Abstract:Abstract: It was discovered that there are certain microorganisms that can use the extraordinary toxic metalloid arsenic (As) to gain energy for their growth, even use arsenic instead of phosphorus to grow. In this article, we reviewed recent advanced research achievements and summarized these microbial arsenic metabolisms in the following 6 aspects: 1. Gaining energy by chemolithoautotrophic As(Ⅲ) oxidation; 2. Gaining energy by chemoorganoheterotrophic As(Ⅲ) oxidation; 3. Gaining energy by respiratory As(Ⅴ) reduction; 4. As(Ⅲ) oxidation coupling with photosynthesis; 5. The interactions among As(Ⅲ) oxidation, As(Ⅴ) reduction and As(Ⅲ) oxidation coupling with photosynthesis; 6. Growth using As(Ⅴ) instead of phosphorus. Gaining information of microbial arsenic metabolisms is fundamental important for better understanding of live creation, biodiversity, evaluation, biogeochemical cycle and bioremediation.

Abstract:Abstract: The research on marine actinobacteria has worldwide interest because of their potential to produce special and new metabolites. Based on the research history of marine actinobacteria, we reviewed the research progress, conception, bio-resources and diversity, secondary metabolites, ecological function, genomics of marine actinobacteria and finally introduced the status of marine actinobacterial research in China.

Abstract:Abstract: Manganese oxides are a type of high-reactive minerals that play an important role in biogeochemical cycling of many major and trace elements. Bacteria are crucial in bio-catalyzing the formation of most naturally occurring Mn minerals. Currently, a variety of bacterial strains isolated from various habitats such as oceans, fresh waters, soils and ores have exhibited the distinctive bio-oxidation activities to convert the soluble Mn(Ⅱ) oxides to the insoluble Mn(Ⅳ) ones. Here we review the research advances regarding the structures and characterization of several classes of bacterial manganese(Ⅱ)-oxidizing genes, their expression regulations, the structures as well as functions of multicopper oxidases they encoded. Some raised mechanistic questions and the future research prospects are also discussed.

Abstract:Abstract:[Objective] The aim of this study was to identify the culturable endophytic bacteria recovered from the Populus euphratica at the disused (122 years ago) ancient Ugan river of middle reachs of Tarim river, and to understand their Phylogenetic diversity and community structure. [Methods] Bacteria were isolated from the storage liquid in the stem of 2 Populus euphratica stands by using 3 types of different cultural medium (Luria -Bertani,Trypticase Soy Agar and Nutrient Agar), followed carry out 16S rDNA identifications and analysis of their biodiversity.[Results] A total of 62 phenotypically different isolates were sequenced and according to their 16S rDNA sequence similarities to type strains of described organisms,they have been placed into four phylogenetic groups (Firmicutes , Gamma Proteobacteria, Actinobacteria and Alpha Proteobacteria) , 18 genera (Bacillus,Pseudomonas,Kocuria,Staphylococcus,Oceanobacillus,Micrococcus,Planomicrobium,Planococcus,Brevibacterium,Pantoea,Macrococcus,Providencia,Halomonas,Rhizobium,Photobacterium,Psychrobacter,Brenneria and Acinetobacter), and 32 species . Among them, Bacillus and pseudomonas were the most widely distributed and predominant ,occupied the majority of isolates 40.32% and 16.13%, respectively. Isolate KTH-63 (HM371419) formed a distinct clade with Macrococcus brunensis in phylogenetic tree based on 16S rDNA sequence among the family Staphylococcaceae ,so it was demonstrated that the KTH-63 represents a potential novel genus and novel species within the family Staphylococcaceae with 92.491％ sequence similarity with the described species Macrococcus brunensis of this family . Isolates KLH-1,KLH-21,KLH-18, KLH-25,KNA-3,KTH-8,KTH-14,KTH-20 and KNA-26 with 96.089%-97.769% sequence similarities to their closely related members were presumed to be potential novel species, and the discovery rate of potential novel species in the endophytic bacterial community of Populus euphratica was reach up to 16.13%. Furthermore ,10 genera(1 potential novel genus and 9 known genera as the plant endophytic bacteria firstly discovered in Populus euphratica stands ) and 18 species (10 potential novel species and 8 known species as the plant endophytic bacteria firstly discovered in Populus euphratica stands) have been added to the plant endophytic bacterial categoria by the data obtained in this work. [Conclusion] The result showed that the cultivable endophytic bacterial diversity in Populus euphratica at Ugan riwer was very abundant and have high percentage of potential novel species, and it have greatly refreshed the plant endophytic bacterial records .The community structure obtained in this study also may be presumed as a miniature of the endophytic bacterial flora in the Populus euphratica during the recent ages before the affect of modern civilization prossessing to Tarim river valley,which deserve further study and exploitation.

Abstract:Abstract: [Objective] In order to obtain the effective plant growth-promoting bacteria, we studied the diversity of siderophore-producing endophytic bacteria in the roots of Cymbidium goeringii. [Methods] We screened for siderophore-producing bacteria from 189 root endophytic strains of Cymbidium goeringii by using chrome azurol S (CAS) agar plate assay. The diversity of siderophore-producing endophytic bacteria was investigated by 16S rRNA gene sequence analysis. [Results] We obtained 47 siderophore-producing strains (24.9% of the total strains). Sequence analysis revealed that 47 strains were members of 31 species of 17 genera in four phylogenetic groups (Alphaproteobacteria, Betaproteobacteria, Firmicutes, Actinobacteria). The dominant group was Actinobacteria (42.6%), and the dominant genera were Bacillus and Variovorax. The strains of Variovorax produced relatively high levels of siderophore. In addition, there may be two novel taxonomic units. [Conclusion] The results show that there are abundant species diversity of siderophore-producing endophytic bacteria in the roots of Cymbidium goeringii.

Abstract:Abstract: [Object] To investigate the function of flavohaemoglobin (HMP) in Staphylococcus aureus RN6390 under the nitrification pressure, we constructed the hmp gene deletion mutant of RN6390 strain. [Methods] According to principle of homologous recombination, we obtained the up stream and down stream sequences of hmp gene by PCR using chromosomal DNA of S. aureus RN6390 as template. Antibiotics pressure and alternating temperature culture were applied for mutant strain selection. We verified the clones screened out by genome PCR and real-time PCR quantification. Sodium nitroprusside (SNP), as nitric oxide (NO) donor, was used for NO resistance evaluation. In addition, we compared the bacteria biofilm formation ability of hmp gene mutant strain with wild type. [Results] We successfully constructed hmp gene mutant strain of S. aureus RN6390. The expression of hmp gene was direct correlate with the concentration of exogenous NO. We found that compared to wild type, the mutant strain was more sensitive to NO and it is prone to form bacteria biofilm. [Conclusions] The successfully constructed hmp gene deletion mutant of S.aureus provided the possibilities to further investigate the biological function of hmp gene in the resistance of S.aureus to NO from host immune system.

Abstract:Abstract: [Objective] To analyze the T-DNA integration pattern in the genome of grey mold Botrytis cinerea. [Methods] T-DNA (Transfer DNA) inserted mutant library of Botrytis cinerea was created by Agrobactirium tumfacience mediated transformation. By using TAIL-PCR (Thermal asymmetric interlaced polymerase chain reaction), we amplified and cloned the chromosomal regions flanking T-DNA insertions. The obtained T-DNA flanking sequences were subjected to alignment with standard T-DNA border sequence for identification and analysis of integration. [Results] Up to 69% T-DNA inserted at noncoding regions and 30% inserted at exons. Recombination including deletion or addition of bases in T-DNA region was observed. The right borders of the T-DNA were frequently truncated, and by contrast the left borders were less prone to degradation and appeared to have been inserted in a relatively integrated manner. Extra sequence additions also occurred in T-DNA integration sites. [Conclusion] Analysis of T-DNA integration pattern in B. cinerea genome will stimulate the functional genomics study of this fungus.

Abstract:Abstract：[Objective] To construct cheA (chemotaxis, che) insertion mutant of Campylobacter jejuni and to observe the role cheA gene plays in coloning mice of Campylobacter jejuni. [Methods] we generated cheA gene insertion mutant of C. jejuni NCTC11168 based on homologous recombination. The cheA mutant was checked by PCR and sequencing. We detected the difference in coloning mice between cheA mutant and wild-type of C. jejuni NCTC11168 by CFU(Colony-Forming Units) counting of C. jejuni in jejunal content. We conformed the role cheA gene plays in coloning mice of Campylobacter jejuni by conplementation analysis. [Results] PCR results reveal that we have successfully construted cheA insertion mutant of C. jejuni NCTC11168. The cheA mutant displayed significantly attenuated colonization on jejunal mucosa of mice compared to wild-type strain (P<0.05). Complementation analysis shows that the complementation of cheA mutant regained its ability in coloning on jejunal mucosa of mice. [Conclusion] The cheA mutant and it’s complementation were successfully construted in our research. The cheA gene may play an important role in coloning of C. jejuni on jejunal mucosa of mice.

Abstract:Abstract: [Objective] To characterize the toxin-antitoxin system (TA system) in Mycobacterium tuberculosis, which consist of MazF homologue gene and its upstream gene. [Methods] Seven M. tuberculosis MazF homologues were induced alone or co-expressed with their upstream genes respectively in Escherichia coli and Mycobacterium smegmatis, to test the toxic effects of the MazF homologues on bacteria growth, and the antitoxic effects of protein encoded by their upstream genes. The RNA cleavage activity of MazF homologous was identified in vitro with Rv0707 mRNA as the substrate. The promoter region of the identified toxin-antitoxin loci in M. tuberculosis was cloned in front of the lacZ reporter gene in pSD5B vector. The promoter activity was measured under the normal or starvation condition. [Results] The growth of either E. coli or M. smegmatis was inhibited by four MazF homologous proteins, among which Rv1102c, Rv1991c and Rv2801c, but not mtPemK, had the RNA cleavage activities. The toxic effects and RNA cleavage activities of Rv1102c, Rv1991c and Rv2801c were inhibited by their corresponding antitoxin Rv1103c, Rv1991a and Rv2801a, respectively. The other three MazF homologues, Rv1942c、Rv0659c and Rv1495, were not toxic to E. coli and M. smegmatis and also could not cleave RNA. It was found that the promoter activities of Rv2801a-2801c and Rv1991a-1991c systems were significantly increased under the complete starvation condition. [Conclusion] Our results demonstrated that Rv1103c-1102c, Rv1991a-1991c Rv2801a-2801c and mtPemI-mtPemK were typical toxin-antitoxin systems in M. tuberculosis. Rv1102c, Rv1991c and Rv2801c were toxin proteins which inhibited cell growth through their RNA cleavage activities, while the mechanism of mtPemK toxin is still unknown. It is possible that Rv2801a-2801c and Rv1991a-1991c systems are involved in the starvation stress response.

Abstract:Abstract: [Objective] In order to provide the basis for application of Streptomyces hygroscopicus BS-112 and its antifungal active substances, we isolated and purified the active substances produced by strain BS-112, and then we examined their chemical structures and determined their inhibitory effects on Aspergillus flavus. [Methods] We extracted and purified the antifungal active substances from strain BS-112 using column chromatography with X-5 macroporous resin and 200–300 mesh silica gel respectively, and then purified them by preparative HPLC with Venusil XBP C18 column. The chemical structures of active components were identified by means of spectroscopic analysis, including mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). Their minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) against Aspergillus flavus were determined by the standard broth microdilution method. [Results] We obtained four active components from the fermentation broth of strain BS-112 and their chemical structures were the same as Tetrins A and B, Tetramycins A and B. Assay using 96-well plate illustrated that the MIC of Tetrin A, Tetrin B, Tetramycin A and Tetramycin B against Aspergillus flavus were 3.13 μg/mL, 12.56 μg/mL, 1.56 μg/mL and 6.25 μg/mL respectively, and their MFC were 6.25 μg/mL, 25.0 μg/mL, 3.13 μg/mL and 12.56 μg/mL respectively. [Conclusion] The antifungal active substances of strain BS-112 were composed of four compounds, Tetrins A and B, Tetramycins A and B. The four active compounds showed strong inhibitory against Aspergillus flavus.

Abstract:Abstract:［Objective］The diversity of culturable lipase-producing bacterial strains from permafrost soils at the terminus of a glacier in the Tianshan Mountains was investigated. Isolation and molecular phylogenetic analysis were performed to expand our knowledge on diversity of psychrotrophic and psychrophilic bacteria. In addition, efforts were made focusing on screening for cold active lipases.［Methods］Lipase-producing bacterial strains were detected on tween 80 and olive oil plates, respectively. Identity and genetic diversity of strains isolated were determined by spatial 16S rRNA gene sequences and rep-PCR fingerprint. The physiological tests were carried out to determine the phonotypic differences between strains showing high similarity of 16S rRNA gene sequences.［Results］Of the total 17 bacterial stains exhibiting cold-adapted lipase activity, we found that only 8 stains were able to hydrolyze olive oil. Based on 16S rRNA gene sequences, the lipase-producing bacterial isolates fell in five phylogenetic groups: subclasses ?, ? and ? of Proteobacteria, Actinobacteria, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum. Nearly 59% of the isolates were affiliated with the genus Pseudomonas.［Conclusion］The results enrich our knowledge on the psychrotrophic bacterial diversity and biogeographic distribution of cold active lipases-producing bacteria in cold environments.

Abstract:Abstract: [Object] We investigated the proteomic profile of Lactobacillus brevis NCL912 under optimum pH and acidic pH in the media without the addition of sodium L- glutamate to characterize the differential expression proteins and function by two-dimensional gel electrophoresis. [Methods] The differential expression proteins were separated and analyzed by two-dimensional gel electrophoresis, mass spectrum and bioinformatics. [Results] The results showed that the two-dimensional gel electrophoresis profiles of L. brevis NCL912 were uniformity, well-resolution and repeatability. 25 proteins were differently expressed in the two profiles. Among them, 8 proteins were identified and analyzed by the mass spectrum and bioinformatics due to the lack of genome sequence data of L. brevis NCL912. These proteins played the roles of the synthesis of protein and DNA, glycolysis and regulating the cellular energy level. [Conclusion] The differential expression proteins might play the important role in the acid stress resistance mechanism which may protect cell against acid stress.

Abstract:Abstract:[Objective] Nitrite accumulation in aquaculture water is toxic to reared animals. One of the solutions to this problem is to apply denitrifying bacteria. This paper is intended to get a strain of phototrophic bacteria for efficient romoval of nitrite from aquaculture water. [Methods] We used soft agar to isolate and purify phototrophic bacteria. We investigated biological characteristics of the isolate by means of light and electronic observations, physical and chemical tests, analized its phylogenetical position based on the sequences of 16S rDNA and The gene that codes for photosynthetic reaction center subunit M (pufM). [Results] A photosynthetic bacterial strain, named wps, showed a high removal efficiency of nitrite, was isolated from the freshwater ponds. Cells were gram-negative, rod-shaped, slightly curved, 0.4-0.6 × 1.5-4.0 μm, motile by means of polar multiple flagella. Intracellular membranes were of the lamellar type. It grew under facultative anaerobic conditions in the light with bacteriochlorophyll a and carotenoid of sppirilloxanthin series as photosynthetic pigment. The optimum growth was obtained at pH 5.5-8.5, in a range of 0-2% salinity and at a temperature of 25-38℃. The similarity of 16S rDNA between strain wps and Rhodopseudomonas palustris was 98.9% and 94.9% for pufM gene. However, there are significant differences between them in the morphological and physiological characteristics, i.e. grew at pH 5.5; no growth photoautotrophicaly with sodium hydrogen carbonate; could not utilize citrate or formate as only carbon source; required thiamine hydrochloride and calcium pantothenate as growth factors. [Conclusion]. Strain wps may represent a novel species in genus Rhodopseudomonas and possibly find its application in the bioremediation of polluted aquaculture water.

Abstract:Abstract: [Objective] We studied the inhibition of infectious bursal disease virus (IBDV) replication in chicken embryos by recombinant avian adeno-associated virus (AAAV)-delivered VP1- and VP2-specific microRNAs (miRNAs). [Methods and Results] We co-transfected AAV-293 cells with the VP1- or VP2 gene-specific miRNA expression vector pAITR-RFPmiVP1 or AITR-RFPmiVP2E, AAAV packaging vector pcDNA-ARC and adenovirus helper vector pHelper, resulting in recombinant virus rAAAV-RFPmiVP1or rAAAV-RFPmiVP2E. We also generated the recombinant viruses rAAAV-RFP (without miRNA expression cassette) and rAAAV-RFPmiVP2con (expressing control miRNA) using the same method as the control purpose. Electron microscopy showed that the recombinant viruses had a typical morphology of AAV. We confirmed the presence of miRNA expression cassette in the recombinant viral genomes by using PCR. Our poly(A)-tailed RT-PCR showed correct expression of the miRNAs in the rAAAV-transduced DF-1 cells. We inoculated the recombinant viruses individually into 8-day-old SPF chicken embryos and then challenged them using Lukert strain IBDV on day 2 after inoculation. Our IBDV titration assay showed that the 50% tissue culture infectious dose (TCID50) of rAAAV-RFP- or rAAAV-RFPmiVP2con-inoculated group was 8.0 log10, whereas the TCID50 of rAAAV-RFPmiVP1-inoculated group decreased to 1.0 and 0.8 log10 on day 3 and 6 after challenge, respectively. Similarly, the TCID50 of rAAAV-RFPmiVP2E-inoculated group decreased to 1.5 and 2.0 log10, respectively. [Conclusion] These data suggest that rAAAV can transduce efficiently chicken embryos and the expressed VP1- and VP2-specific miRNAs can inhibit the replication of IBDV efficiently.

Abstract:Abstract: [Objective] To study the diversity of culturable actinomycetes isolated from the sea deposit of Tiger beach at Bohai bay, Dalian, China. [Methods] By using five different media strains we isolated actinomycetes strains from the sea deposit of Tiger beach at Bohai bay, Dalian, China. Partial 16S rRNA sequences were carried out to characterize the diversity of culturable actinomycetes. [Results] A total of 1215 colonies with phenotypical actinomycetes were isolated, of which 271 were classified by 16S rRNA phylogenetic analysis. The data showed that 251 strains were homologous with Actinobacteria (92.26%) including 15 genera of 11 families, and the remaining 20 strains were possible members of the phylum Firmicutes and Proteobacteria. There were 7 strains (3-166, 3-117, 3-435, 5-186-2, 5-9, 5-171, 3-134) to be preliminarily identified as unreported species. [Conclusion] A high diversity of culturable actinomycets both in terms of the number of species and phylogenetic composition was presented from the sea deposit of Tiger beach in Bohai bay.

Abstract:Abstract: [Objective] Development of a method of transforming Aspergillus niger conidiospores with Agrobacterium tumefaciens T-DNA system and using it as a tool for genome annotation via construction a T-DNA insertion mutant library of A. niger. [Methods] A. tumefaciens EHA105 carrying the binary vector pCAMBIA1301 was used to transform A. niger conidiospores under induction conditions. Hygromycin resistant transformants were screened on the selected medium. Sequence analysis was performed usimg inverse PCR method in stable mutants. [Results] In total, 193 hygromycin resistant stable transformants were obtained and the transformation rate was about 5.6?102 transformants /108 conidiospores. Obvious morphological changes were observed in some mutants and sequence analysis of T-DNA inserted site was performed in one conidiation-defective mutant. The result showed that the interrupted gene belonged to major facilitator superfamily (MFS). [Conclusion] Our results indicated that the transformation system of A. niger conidiospores set up in this study, combing with sequence analysis of T-DNA insertion site, might be an effective way for genome annotation in A. niger.

Abstract:Abstract: [Objective] In order to reveal why SEF14 fimbriae are restrictively expressed on strains of serogroup D salmonella, mainly S. enteritidis and S. dublin, the difference and variation of the sef14 operon gene clusters in S. enteritidis and related serogroup-D Salmonella were analyzed. [Methods] The genes encoding subunits of sefA, sefD and sefR in S. pullorum, S. enteritidis and S. dublin were amplified by PCR method and then sequenced to analyze the the difference and variation, respectively. [Results] The results of PCR amplification showed that prevalence of sefA, sefD and sefR genes in S. enteritidis and S. dublin was 100%. In 18 isolates of S. pullorum, the prevalence of sefA gene was 100%, while the prevalence of sefD and sefR genes was 38.9% (7/18), and 11 strains isolated after 1980s did not contain any gene sefD or sefR. The sequencing data of PCR products revealed that sequences of sefA, sefD and sefR genes in S. enteritidis and S.dublin were identical with those those from NCBI GenBank data which accession number were L11008, U07129 and AF233854, respectively. Interestingly, among the 7 strains of S. pullorum before 1980s, the sefD sequence has a missing base pair at position 196 and caused open reading frame (ORF) shift, resulting in a stop codon (TAG) at position 71 amino acid residual (Leu of TTA at position 214-216 shift into stop codon of TAG at position 215-217). Unlike S. pullorum, all S. enteritidis and S. dublin tested could express SEF14 fimbriae in vitro. [Conclusion]Based on the data of the difference and variation of sef14 operon gene clusters between S. enteritidis and S. pullorum, we may explain why SEF14 fimbriae in S. pullorum could not be expressed.

Abstract:Abstract: Actinobacteria is one of the best studied taxa of prokaryotes due to its great importance in biotechnology, medical science, ecology and etc.. Modern actinobacterial taxonomy is polyphasic taxonomy, which is based on the phylogenetic analysis of sequences of 16S rRNA genes and other conservative molecular sequences, and employs a variety of microbial information for polyphasic systematic study. Currently, with the development of large-scale sequencing, over 100 actinobacterial genomes have been finished. A comprehensive, detailed and robust phylogeny of actinobacteria is thus needed for understanding how this group emerged and maintained such a vast diversity throughout evolution and how every subgroup related to each other from various habitats. “Phylogenomics” and “Genomic Encyclopedia of Bacteria and Archaea, GEBA project” indicated that actinobacterial taxonomy stepped into the era of genomics. This review summarizes the actinobacterial taxonomic methodology of the genomic era, and results from recent studies.