 after induced by IPTG，and then used to evaluate its immunogenicity in mice and the potential application in ELISA assay for the detection of bovine tuberculosis.［Results］SDS-PAGE and Western blot results show that MPT83 fusion protein was expressed successfully and possessed a good immunological reactivity．Flow cytometry (FCM) analysis displayed decreased expression of CD80 on dendritic cells and up-regulation of CD69 expression on both splenic CD4 + and CD8 + T cells. Meanwhile，more IL-4 specific secreting cell spots rather than those of IFN-γ were detected by ELISPOT assay in C57BL/6 mice injected with the fusion protein．Total 200 serum samples were detected by indirect ELISA based on MPT83 as antigen and the results showed 48.6% positive coincidence rate and 90% negative’s compared to results of peripheral blood specific IFN-γ release assay in bovine tuberculosis detection．[Conclusions］MPT83 fusion protein was expressed successfully with capability of eliciting Th2 immune response in mice and could be used for ELISA assay to detect bovine tuberculosis as a serological diagnosis antigen.

Abstract:Abstract:［Objective］We studied the serological response between the special regions on the Torque teno sus virus 2 (TTSuV2) ORF1 coded protein and the porcine sera from conventional pigs.［Methods］Based on a Chinese TTSuV2 strain from Guangdong province，two overlapped virus proteins were expressed from Escherichia coli．Then，purified recombinant TTSuV2 ORF1a and TTSuV2 ORF1ab proteins were used as the antigens in the Western Blotting and ELISA assay.［Results］The recombinant TTSuV2 ORF1a and TTSuV2 ORF1ab proteins were identified with the special tag monoclonal antibody．The results of the ELISA tests shown that there were significant relationships between two groups of dates from the recombinant TTSuV2 ORF1a and TTSuV2 ORF1ab proteins antigenic assay．The results of the following Western Blotting assay indicated that the TTSuV2-specific IgG antibodies were contained in pig sera． ［Conclusion］The truncated TTSuV2 ORF1a protein (positions 168 to 346 corresponding to TTSuV2 GDIMA1) contains important B cell epitopes which can stimulate immune system antibody secretion．The truncated TTSuV2 ORF1a protein could be effective in TTSuV2 immunodiagnosis.

Abstract:Abstract:［Objective］Bursopentin (BP5) is a multi-functional bioactive peptide with functions of immunomodulatory，antioxidant and antitumor. However，the antitumor mechanism of BP5 is still unclear.［Methods］We constructed T7 phage cDNA library of DT40 cells，and the proteins interacted with BP5 were identified. Then，the expression profile of BP5-treated DT40 cells were analyzed using gene microarray，p53 Luciferase activity was detected.［Results］The results of the expression profiling revealed that BP5 regulated expression of 1078 genes，of which 537 were up-regulated and 541 were down-regulated．Differentially expressed genes involved in various pathways were identified，of which 25 pathways were associated with immune responses and tumorigenic processes，including the p53 signaling．Furththmore，BP5 significantly enhanced p53 luciferase activity and stimulated expression of p53 protein in HCT116 cells.［Conclusion］These results suggest that BP5 exerted antitumor activity through p53 signaling and that this study provides novel insights on the antitumor mechanism of BP5.

Abstract:Abstract:In this paper，we provided an overview of proposals submitted and projects funded in 2014 at the Division of Microbiology，Department of Life Sciences，National Natural Science Foundation of China．The traits and problems in different sub-disciplines were analyzed，the background，results and analysis of internet voting before panel meetings in Microbiology discipline were also introduced．The information will provide references for Chinese researchers to apply funding in microbiology discipline in the future．