Abstract:[Objective] The substrate-binding mechanism of D-psicose 3-epimerase from Ruminococcus sp. has been studied. [Methods] The residues involved in substrate-binding were selected using homology modelling and sequence alignment. Mutants were constructed by site-directed mutagenesis and studied by kinetic analysis. [Results] Two residues, Y6 and A109, were selected. Four mutants, Y6F, Y6I, A109P and A109L were constructed. [Conclusion] Y6 was involved in both catalysis and substrate-binding by the aromatic ring of the amino acid, while the -OH was important for substrate-binding. A109 was an important site that affected binding rather than catalyzing. This research would contribute to the study of catalytic mechanism and molecular modification of D-psicose (D-tagatose) 3-epimerase.

Abstract:[Objective] To analyse the taxonomic status of the red-coccus bacterium conserved in our laboratory, and the compositions of pigments extract were determined. [Methods] This bacterium was classified based on morphological features, 16S rRNA gene sequences and physiological tests. Total pigments were extracted from biomass using ethanol-acetone. Preliminary separation of pigments was achieved by thin layer chromatography, and UV/visible scanning spectra and mass spectra were used for the qualitative analysis of each composition. [Results] This bacterium was classified to the genus of Arthrobacter sp.. There were main four compositions on the silica-gel plate. Only one next to the solvent front was yellow, and others were orange. The spectroscopic data and mass spectra showed that the yellow and red components might be the β-carotene and spirillloxanthin series respectively. [Conclusion] The production of carotenoids by the strains of Arthrobacter sp. was observed, when cheap materials such as molasses and corn steep liquor were used as nutrient medium, and further investigations will have practical values.

Abstract:[Objective] To clarify the removal of ammonium, nitrate and nitrite by a marine purple sulfur bacterium capable of growing on nitrite as sole nitrogen source. [Methods] Ammonium, nitrate and nitrite concentration in simulated wastewater were determined using the Nessler’s reagent spectrophotometry, N-(1-naphthyl)-1,2-diaminoethane dihydrochloride spectrophotometry, UV spectrophotometry, respectively. [Results] The removal of ammonium, nitrate and nitrite as well as bacterial biomass and pH increased with time, and then tended to equilibrium. Strain YL28 exhibited effective ammonium removal ability, with a maximal removal and tolerance of 9.64 mmol/L and 36.64 mmol/L, respectively. The removal rate exceeded 97.61% when the concentration of ammonium was less than 3.21 mmol/L. Compared to ammonium, the cell growth rate, biomass and pH of wastewater enhanced slowly while using nitrate and nitrite as sole nitrogen source. However, the removal of nitrate and nitrite were higher than that of ammonium. Nitrate and nitrite in wastewater could be completely removed when their concentrations were up to 13.50 mmol/L and 22.90 mmol/L, respectively. When the three inorganic nitrogen existed simultaneously in wastewater, ammonium, nitrate and nitrate could be removed by strain YL28, the removal of nitrate and nitrite was higher than that of ammonium. [Conclusion] Strain YL28 would be a promising candidate for bioremediation of polluted aquaculture wastewater, especially for nitrite-polluted marine culture wastewater.

Abstract:[Objective] To isolate strains producing glucose oxidase from the sample of sea mud and sea sand in Yellow Sea. [Methods] The strain isolated by plate culture and enyme activity determination, which was identified based on its morphology characteristics, physiobiochemical characteristics and 16S rRNA gene sequence analysis, parts of the glucose oxidase properties were studied, also. [Results] The strain GOD2 (glucose oxidase) was identified as Pseudomonas (Pseudomonas migulae), and the optimum reaction temperature of the enzyme was 20 °C, with the poor thermal stability, the relative activity of the remaining 80% at 40 °C. Enzyme activity decline rapidly above 40 °C. [Conclusion] GOD2 had a highly research value, and there is no report about it to produce glucose oxidase, at present.

Abstract:[Objective] The purpose of this paper is to reveal the genetic and physiological diversity of atrazine-degrading bacteria isolated from industrial wastewater, to provide new insights for molecular mechanism of atrazine biodegradation. [Methods] Atrazine-degrading genes of 27 atrazine-degrading strains were detected by the conventional PCR and their composition of atrazine-degrading genes was analyzed. The genome composition of these bacteria was analyzed by repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprinting. Triazine hydrolase TrzN in bacteria, the first enzyme for atrazine-degrading pathway, was detected by Western blot analysis. Nitrogen source (atrazine, ametryn, atratone, cyanazine, prometryn, simazine and cyanuric acid) and carbon source (glucose, sucrose, maltose, lactose, sodium citrate, sodium succinate and sodium acetate) used by these bacteria were analyzed by measuring absorbance of the culture at 600 nm. [Results] The conventional PCR experiments indicated that three main atrazine-degrading gene combinations (i) trzN-atzBC, (ii) trzN-atzABC, and (iii) atzADEF were observed respectively from either strain of 27 atrazine-degrading strains. rep-PCR analysis indicated that these bacteria could be differentiated into 7 clusters. Western blot analysis proved that 24 strains contained TrzN protein. Nitrogen source experiments showed that two strains could use all of the 7 compounds as nitrogen source and other 25 strains only could use 2?6 compounds as nitrogen source. Carbon source experiments showed that 10 strains could use all of the 7 compounds as carbon source and other 17 strains only could use 3?6 comlounds as nitrogen source. [Conclusion] These 27 atrazine-degrading strains isolated from industrial wastewater have their genetic and physiological diversity.

Abstract:[Objective] To construct a secretory expression vector of Trichoderma reesei and prove its feasibility by recombinant expression of enhanced green fluorescence protein (eGFP), and observe the process of the expression of eGFP, spontaneously. [Methods] Construct the expression vector pPth15 by successively ligate the promoter of the CBH1 gene of T. reesei, the signal peptide sequence, the terminator and the hygromycin resistance gene into the backbone plasmid pUC19. Insert eGFP gene into pPth15 to aquire the recombinant plasmid pPth15-eGFP, and transformate it into the T. reesei protoplasts. Screening positive transformants via hygromycin resistance and PCR amplification. [Results] After culturing the positive transformants on the PDA medium for 2 to 3 days, green fluorescence distribution was emerged on the hyphae tips, the septum and the extracellular medium. [Conclusion] The constructed expression vector could be used for eGFP expression, and this work makes contribution to the follow-up study of heterogeneous protein expression in T. reesei and provides a reference in the research of extracellular protein expression of T. reesei.

Abstract:[Objective] To aquire compounds with novel structures or potent antimicrobial activities through research on metabolites of endolichenic fungus Elaphocordyceps sp. isolated from Leptogium sp.. [Methods] We confirmed taxonomy status of the endolichenic fungus by PCR amplification, evaluation of the ITS (internal transcribed spacer) sequence of nrDNA, sequence comparision in GenBank Database and construction of the phylogenetic tree. Constitutes were isolated by the gel column chromatography, sephadex LH-20 and HPLC methods. Structures were elucidated by physicochemical properties and spectral data analysis. Double dilution method was adopted to evaluate antifungal activity. [Results] The testing strain was identified as Elaphocordyceps sp.. Three compounds were isolated and purified from the fermentation of the endolichenic fungus. Their structures were elucidated to be ergosterol, 4,8-dihydroxy-1-tetralone and De-O-methyldiaporthin. The three compounds show weak inhibition on Candida albicans sc5314. De-O-methyldiaporthin exhibits very strong phytotoxic activity, with leaf sensitivity concentration of 4 nmol. The concentration of De-O-methyldiaporthin in fermentation broth of Elaphocordyceps sp. reached 0.75 mg/L (3.2′103 nmol), much higher than the leaf sensitivity concentration of 4 nmol. [Conclusion] De-O-methyldiaporthin produced by the fungus of Elaphocordyceps sp. can be a microbial herbicide.

Abstract:[Objective] The healthy and huanglongbing-affected Catharanthus roseus under manual-grafting condition were used as materials for research. The endophytic bacterial community structure and function of different parts of healthy and huanglongbing-affected Catharanthus roseus were analysisied, which provided a basis theoretical foundation for correlative study between citrus huanglongbing and Catharanthus roseus endophytic bacterial. [Methods] The endophytic bacterial communities were analysised based on the method of facultative anaerobic culture techniques compared with endophytic bacteria features analysis. [Results] The endophytic bacteria were isolated from sterilized-surface midribs of leaves and phloems, which from stems and roots. With the method of artificial anaerobic culturing, 67 strains were obtained and identified as 29 genera based on the blast results of 16S rDNA. The dominant bacterial population of diseased and healthy C. roseus were mainly came from Curtobacterium sp., Erwinia sp., Bacillus cereus, Brevundimonas sp. and Bacillus sp., While, the common dominant population of both diseased and healthy C. roseus was belonged to Staphylococcus equorum. Performing the methods of physiological and biochemical identification, total of 6 bacterial isolates showed a minimum of four traits related to indole acetic acid (IAA) synthesis, nitrogen (N) fixation and siderophore production. production of 1-amino-cyclopropane-1-carboxylate deaminase, production of antibiotic, amylolysis and protease activity were characterized. Dominant isolates were characterized as 6 genera by functional analysis: Staphylococcus equoru subsp., Bacillus subtilis, Bacillus megaterium, Curtobacterium sp., Morganella morganii, and Lysobacter sp.. These traits may be involved in HLB-affected C. roseus. [Conclusion] The results of bacterial community of the identified endophytic bacteria of Catharanthus roseus and their functional analysis showed the potential role on biological control of HLB pathogen.

Abstract:[Objective] The safety assessment of 7 strains of lactic acid bacteria (Enterococcus spp.) isolated from mare? s milk and its products (koumiss) was investigated in a mice feeding trial. Acute toxicity, thirty days feeding and bacterial translocation were conducted. [Methods] Different dose of Enterococcus JHZ9, JHZ15, JHZ17, JHZ22, JHZ25, JHZ28 and JNN1 were administrated to treatment groups separately by gavage for 7 days and 30 days, while the general appearances, body weight and food consumption were observed and recorded daily. Mice were killed after 30 days and the kidney, spleen, liver, heart and lungswere observed and weighted, then collected for bacterial translocation experiment. [Results] Except that test bacterial strains JHZ17 in the high dose group had significant difference with control group (P<0.05). In both acute toxicity and thirty days feeding experiments, no significant differences (P>0.05) were found between the treatment group and the control group in the weight of mice, food intake and the internal organ (heart, liver, spleen, lung and kidney) indices. The maximum tolerated dose (MTD) of acute toxicity is higher than 1010 CFU/(mL bw) 20 mL/(kg·d), within the range of non-toxic. No bacterial translocation was observed. [Conclusion] Lactic acid bacteria JHZ9, HZ15, JHZ17, JHZ22, JHZ25, JHZ28 and JNN1 have no toxicity and side effects.

Abstract:[Objective] We used UV induced protoplast mutagenesis to study breed a new high-yielding lipid-producing strain which could use cheap carbon source. [Methods] Get the 1.5% glusulase and 1.0% cellulose solution. Hydrolyze to remove the cell wall and obtain the protoplast of 2A00015 (Candida parapsilosis). Put it under the ultraviolet lamp for mutagenesis and cultivate regenrated wall. Then screen to get the high oil generated yeast which could be fermented by low-cost carbon source. Determine its fat acid components by gas chromatography with mass spectrometric (GC-MS). [Results] Cultivated the mutant strain with best mutation 2A00015/25 in the glucose. We found that the biomass, oil yield rate and oil production are separately 17.77 g/L, 58.12% and 10.32 g/L, which are separately 12.45%, 23.32% and 38.68% higher than the original strain. We have also cultivated the mutant strain in the waste molasses and found the biomass, oil yield rate and oil production are separately 18.54 g/L, 49.44% and 9.17 g/L, which are separately 9.09%, 21.16% and 32.18% higher than the original strain. The oil yield rate is lower in the waste molasses cultivation than that in glucose cultivation. However, in consideration of environment protection and cost of raw materials, the waste molasses is of much more advantages. It is tested that the fat generated from the waste molasses fermentation consists of eight kinds of fat acid. Its fat acid components are similar to the vegetable oil, in which the content of unsaturated fatty acid comprised 82.4% of the total fatty acid. [Conclusion] Basing on UV induced protoplast mutagenesis, we have successfully bred a new high-yielding oil strain which can make use of the low-cost carbon source, with its oil production rate 49.4% which has increased by 21.2%.

Abstract:[Objective] We constructed Bti engineering strains that can highly express chitinase genes constitutively, so that the strains can inhibit the growth of fungi more effectively. [Methods] The full length and series of deletion promoter fragments of chitinase gene chiB from Bacillus thuringiensis were cloned into a shuttle promoter-probe vector pCB, and all of the constructed plasmids were transformed into Bti75 strains. A 190 bp-length high constitutive expression promoter was identificated based on the results of β-galactosidase activity and real time-PCR. By using this promoter, we constructed Bti75 engineering strains that express chitinase gene chiMY of Bacillus licheniformis and chiA gene of Bti75 itself. SDS-PAGE and zymogram analysis were used to examine chitinase expression. In order to evaluate the antifungal activity assay of engineering strains against 3 plant pathogenic fungi, mycelium growth and spore germination inhibition assay were done. [Results] Without induction, β-galactosidase activity and mRNA contents of β-galactosidase of Bti75(pBPΔ7) was about 7-fold and 2.5-fold high than that in Bti75(pBP), respectively. Without chintin induction, the chitinase activity of Bti75(pDA) and Bti75(pDM) was up to 3.5-fold higher compared with the parent strain. The results of SDS-PAGE and zymogram analysis confirmed that chitinase could be highly expressed in engineered strains. The results of antifungal activity assay indicated that the two engineered strains achieved a significant improvement in inhibiting three plant pathogenic fungi species. [Conclusion] The 190 bp deletion mutant promoter could drive the expression of different chitinase genes constitutively and efficiently. Without induction, the two engineered strains with the deletion promoter exhibited high antifungal activity.

Abstract:[Objective] To study the role of the nucleotide binding oligomerization domain-like receptor (NODs) and their signaling pathways in the pathogenesis of invasive pulmonary aspergillosis (IPA) in mice. [Methods] Mice were randomly divided into three groups: negative control, mice infected with Aspergillus fumigatus only and mice of combination of immunosuppression and Aspergillus fumigatusinfection (IPA group). Only IPA mice were immunosuppressed with Cyclophosphamide (100 mg/d) for 2 d before Aspergillus fumigatus spores were inhaled into mice of different groups, normal saline was used instead for negative control. Then mice were killed at different time points and lung tissues were collected for biopsy and Aspergillus fumigatus colony count. RT-PCR and Western blot were used to measure NOD1, NOD2, RIP2 mRNA level and TNF-α protein expression in lung tissues, respectively. [Results] At 72 h of post-infection, severe inflammation and a large number of hyphae were observed in lung tissues of IPA mice. Their Aspergillus fumigatus load at all time points were higher when compared with mice infected with Aspergillus fumigatus only, and their NOD1 and RIP2 mRNA levels were continuously lower. While NOD2 mRNA was abnormally highly expressed at 24 h and stayed at low expression level afterwards. TNF-α was highly expressed in mice infected with Aspergillus fumigatus only and reached the peak at 48 h and 72 h post-transfection. However, TNF- α was released slowly and lowly in IPA mice. [Conclusion] Persistently inhibited NOD1 and RIP2 expression along with late-stage inhibited NOD2 expression in IPA mice resulted in the low-expression of proinflammatory cytokine, which may led to the development of invasive pulmonary aspergillosis.

Abstract:[Objective] To screen and identify a microorganism with zearalenone (ZEN) detoxification ability from fermented foods, and determine its decomposing efficiency and the properties of producing enzymes. [Methods] Solid media with toxin analogue (PL) were used to obtain toxin analogue resistant strains from 25 materials of fermented foods. Through the second screening of ZEN as the selective stress, a ZEN detoxified microorganism with high decomposition efficiency was confirmed. Its degradation efficiency was further evaluated in analysis of ZEN residue of bacteria suspension using high performance liquid chromatography (HPLC). Then we analysed the properties of producing cellulase, xylanase and β-glucosidase separately. The strain was identified for the taxonomical position using morphological observation and molecular identification. [Results] A ZEN detoxified strain BF-B-3 was screened and identified as Bacillus subtilis. Its degradation rate reached 62.48%. The cellulase, xylanase and β-glucosidase activity of Bacillus subtilis BF-B-3 reached 160.38 U/mL, 84.51 U/mL and 4.14 U/mL separately. [Conclusion] Bacillus subtilis is a kind of feed microorganism. Bacillus subtilis BF-B-3 has favorable security for detoxifying ZEN, and could grow under different carbon sources. Bacillus subtilis BF-B-3 has potential values for preventing mould pollution of crops and feed.

Abstract:Engineered nanomaterials are widely used in industries and daily life due to their unique physical and chemical properties. However, comprehensive and systematic reports concerning the possible impacts of nanomaterials on wastewater treatment microorganisms are limited. Therefore, this review summarizes the potential influences of widely used nanomaterials on wastewater biological treatment, such as biological removal of carbon, nitrogen, and phosphorus, methanation, and functional microbial communities. Also, two approaches to reduce the toxicity of silver nanoparticles are discussed. The results of this review provide an important theoretical basis on further investigation of the potential effects of nanomaterials on biological sewage treatment.

Abstract:Gaps in microbial genome sequences may lead to loss of important biological information and cause trouble for further interpretation of genetic information. Gap closure is thus a critical step in completely genome finishing of microorganisms. In this review, we critically summarize six major strategies for gap filling of microbial genomes, including reference alignment, multiplex PCR, genome walking, genome library clone-end sequence, paired-end and whole genome mapping.

Abstract:The microbial flora located in human nasopharynx keeps a dynamic balance with the human body. It plays an important role in maintaining the human health, and related with various upper respiratory tract diseases. The relationship within nasopharynx microbes, and between microbes and host, is the key cause of human upper respiratory tract diseases. The development of culture-dependent and-independent molecular methods in microbial communities research provide a useful tool to learn more about the composition and structure of human nasopharyngeal microbial communities. It demonstrated that the dominant floras located in human nasopharynx were Streptococcus pneumoniae, Haemophilus influenzae, which were considered as potential pathogens in nasopharynx. In this work, the development in nasopharyngeal microbial community research was reviewed in the following three aspects: the balance between nasopharyngeal microbe and the human body; method in nasopharyngeal microbial community research; and the composition of nasopharyngeal microbial community and the internal relationship among them. This review summarized the progress on this field for the reference of the future work.

Abstract:Succinic acid is an important four-carbon platform compound that is widely applied in the pharmaceutical, agricultural and food industry. Biological processes for succinic acid production show a good prospect for its social, environmental and economic benefits. Corynebacterium glutamicum is broadly used for industrial production of value-added chemicals such as amino acid and nucleotide. Under anaerobic conditions, C. glutamicum cell growth is arrested, but the cells retain the capability to metabolize sugars to various organic acids efficiently. It is thus becoming a desired succinate-production strain by means of metabolic engineering. Combining with the latest achievements of succinic acid production with C. glutamicum, this mini-review summarized the metabolic engineering strategies of constructing an efficient succinate-production strain, the expansions of substrate utilization, and the prospects of future research.

Abstract:ATP-binding cassette (ABC) transporters are members of a protein superfamily that is one of the largest and most ancient families with great variety of functions. They are involved in transporting different substrates across the plasma membrane. As more ABC transporters were identified from the gene clusters for antibiotic biosynthesis, recent studies have begun to focus on their biological functions. The polyene antibiotics represent a class of biologically active fungal metabolites synthetized by the genus Streptomyces. As drug resistance to polyene remains rare, polyene antibiotics have been the cornerstone of therapy for critically ill patients with invasive fungal infections. We reviewed the latest research progresses on ABC transporters of polyene antibiotic biosynthesis gene clusters, including their structure characteristics and biological functions. The potential relationship between the secondary structure and their function is also discussed.

Abstract:More and more antibiotic analogs are biosynthesized by combinatorial biological techniques. Eleven FK506 analogs are biosynthesized through replacement of allylmalonyl group and 4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid group with heterologous building blocks, and replacement of allylmalonyl biosynthetic elements with heterologous modular biosynthetic elements. These studies will expand our understanding of heterologous building blocks and modular biosynthetic elements and may help us generate more new antibiotic analogs.

Abstract:Bacillus subtilis and closely related species, as members of the “subtilis-group”, are composed of rod-shaped, endospore-forming bacteria with high degree of phenotypic and biochemical similarity. The “subtilis-group” includes B. subtilis, B. licheniformis, B. pumilus, B. amyloliquefaciens, B. atrophaeus, B. mojavensis, B. vallismortis, B. sonorensis, B. tequilensis, B. siamensis. Strains of the group have been exploited for biotechnological, industrial and agricultural applications; therefore, their rapid and accurate identification is important in practice. Here we reviewed traditional and molecular methods to identify these species. The combination of the physiological and biochemical characteristics, sequencing analysis and species-specific PCR would allow us to rapidly and accurately differentiate the strains of the group.

Abstract:Imaging agents for bacterial infection are composed of some tracer groups including radionuclides and fluorescent dyes conjugated to some guides which specifically target bacteria. By detecting the tracer signals, bacterial infection sites can be positioned. So far, on the basis of the specific binding sites of bacteria (such as cell wall, enzyme and receptor), a lot of imaging agents are constructed for bacterial infection. These agents include radiolabelled antibiotics, bacteriophage, antimicrobial peptides, nucleosides and nucleoside analogs and fluorochrome labeled sugars. They are promising to detect clinical infection disease for their highly specifically accumulation in the infection sites, but not in the non-bacterial induced inflammation sites and normal tissues. Here we reviewed the types, imaging mechanisms and the latest progress of these imaging agents for bacterial infection. Based on this, characteristics of ideal imaging agents for bacterial infection are also summarized, and it can be useful for further study and development of new imaging agents.

Abstract:Helicobacter pylori (H. pylori) infection can cause peptic ulcer disease, gastric mucosa associated lymphoid tissue lymphoma (GMALTL) and even gastric cancer. With increasing problem of antibiotic resistance, study on mechanisms of which is promoted deeply. Molecular detection methods, especially the nucleic acid detection technologies, can be used to detect antibiotic resistance genes or mutations efficiently, rapidly and accurately, hence guiding the clinical treatment of H. pylori infection and helping to investigate antibiotic resistance rate timely and effectively in a large area. In this review, H. pylori antibiotic resistance mechanisms of the most commonly used antibiotics were discussed and associated resistance genes or mutations were emphatically reviewed.

Abstract:A triangular bilingual learning model of “listening-discussion-presentation”, as well as a dynamic management model of “learning-feedback-revising” has been established in the course of microbiology with the assistance of Blackboard Learning System. Given the bilingual lecturing, well-developed discussion forum, and the teacher’s guidance both in classroom and online, students’ comprehensive performance and the results of postgraduate qualifying examination are both greatly increased. Students’ innovative ability is accordingly enhanced. Students who are for this synthesized reform account for 90 percent.

Abstract:Water Treatment Microbiology is an important specialized foundation curriculum in applied chemistry profession. In order to meet the training needs of excellent engineers education program, Water Treatment Microbiology curriculum was reformed and practiced in four aspects containing the formulation of teaching program, optimization of teaching content, reformation of teaching methods and means and construction of curriculum examination evaluation system according to existing problems in the teaching. The results indicated that the engineering practices and innovative capability of students were increased and the comprehensive quality of students was improved based on the curriculum reformation and practice, which could lay a foundation for the successful training of excellent engineers.

Abstract:[Objective] To establish a multiplex PCR assay on the basis of the SXI1α gene located at the mating type α (MATα) locus and SXI2a gene located at the mating type a (MATa) locus for rapid identification of the mating types of Cryptococcus gattii. [Methods] The primers specific for the alleles of SXI1α gene fragment within the MATα locus of Cryptococcus gattii was designed in combination with the primers specific for the alleles of SXI2a gene fragment within the MATa locus for multiplex PCR analysis of mating types of Cryptococcus gattii. The results were compared with those obtained from regular PCR using primers MFα and STE12α specific for the MATα locus, and primers STE20a and STE3a specific for the MATa locus reported previously, as well as mating assays. [Results] The mating types of all strains including four major genotypes, VGI, VGII, VGIII and VGIV, were successfully identified by multiplex PCR analysis based on the SXI1α and SXI2a genes. However, the mating types of a part of strains could not be determined by regular PCR assays with primer STE12α, STE20a or STE3a; the mating types of 66.7% percent of strains involved here could not be determined due to their inability to mate with the tester strains. [Conclusion] The multiplex PCR analysis is superior to regular PCR or mating assay reported previously.

Abstract:

Abstract:

Abstract:[Objective] Based on the genome sequences of two super extensive drug resistant isolates of Mycobacterium tuberculosis, we attempted to identify the lineage-specific SNPs and found the drug resistant-associated mutations in them. [Methods] By using the next-generation sequencing technology on two super-extensive drug resistant isolates of mycobacterium tuberculosis (FJ05194 and GuangZ0019), we compared them with the reference sequence to identify the single nucleotide polymorphisms (SNP) and divided the SNPs into lineage-specific and isolate-specific SNPs. Check the coordinate of SNPs on the genome, then we did the enrichment analysis of the lineage-specific SNPs, and were able to found out the drug resistant mutations from the isolate-specific SNPs by overlapping with the candidate drug resistance associated regions. [Results] The Lineage2-specific SNPs was significantly enriched on ABC transpoters and nucleotide excision repair KEGG pathways, and the extensive drug resistance were mainly attributed to the mutation of the well-known genes (rpoB, katG, rpsl, gyrA, gyrB, embB, ethA and so on), but the capreomycin and kanamycin resistance was probably due to some candidate resistant-associated genes (Rv1393c, Rv0265c, narX) and the efflux pump genes (pstB, Rv2333c and Rv2687c). [Conclusion] The Lineage2-specific-SNPs possible result in the high mutation rate and drug resistance of the Lineage2. And the mechanism of super extensive drug resistance is more complex, it not only involve with the drug target and drug activation genes, but also some unclear candidate genes such as compensatory genes and efflux pump genes.

Abstract:[Objective][Methods] Box-Behnken design combing with response surface modeling was used to study the relationships between cell growth of a moderate thermophilic Bacillus subtilis BY25 and its thermostable protease yield under different carbon (glucose) and/or nitrogen (soybean waste powder) sources as well as pH values. [Results] The higher values of the coefficients of determination, lack-of-fit<4 and p value<0.05 were in good agreement with the optimum combination of process parameters, which proved the fitness of the selected model for analyzing the experimental data. Data revealed that both low and medium nitrogen applications greatly contributed to the growth of Bacillus subtilis, which however inhibited the yield of thermostable protease; under high nitrogen application both bacterial growth and protease yield were stimulated. Contour plots displayed the maximal enzyme yield was accompanied with 47.3% and 61.4% maximal biomass under low and medium nitrogen applications, but with 96.3% maximal biomass under high nitrogen application. [Conclusion] The higher nitrogen concentration could increase the specific enzyme productivity, and soybean product waste could contain a better inducer for protease synthesis in Bacillus subtilis.

Abstract: