ycjX和ycjF基因在布鲁氏菌热应激中的作用

【背景】细菌应对环境压力的能力对其存活和增殖及引起感染具有重要意义。充分揭示布鲁氏菌应激机制,可为防控布鲁氏菌病提供理论依据。研究发现,ycjX基因在细菌热应激时高表达且可能受σ³²调节,ycjF基因在细菌败血症期间表达显著上升,二者在革兰阴性菌中可能构成操纵子。【目的】研究ycjX和ycjF基因在布鲁氏菌热应激中的作用。【方法】对布鲁氏菌(Brucella)及其ycjX和ycjF双基因缺失株(△ycjXF)和回补株(C△ycjXF)进行热应激试验,计算3株菌的存活率。利用RT-PCR和β-半乳糖苷酶活性检测试验鉴定ycjX和ycjF的操纵子模式和启动子区域活性。利用ChIP试验分析σ³²与ycjX和ycjF的靶向调节关系。表达并纯化pGEX-4T-1-σ³²重组蛋白,凝胶电泳迁移试验分析σ³²与ycjX和ycjF之间的结合关系。【结果】热刺激后△ycjXF存活显著低于Brucella suis S2和C△ycjXF。明确布鲁氏菌ycjX和ycjF为同一转录本,确定其启动子区域具有活性。ChIP试验表明,σ³²可靶向富集在ycjXF启动子区,凝胶电泳迁移试验确定σ³²可与ycjXF体外直接结合。【结论】在σ³²的调节下,ycjX和ycjF在布鲁氏菌热应激中发挥正向作用。     

一株糖降解噬糖菌FZY0027的多糖水解活性及基因组分析

【目的】潮间带海水中分离获得一株具有水解多糖能力的菌株FZY0027,分析其对不同多糖的水解能力和基因组特征。【方法】通过形态观察、16S rRNA基因测序和基于Illumina NovaSeq和OxfordNanopore PromethION测序技术全基因组测序对菌株FZY0027进行鉴定。使用dbCAN、EasyCGTree、BRIG和Easyfig等生物信息学软件将菌株FZY0027和降解糖噬糖菌(Saccharophagus degradans) 2-40^T进行比较。使用3,5-二硝基水杨酸(3,5-dinitrosalicylic acid, DNS)法测定多糖水解活性。【结果】菌株FZY0027与S. degradans 2-40^T的16S rRNA基因序列相似度达到99.9%,初步鉴定为降解糖噬糖菌(S. degradans) FZY0027。该菌株在水解淀粉、木聚糖和甘露聚糖时产生的还原糖浓度最高,分别为2.28、1.75和1.10 mg/mL。菌株FZY0027基因组全长5 178 381 bp,共编码4 156个基因,G+C含量为45.8%。菌株FZY0027与S. degradans 2-40^T的平均核苷酸一致性(average nucleotide identity, ANI)、平均氨基酸一致性(average amino acid identity, AAI)和DNA-DNA分子杂交(digital DNA-DNA hybridization, dDDH)值分别为96.5%、96.7%和70.0%。经碳水化合物活性酶数据库注释获得303个基因,其中,菌株FZY0027和S. degradans 2-40^T分别有糖苷水解酶(glycoside hydrolases, GHs)结构域的基因137个和130个。菌株FZY0027具有多个参与淀粉、木聚糖等多糖水解的基因,这与菌株FZY0027对淀粉和木聚糖的水解能力强的结果一致。然而,与S. degradans 2-40^T相比,菌株FZY0027在实验条件下只能水解少数多糖,这可能需要特定的诱导条件才能充分发挥其多糖水解能力。【结论】菌株FZY0027是一株多能型多糖水解菌,具有潜在开发价值。     

稻田除草剂二氯喹啉酸降解菌15#的分离、鉴定及降解特性

[背景] 二氯喹啉酸（Quinclorac,QNC）是一种高选择性、激素类、低毒性除草剂,主要用于防治稻田稗草,持效期长,易于在土壤中积累而影响后茬作物的生长发育,而且环境中残留的QNC可对动物生长发育产生不良影响,并影响微生物的群落结构和丰度。[目的] 从稻田土壤中分离筛选出一株可降解除草剂QNC的菌株,鉴定并明确其降解特性。[方法] 通过形态学、生理生化试验、磷脂脂肪酸（Phospholipid Fatty Acid,PLFA）微生物鉴定、16S rRNA基因测序及分析鉴定菌株。通过单因素实验探究菌株的降解特性。[结果] 筛选得到一株编号为15^#的QNC降解菌,被鉴定为无色杆菌属菌株（Achromobacter sp.）。降解特性研究结果表明,菌株15^#的最佳培养条件为：30℃、pH为6.0、初始QNC浓度为100 mg/L、接种量为7%、添加质量分数为0.1%的酵母浸粉、氮源为蛋白胨,在此条件下培养21 d后QNC的降解率可达43.0%。[结论] 筛选到降解QNC新菌株并为该菌株的进一步研究提供理论基础。     

一株深海热液环境来源的PAHs降解菌TVG9-Ⅶ的系统发育与降解基因

【目的】对一株深海热液环境来源的多环芳烃(PAHs)降解菌进行系统发育分析并对其降解特性和降解机制进行研究。【方法】对16S rRNA基因进行扩增和测序,进行基于16S rRNA基因序列的系统发育分析;利用GC-MS测定其对PAHs的降解率;通过构建基因组Fosmid文库,克隆PAHs降解基因簇;并利用RT-PCR和qPCR研究关键降解酶基因在不同PAHs诱导下的表达情况。【结果】从西南太平洋劳盆地热液沉积物中分离到一株PAHs降解菌株TVG9-Ⅶ,系统发育分析结果表明,该菌株属于新鞘氨醇杆菌属(Novosphingobium),与该属的Novosphingobium indicum H25T系统发育关系最为密切,它们的16S rRNA基因序列相似性高达99.7%。该菌株在21 d内对菲、荧蒽和芘的降解率分别为95.2%,57.3%和69.6%。从Fosmid文库中筛选得到一个负责PAHs降解的上游基因簇,包含了PAHs起始降解双加氧酶大小亚基(pheA1a/b)基因和一个脱氢酶基因;RT-PCR和qPCR实验表明,双加氧酶大亚基基因pheA1a在菲的诱导下上调表达4.2倍,而在萘及高环荧蒽和芘的诱导下无上调。【结论】菌株TVG9-Ⅶ是Novosphingobium属深海热液来源的PAHs降解菌,具有良好的降解特性,特别是对高环PAHs的降解效果较好。     

Bacillus sp.ZJS3基因组测序及砷胁迫下相关基因表达分析

【背景】环境中高毒性As³⁺的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As³⁺耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As³⁺胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As³⁺耐受细菌的研究提供基础数据。【方法】使用单分子实时测序（single-molecule real-time sequencing,SMRT）及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As³⁺胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZ、arsA、arsB、arsC等基因的存在表明该菌株具有As³⁺外排和还原As⁵⁺的能力,phoUpstBACS的存在表明菌株可以吸收As⁵⁺,但菌株受到外界环境As³⁺胁迫时arsB表达水平降低。     

趋化信号转导基因cheA突变对Pseudomonas putida DLL-1甲基对硫磷的趋化性及原位降解的影响

Pseudomonas putida DLL-1是一株甲基对硫磷(MP)高效降解菌株,同时对MP具有趋化性。cheA基因是菌株趋化信号转导过程中负责编码组氨酸激酶的基因,为了研究菌株趋化性在农药原位降解中的作用,通过基因打靶的方式使P.putida DLL-1染色体上单拷贝的cheA基因失活,成功地获得了MP的趋化突变株P.putida DAK,突变株与野生菌株生长能力没有显著差异。通过土壤盆钵试验(MP浓度为50mg/kg),发现在灭菌与未灭菌土壤中趋化突变株对MP的降解能力低于原始出发菌株DLL-1约20%~30%,说明菌株DLL-1趋化性的丧失会减慢其对农药的降解,趋化性在农药的原位降解过程中发挥重要作用。     

p14ARF对人黑色素瘤细胞增殖的影响及其作用机理的初探

ARF(alternative reading frame)作为INK4a/ARF的β转录产物,能够稳定p53, 诱导细胞周期阻断或凋亡.利用高表达p14^ARF的人黑色素瘤细胞模型,探讨了ARF抑制细胞增殖的分子作用机理.研究发现p14^ARF高表达能将细胞周期阻断在G1和G2期, p53, p21^cip1和p27^kip1蛋白水平明显增强, 而p-ERK1/2,CyclinD1和CyclinE蛋白水平下降, 明显抑制细胞生长. 提示p14^ARF能通过ERK(extracellular signal-regulated kinase)信号通路相互协调作用抑制A375细胞增殖.     

多环芳烃降解菌的筛选、鉴定及降解特性

【目的】多环芳烃(PAHs)是一类普遍存在于环境中且具有高毒性的持久性有机污染物,高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。研究拟从供试菌株中筛选多环芳烃高效降解菌,并分析其降解特性,为多环芳烃污染环境的微生物修复提供资源保障和科学依据。【方法】采用平板法从25株供试菌株中筛选出以菲和芘为唯一碳源和能源的高效降解菌,经16S rRNA基因序列进行初步鉴定,通过单因素实验法分析其在液体培养基中的降解特性。【结果】筛选出的3株多环芳烃高效降解菌SL-1、02173和02830经16S rRNA基因序列分析,02173和02830分别与假单胞菌属中的Pseudomonas alcaliphila和Pseudomonas corrugate同源性最近,SL-1为本课题组发表新类群Rhizobium petrolearium的模式菌株;降解实验表明,菌株SL-1 3 d内对单一多环芳烃菲(100 mg/L)和芘(50 mg/L)的降解率分别达到100%和48%,5 d后能够降解74%的芘;而其3 d内对混合PAHs中菲和芘的降解率分别为75.89%和81.98%。菌株02173和02830 3 d内对混合多环芳烃中萘(200 mg/L)、芴(50 mg/L)、菲(100 mg/L)和芘(50 mg/L)的降解率均分别超过97%。【结论】筛选出的3株PAHs降解菌SL-1、02173和02830不仅可以高效降解低分子量PAHs,还对高分子量PAHs具有很好的降解潜力。研究表明,由于共代谢作用低分子量多环芳烃可促进高分子量多环芳烃的降解,而此时低分子量多环芳烃的降解将受到抑制。     

金属离子胁迫对Sphingomonas sp. Y2降解壬基酚聚氧乙烯醚特性的影响

壬基酚聚氧乙烯醚（NEPOs）是全球应用量最大的非离子型表面活性剂之一,具有环境雌激素毒性。NPEOs的中间代谢产物种类多、难降解,且毒性远高于其母系化合物。为研究金属离子对功能微生物Sphingomonas sp. Y2降解NPEOs特性的影响,分析了金属离子的最低抑制浓度（MIC）、细菌形态、NPEOs降解效率及代谢产物组成等变化。结果显示,菌株Y2对多种金属离子具有耐受性,在重金属培养基中对Mn²⁺、Zn²⁺具有较高的耐受性,MIC分别为500、90 mg/L;在500 mg/L Mn²⁺胁迫下,菌株Y2对NPEOs降解率为100.00%（3 d）;在90 mg/L Zn²⁺胁迫下,菌株Y2对NPEOs的降解率为20.62%（5 d）;两种离子双重胁迫下NPEOs降解率为15.65%（5 d）;Mn²⁺胁迫下菌株Y2细胞表面结构和形态发生明显变化,且改变了NPEOs代谢产物中组分的含量组成,其中短链NPEOs与短链壬基酚聚氧乙烯酸（NPECs）的比例为0.68,与对照相比,抑制/减缓了短链NPEOs的羧化反应。结果表明,菌株Sphingomonas sp. Y2对多种金属离子具有耐受性,Mn²⁺胁迫对细胞表面超微结构及NPEOs中间代谢产物组分组成产生显著影响。该研究将为表面活性剂类污染物的生物降解及相应代谢产物在环境中的毒性评价提供理论依据。     

来源于中国南海的微生物新种乙醇速生杆菌(Celeribacter ethanolicus)多相分类研究

【目的】研究来源于南海海水的一株速生杆菌属菌株NH195^T 的多相分类。【方法】采用表型、基因型和化学分类方法,并综合系统发育关系结果,分析菌株NH195^T 的分类学地位。【结果】菌株NH195^T 是一株革兰氏阴性、好氧、杆状、无运动性细菌;能积累Poly-β-hydroxybutyrate (PHB);能在0.5%-10.0% (质量体积比) NaCl 浓度,pH 5.0-9.0 和20-40 ℃ 条件下生长,最适NaCl 生长浓度为1.0%-3.0%;氧化酶、触酶和脲酶反应结果阳性。菌株NH195^T 主要呼吸醌为Q-10,主要脂肪酸为C_18:1ω7c、C_18:1ω6c 和11 methyl C_18:1ω7c,主要极性脂为磷脂酰胆碱、磷脂酰甘油、双磷脂酰甘油、一个未知的氨基脂和两个未知脂。基因组G+C 含量为61.3 mol%。基于16S rRNA 基因的系统发育结果显示,菌株NH195^T 隶属于速生杆菌属;其与速生杆菌属标准菌株的16S rRNA 基因相似性范围为94.4%-97.7%。菌株NH195^T 与速生杆菌属标准菌株C. halophilus ZXM137^T 和C. indicus P73^T 的平均核苷酸一致性(ANI)分别为78.6%和78.0%;基于基因组数据计算所得的DNA 杂交同源率分别为26.1%和23.0%。【结论】基于表型和基因型结果,菌株NH195^T 代表了速生杆菌属一个新物种,命名为Celeribacter ethanolicus,标准菌株为NH195^T (CGMCC 1.15406^T=JCM 31095^T)。     

Synthesis of N-monomethylagmatine, a decarboxylation product of N-monomethyl- -arginine

N^G-Monomethylagmatine, a decarboxylation product of N^G-monomethyl- -arginine, has been synthesized by reacting putrescine with N,S-dimethylthiopseudouronium iodide. The structural identity of the product was confirmed by proton NMR and mass spectroscopy, and its properties were determined on thin-layer and electrophoretic chromatography.     

(Na + K)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K⁺-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K⁺-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight)⁻¹ · min⁻¹ were obtained, the value decreasing with age and K⁺-deficiency. Complete inhibition of the K⁺-dependent phosphatase was obtained with 10⁻³ M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K⁺-dependent phosphatase activity and (Na⁺ + K⁺)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min⁻¹ was estimated from simultaneous determination of K⁺-dependent phosphatase activity and [³H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [³H]ouabain binding sites measured in intact muscles or biopsies hereof.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Mathematical modelling of ATP, K and Na interactions with (Na + K)-ATPase occurring under equilibrium conditions

The controlling effect of ATP, K⁺ and Na⁺ on the rate of (Na⁺ + K⁺)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.

1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.

2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K⁺ and Na⁺ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.

3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)₂ complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K⁺ and Na⁺ were absent or K⁺ and K⁺ plus Na⁺, respectively, were present in the experiments.

4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na⁺ + K⁺)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.

The phospholipid requirement of the (Ca + Mg)-ATPase from human platelets

The phospholipid requirement of the (Ca²⁺ + Mg²⁺)-ATPase present in a membrane fraction from human platelets was studied using various purified phospholipases. Only those phospholipases, which hydrolyse the negatively charged phospholipids, inhibited the (Ca²⁺ + Mg²⁺)-ATPase activity. The ATPase activity could be restored by adding mixed micelles of Triton X-100 and phosphatidylserine or phosphatidylinositol. Micelles with phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine or sphingomyelin could not be used for reconstitution and inhibited the activity of the native enzyme.     

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H⁺-ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H⁺-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.     

Inhibition of NTPDase, 5′-nucleotidase, Na/K-ATPase and acetylcholinesterase activities by subchronic treatment with Casearia sylvestris

The aqueous extract of Casearia sylvestris was tested in cortical membrane preparations. C. sylvestris was obtained commercially from two different sources, designated as Sample A and Sample B. The enzymes studied in this work were NTPDase-like, 5'-Nucleotidase, Na(+)/K(+)-ATPase and acetylcholinesterase (AChE). Adult rats received aqueous extracts from C. sylvestris in a dose of 20mg/kg body wt. daily for a 75-day-period, by oral administration (gavage). Our study showed that this treatment caused an inhibition of NTPDase-like activity with both, ATP (19.41% with Sample A and 25.03% with Sample B) and ADP (41.57% with Sample A and 31.20% with Sample B) as substrates. This treatment also caused an inhibition of 5'-nucleotidase activity (28.34% with Sample A and 31.46% with Sample B) and Na(+)/K(+)-ATPase (25.08% with Sample A and 24.81% with Sample B). The rate of acetylcholine degradation was reduced, as shown by the inhibition of AChE (31.65% and 26.74%, Samples A and B, respectively). These results suggest that extracts of C. sylvestris can cause neurochemical alterations in the purinergic and cholinergic systems of the central nervous system.