Pip介导铜绿假单胞菌吩嗪基因簇phz2的表达

[目的]为了确定铜绿假单胞菌调控因子Pip对两个不同吩嗪合成基因簇(phz1和phz2)的具体调控方式与可能的调控机制.[方法]根据基因比对结果,采用同源重组技术构建Pip调控因子缺失突变株PA-PG以及克隆ip基因作互补分析;再以已构建的吩嗪基因簇缺失突变株PA-Z1G和PA-Z2K为受体菌,构建突变株PA-PD-Z1G和PA-PG-Z2K,测定并比较野生株及相关突变株的吩嗪-1-羧酸和绿脓菌素的合成量,推定Pip对两个不同吩嗪合成基因簇的调控方式.[结果]在GA培养基中,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都比野生型明显减少;互补分析显示,突变株PA-PG的吩嗪-1-羧酸和绿脓菌素都显著提高并恢复到野生株PAO1水平;突变株PA-Z1G的吩嗪-1-羧酸和绿脓菌素合成量因Pip缺失而显著减少;而突变株PA-Z2K的吩嗪-1-羧酸和绿脓菌素合成量在Pip缺失后仍保持不变.[结论]初步推定,转录调控因子Pip对铜绿假单胞菌吩嗪合成代谢的确具有促进作用;Pip通过正向调控吩嗪基因簇phz2的合成功能实现对吩嗪合成代谢的调控.     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

不同成熟度煤样产甲烷潜力

摘要:【目的】评估不同类型煤炭生物降解转化为甲烷的潜力,研究原位煤层的微生物群落结构特征。【方法】分别在原位模拟、补加烃降解产甲烷菌系和补加碳源下厌氧培养煤样,利用气相色谱监测甲烷产生趋势,及高通量测序技术研究原位煤层的细菌和古菌群落。【结果】10个样品中有3个高成熟度煤样可以被厌氧降解转化为甲烷。通过生物强化和添加外源底物可以促进HF煤样的产甲烷潜力。其中SL 煤样中的古菌类群主要是氢营养型产甲烷菌Methanoculleus和乙酸营养型产甲烷菌Methanosaeta为主,细菌类群主要 属于Firmicutes(54.4%)、Proteobacteria(30.9%)、未培养微生物(10.8%)、Caldiserica(1.5%)及Thermotogae(1.3%)。【结论】不同成熟度煤样降解产气潜力不同,在部分原位煤层中可能存在参与烃降解与甲烷产生的功能菌。     

Streptomyces muensis sp. nov.

A strain of Streptomyces, MBRL 179^T, isolated from a sample from a Limestone quarry located at Hundung, Manipur, India, was characterized by polyphasic taxonomy. The strain formed a monophyletic clade with Streptomyces spinoverrucosus NBRC 14228^T (16S rRNA gene sequence similarity of 99.3 %) in the Neighbour-joining tree. DNA–DNA hybridization experiment gave a DNA–DNA relatedness value of 34.7 % between MBRL 179^T and S. spinoverrucosus NBRC 14228^T. Strain MBRL 179^T contained LL-diaminopimelic acid, xylose, glucose, and mannose in the whole cell-wall hydrolysates along with small amount of ribose. The major polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositolmannoside, with other unknown phospholipids and aminophospholipid. MK-9(H₆), MK-9(H₈) and MK-9(H₄) were the predominant menaquinones detected. The major fatty acids were anteiso-C_16:0 (28.1 %), iso-C_16:0 (20.3 %), C_16:0 (9.4 %) and anteiso-C_17:0 (8.3 %). The G+C content of the genomic DNA was 71.1 %. Based on the polyphasic experiment results, the strain MBRL 179^T merits recognition as a representative of a novel species of the genus Streptomyces for which the name Streptomyces muensis sp. nov. is proposed; the type strain is MBRL 179^T (=JCM 17576^T = KCTC 29124^T).     

Immobilization of unraveled immunoglobulin G using well-oriented ZZ–His protein on functionalized microtiter plate for sensitive immunoassay

Highly efficient protein immobilization is extremely crucial for solid-phase immunoassays. We present a strategy for oriented immobilization of functionally intact immunoglobulin G (IgG) on a polystyrene microtiter plate via iminodiacetic acid (IDA)–Ni²⁺ and ZZ–His protein interaction. We immobilized a ZZ–EAP (Escherichia coli alkaline phosphatase)–His fusion protein, which exhibits Fc binding, His tag, and intrinsic AP activities, and analyzed it against the interaction between rabbit IgG anti-horseradish peroxidase (anti-HRP) and its binding partner HRP to investigate the specificity and efficacy of this method. We compared the IDA–Ni²⁺–(ZZ–His) method with ZZ–EAP random immobilization using sandwich enzyme-linked immunosorbent assay, and the results showed that the former method had an enhanced signal, 10-fold higher sensitivity, and a wider linear range. Thus, the proposed method allows a broad range of oriented immobilized functionally intact IgG antibodies on polystyrene plates using only one type of IDA–Ni²⁺ chelate surface because the ZZ protein can bind to the Fc region of various IgGs.     

Halomonas zhaodongensis sp. nov., a slightly halophilic bacterium isolated from saline–alkaline soils in Zhaodong, China

A slightly halophilic bacterium (strain NEAU-ST10-25^T) was isolated from saline–alkaline soils in Zhaodong City, Heilongjiang Province, China. The strain is a Gram-negative, aerobic motile rod. It accumulates poly-β-hydroxyalkanoate and produces exopolysaccharide. It produces beige-yellow colonies. Growth occurs at NaCl concentrations (w/v) of 0–15 % (optimum 3 %), at temperatures of 4–60 °C (optimum 35 °C) and at pH 6–12 (optimum pH 9). Its G+C content is 53.8 mol%. Phylogenetic analyses based on the separate 16S rRNA gene and concatenation of the 16S rRNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphila DSM 16354^T, with which strain NEAU-ST10-25^T showed 16S rRNA, gyrB and rpoD gene sequence similarities of 99.2, 82.3 and 88.2 %, respectively. The results of DNA–DNA hybridization assays showed 60.47 ± 0.69 % DNA relatedness between strain NEAU-ST10-25^T and H. alkaliphila DSM 16354^T, 42.43 ± 0.37 % between strain NEAU-ST10-25^T and Halomonas venusta DSM 4743^T and 30.62 ± 0.43 % between strain NEAU-ST10-25^T and Halomonas hydrothermalis DSM 15725^T. The major fatty acids are C_18:1 ω7c (62.3 %), C_16:0 (17.6 %), C_16:1 ω7c/C_16:1 ω6c (7.7 %), C_14:0 (2.9 %), C_12:0 3-OH (2.8 %), C_10:0 (2.1 %) and C_18:1 ω9c (1.6 %) and the predominant respiratory quinone is ubiquinone 9 (Q-9). The proposed name is Halomonas zhaodongensis, NEAU-ST10-25^T (=CGMCC 1.12286^T = DSM 25869^T) being the type strain.     

Protection against cardiac injury by small Ca2 +-sensitive K+ channels identified in guinea pig cardiac inner mitochondrial membrane

We tested if small conductance, Ca^2 +‐sensitive K⁺ channels (SK_Ca) precondition hearts against ischemia reperfusion (IR) injury by improving mitochondrial (m) bioenergetics, if O₂‐derived free radicals are required to initiate protection via SK_Ca channels, and, importantly, if SK_Ca channels are present in cardiac cell inner mitochondrial membrane (IMM). NADH and FAD, superoxide (O₂^?), and m[Ca^2 +] were measured in guinea pig isolated hearts by fluorescence spectrophotometry. SK_Ca and IK_Ca channel opener DCEBIO (DCEB) was given for 10 min and ended 20 min before IR. Either TBAP, a dismutator of O₂^?, NS8593, an antagonist of SK_Ca isoforms, or other K_Ca and K_ATP channel antagonists, were given before DCEB and before ischemia. DCEB treatment resulted in a 2-fold increase in LV pressure on reperfusion and a 2.5 fold decrease in infarct size vs. non-treated hearts associated with reduced O₂^? and m[Ca^2 +], and more normalized NADH and FAD during IR. Only NS8593 and TBAP antagonized protection by DCEB. Localization of SK_Ca channels to mitochondria and IMM was evidenced by a) identification of purified mSK_Ca protein by Western blotting, immuno-histochemical staining, confocal microscopy, and immuno-gold electron microscopy, b) 2-D gel electrophoresis and mass spectroscopy of IMM protein, c) [Ca^2 +]‐dependence of mSK_Ca channels in planar lipid bilayers, and d) matrix K⁺ influx induced by DCEB and blocked by SK_Ca antagonist UCL1684. This study shows that 1) SK_Ca channels are located and functional in IMM, 2) mSK_Ca channel opening by DCEB leads to protection that is O₂^? dependent, and 3) protection by DCEB is evident beginning during ischemia.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Deep Whole-Genome Sequencing of 100 Southeast Asian Malays

Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.