微紫青霉菌Penicillium janthinellum菌株GXCR吸附废水中Cd2+的研究

研究了5种不同预处理方式对丝状真菌微紫青霉菌Penicillium janthinellum菌株 GXCR的Cd2+吸附的影响。结果表明,高温（80℃）、去离子水中的匀浆化、匀浆+碱化（NaOH,0.5mol/L）（简称匀浆碱化）和匀浆+30%二甲基亚砜处理均能提高菌体的吸附率,其中匀浆碱化处理后菌体的吸附效果最佳,吸附增量达到117.96%;匀浆+酸化（H2SO4, 0.5mol/L）处理则导致菌体的Cd2+吸附能力显著下降。匀浆碱化菌体吸附符合典型的Langmuir方程,表明该菌对Cd2+的吸附可能是以表面吸附为主的吸附行为。在吸附-解吸附循环4次后匀浆碱化菌体的Cd2+的吸附效率为58.01%。红外光谱分析显示匀浆碱化处理主要影响菌体表面分子的–OH和C=O基团,其中与Cd2+结合的主要基团是–OH。结果也表明,匀浆碱化菌体具有处理电镀废水的潜能。     

微紫青霉CBHI酶的CBD蛋白编码区在大肠杆菌中的亚克隆及表达

对插入质粒pUC18－181上的微紫青霉（Penicilliumjanthinellum）CBHI酶的cDNA基因进行一系列DNA体外操作,包括进行序列定向缺失,最后将两末端修饰为平端后进行连接使质粒环化。用得到的产生序列定向缺失的重组质粒转化大肠杆菌JM109。利用CBD能吸附到结晶纤维素上的特性,从随机选取的24个缺失转化子中筛选到一株含CBD编码区的转化子JM109（pUC18C）,所表达的CBD融合蛋白分子量为21kD．JM109（pUC18C）所产生的LacZ-CBD融合蛋白可通过对纤维素的吸附-解吸附过程一步纯化。其IPTG诱导的pNPC酶活力为零,表明该菌已不再具有CBHI酶活力。     

大豆连作障碍研究 Ⅱ.大豆连作减产机理及对土壤紫青霉菌毒素的调控对策

研究分析了土壤紫青霉菌分泌毒素对连作大豆全生育过程的危害．结果表明,在大豆种子萌动期毒素致毒作用已经开始;在大豆幼苗期及分枝期,较高浓度毒素（×１０３倍稀释）使大豆完全不结瘤,极低浓度（×１０５倍稀释）仍然抑制４０％结瘤,致使重茬大豆光合固氮能力显著降低．而大豆残根、凋落物等通过刺激紫青霉菌的增长,造成对大豆进一步的危害．对连作大豆土壤紫青霉毒素的调控研究表明,使用土壤放线菌ＭＢ生物防治,可使重茬大豆增产８．４～１８．９％;使用海洋放线菌ＭＢ９７生物防治可使重茬大豆增产３０．５％．应用ＭＢ９７制剂对连作大豆全生育过程的防病、减毒控制的综合调控,可使重茬５年大豆田间微区试验折合大豆产量达到４５７５ｋｇ·ｈｍ－２．     

大豆连作障碍研究 Ⅱ. 大豆连作减产机理及对土壤紫青霉菌毒素的调控对策

研究分析了土壤紫青霉菌分泌毒素对连作大豆全生育过程的危害。结果表明,在大豆种子萌动期毒素致毒作用已经开始; 在大豆幼苗期及分枝期,较高浓度毒素(×10³倍稀释)使大豆完全不结瘤,极低浓度(×10⁵倍稀释)仍然抑制40%结瘤,致使重茬大豆光合固氮能力显着降低。而大豆残根、凋落物等通过刺激紫青霉菌的增长,造成对大豆进一步的危害。对连作大豆土壤紫青霉毒素的调控研究表明,使用土壤放线菌MB生物防治,可使重茬大豆增产8.4～18.9%; 使用海洋放线菌MB-97生物防治可使重茬大豆增产30.5%. 应用MB97制剂对连作大豆全生育过程的防病、减毒控制的综合调控,可使重茬5年大豆田间微区试验折合大豆产量达到4575kg·hm^-2.     

甘蔗金属硫蛋白基因(ScMT2-1-4)的克隆及表达分析

从甘蔗热带种Badila(Saccharum officinarum L.)中克隆获得1个2型金属硫蛋白基因的c DNA序列,命名为Sc MT2-1-4(Gen Bank登录号:KJ504375)。生物信息学分析显示,Sc MT2-1-4基因c DNA长459 bp,开放读码框为243 bp,编码80个氨基酸,富含14个半胱氨酸残基。推导的Sc MT2-1-4蛋白为亲水性蛋白,分子量为7.82 k D,等电点为5.59,该蛋白的二级结构由无规则卷曲和延伸链构成。实时荧光定量PCR检测结果显示,Sc MT2-1-4基因是重金属胁迫的快速响应基因,其对Cu2+、Zn2+和Cd2+胁迫的应答模式提示了:甘蔗不同组织中Sc MT2-1-4对Cu2+胁迫应答的分功不同;Sc MT2-1-4对甘蔗抵御Zn2+胁迫起积极的作用;但该基因不直接参与甘蔗对Cd2+的螯合和解毒的过程。研究结果有助于进一步深入探究MT2基因在甘蔗应答重金属胁迫过程中的作用,为阐明甘蔗富集和耐受重金属的分子机制研究奠定基础。     

拟康氏木霉和微紫青霉纤维二糖水解酶基因Ⅰ(Cbh1)启动子的初步分析

应用 PCR技术 ,分别扩增拟康氏木霉 (T.pseudokoningii S38)和微紫青霉 (P.janthinellumAs3.51 0 ) Cbh1启动子的 2 84bp和 2 1 5bp两个片段 ,并通过凝胶阻滞试验 ,初步分析了在槐糖和葡萄糖两种培养条件下 Cbh1启动子上参与 Cbh1基因表达的调控信息 .凝胶阻滞试验结果表明 ,S38Cbh1启动子片段与葡萄糖和槐糖培养 /诱导条件下的无细胞提取物 ,均形成了蛋白质 - DNA复合物 ,而 As3.51 0 ,只有葡萄糖培养条件下的无细胞提取物与其启动子片段 ,形成了蛋白质 -DNA复合物 .表明在 S38Cbh1启动子的 - 30 2 /- 1 8bp处 ,可能存在与槐糖和葡萄糖介导的调控蛋白相互作用的特定核酸基元 ,而在 As3.51 0 Cbh1启动子上的 - 2 80 /- 65bp处有与葡萄糖介导的阻遏蛋白相互作用的特定核酸基元 .竞争性阻滞试验结果表明 ,S38和 As3.51 0 Cbh1启动子片段与胞内调控蛋白的相互作用是特异性的 .     

拟康氏木霉和微紫青霉纤维二糖水解酶基因Ⅰ(Cbh1)启动子的初步分析

应用 PCR技术 ,分别扩增拟康氏木霉 (T.pseudokoningii S38)和微紫青霉 (P.janthinellumAs3.51 0 ) Cbh1启动子的 2 84bp和 2 1 5bp两个片段 ,并通过凝胶阻滞试验 ,初步分析了在槐糖和葡萄糖两种培养条件下 Cbh1启动子上参与 Cbh1基因表达的调控信息 .凝胶阻滞试验结果表明 ,S38Cbh1启动子片段与葡萄糖和槐糖培养 /诱导条件下的无细胞提取物 ,均形成了蛋白质 - DNA复合物 ,而 As3.51 0 ,只有葡萄糖培养条件下的无细胞提取物与其启动子片段 ,形成了蛋白质 -DNA复合物 .表明在 S38Cbh1启动子的 - 30 2 /- 1 8bp处 ,可能存在与槐糖和葡萄糖介导的调控蛋白相互作用的特定核酸基元 ,而在 As3.51 0 Cbh1启动子上的 - 2 80 /- 65bp处有与葡萄糖介导的阻遏蛋白相互作用的特定核酸基元 .竞争性阻滞试验结果表明 ,S38和 As3.51 0 Cbh1启动子片段与胞内调控蛋白的相互作用是特异性的 .     

拟康氏木霉和微紫青霉纤维二糖水解酶基因I(Cbh1)启动子的初步分析

应用PCR技术,分别扩增拟康氏木霉(T.pseudokoningii S38)和微紫青霉(P.janthinellumAs3.510)Cbh1启动子的284 bp和215 bp两个片段,并通过凝胶阻滞试验,初步分析了在槐糖和葡萄糖两种培养条件下Cbh1启动子上参与Cbh1基因表达的调控信息.凝胶阻滞试验结果表明,S38 Cbh1启动子片段与葡萄糖和槐糖培养/诱导条件下的无细胞提取物,均形成了蛋白质-DNA复合物,而As3.510,只有葡萄糖培养条件下的无细胞提取物与其启动子片段,形成了蛋白质-DNA复合物.表明在S38 Cbh1启动子的一302/一18 bp处,可能存在与槐糖和葡萄糖介导的调控蛋白相互作用的特定核酸基元,而在As3.510 Cbh1启动子上的一280/一65 bp处有与葡萄糖介导的阻遏蛋白相互作用的特定核酸基元.竞争性阻滞试验结果表明,S38和As3.510 Cbh1启动子片段与胞内调控蛋白的相互作用是特异性的.     

24株深海链霉菌耐(嗜)盐性及抗MRSA活性的初步研究

对24深海链霉菌的耐(嗜)盐性及抗MRSA活性筛选,结果显示,耐NaCl盐浓度在10%以上的有23株,中等嗜盐菌2株,16株具有抗MRSA活性,占66.7%.表明深海链霉普遍能耐受10%以上的NaCl浓度,而且大多在高盐浓度下代谢抗MRSA活性物质.     

柽柳金属硫蛋白基因的克隆及序列分析

用木麻黄(Casuarina glauca)的金属硫蛋白基因(metallothionein 1)氨基酸序列对柽柳ESTs序列本地数据库进行tBlastn检索,获得了柽柳金属硫蛋白基因全长cDNA序列,去除polyA后该基因全长366 bp,其中5′非翻译区97 bp,3′非翻译区59 bp,开放读码框(ORF)长210 bp,编码70 个氨基酸组成的多肽,蛋白分子量为6.793 kD,理论等电点为4.99,含10个Cys,集中分布在肽链的N端和C端。BlastP同源性分析表明该基因与花生同源性最高,与小豆同源性最低。该基因的EST序列在GenBank登录(登录号：CV792539)。     

Mutational analysis of the Menkes copper P-type ATPase (ATP7A)

The Menkes protein (ATP7A; MNK) is a ubiquitous human copper-translocating P-type ATPase and it has a key role in regulating copper homeostasis. Previously we characterised fundamental steps in the catalytic cycle of the Menkes protein. In this study we analysed the role of several conserved regions of the Menkes protein, particularly within the putative cytosolic ATP-binding domain. The results of catalytic studies have indicated an important role of 1086His in catalysis. Our findings provide a biochemical explanation for the most common Wilson disease-causing mutation (H1069Q in the homologous Wilson copper-translocating P-type ATPase). Furthermore, we have identified a unique role of 1230Asp, within the DxxK motif, in coupling ATP binding and acylphosphorylation with copper translocation. Finally, we found that the Menkes protein mutants with significantly reduced catalytic activity can still undergo copper-regulated exocytosis, suggesting that only the complete loss of catalytic activity prevents copper-regulated trafficking of the Menkes protein.     

Characterization of a P-type Copper-Stimulated ATPase from Mouse Liver

Mouse liver microsomes treated with octylthioglucoside (OTG-microsomes) were examined for copper-stimulated ATPase activity. The activity was about 1 μmol Pi/mg protein/hr under optimal conditions [300 mm KCl, 3 mm MgSO₄, 10 mm GSH, 0.5 μm CuSO₄, 3 mm ATP and 50 mm acetate buffer at pH5.0]. A reducing agent such as GSH or dithiothreitol was required for the activity, and removal of Cu⁺ from the reaction mixture by bathocuporinedisulfonate resulted in a complete loss of copper-stimulated ATPase activity. Vanadate inhibited the copper-stimulated ATPase activity. The OTG-microsomes were phosphorylated in a hydroxylamine-sensitive and copper-stimulated way. Iron used instead of copper also stimulated both ATPase and phosphorylation. These results suggest that microsomes from mouse liver contain copper/iron-stimulated P-type ATPase. Received: 2 September 1998/Revised: 16 March 1999     

Kinetic characterization of P-type membrane ATPase from Streptococcus mutans

The proton translocating membrane ATPase of oral streptococci has been implicated in cytoplasmatic pH regulation, acidurance and cariogenicity. Studies have confirmed that Streptococcus mutans is the most frequently detected species in dental caries. A P-type ATPase that can act together with F₁F_o-ATPase in S. mutans membrane has been recently described. The main objective of this work is to characterize the kinetic of ATP hydrolysis of this P-type ATPase. The optimum pH for ATP hydrolysis is around 6.0. The dependence of P-type ATPase activity on ATP concentration reveals high (K_0.5=0.27 mM) and low (K_0.5=3.31 mM) affinity sites for ATP, exhibiting positive cooperativity and a specific activity of about 74 U/mg. Equimolar concentrations of ATP and magnesium ions display a behavior similar to that described for ATP concentration in Mg²⁺ saturating condition (high affinity site, K_0.5=0.10 mM, and low affinity site, K_0.5=2.12 mM), exhibiting positive cooperativity and a specific activity of about 68 U/mg. Sodium, potassium, ammonium, calcium and magnesium ions stimulate the enzyme, showing a single saturation curve, all exhibiting positive cooperativities, whereas inhibition of ATPase activity is observed for zinc ions and EDTA. The kinetic characteristics reveal that this ATPase belongs to type IIIA, like the ones found in yeast and plants.     

The K+-translocating KdpFABC complex from Escherichia coli: A P-type ATPase with unique features

The prokaryotic KdpFABC complex from the enterobacterium Escherichia coli represents a unique type of P-type ATPase composed of four different subunits, in which a catalytically active P-type ATPase has evolutionary recruited a potassium channel module in order to facilitate ATP-driven potassium transport into the bacterial cell against steep concentration gradients. This unusual composition entails special features with respect to other P-type ATPases, for example the spatial separation of the sites of ATP hydrolysis and substrate transport on two different polypeptides within this multisubunit enzyme complex, which, in turn, leads to an interesting coupling mechanism. As all other P-type ATPases, also the KdpFABC complex cycles between the so-called E1 and E2 states during catalysis, each of which comprises different structural properties together with different binding affinities for both ATP and the transport substrate. Distinct configurations of this transport cycle have recently been visualized in the working enzyme. All typical features of P-type ATPases are attributed to the KdpB subunit, which also comprises strong structural homologies to other P-type ATPase family members. However, the translocation of the transport substrate, potassium, is mediated by the KdpA subunit, which comprises structural as well as functional homologies to MPM-type potassium channels like KcsA from Streptomyces lividans. Subunit KdpC has long been thought to exhibit an FXYD protein-like function in the regulation of KdpFABC activity. However, our latest results are in favor of the notion that KdpC might act as a catalytical chaperone, which cooperatively interacts with the nucleotide to be hydrolyzed and, thus, increases the rather untypical weak nucleotide binding affinity of the KdpB nucleotide binding domain.     

Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis

The K⁺-translocating KdpFABC complex from Escherichia coli functions as a high affinity potassium uptake system and belongs to the superfamily of P-type ATPases, although it exhibits some unique features. It comprises four subunits, and the sites of ATP hydrolysis and substrate transport are located on two different polypeptides. No structural data are so far available for elucidating the correspondingly unique mechanism of coupling ion transport and catalysis in this P-type ATPase. By use of electron microscopy and single particle analysis of negatively stained, solubilized KdpFABC complexes, we solved the structure of the complex at a resolution of 19 Å, which allowed us to model the arrangement of subunits within the holoenzyme and, thus, to identify the interfaces between subunits. The model showed that the K⁺-translocating KdpA subunit is in close contact with the transmembrane region of the ATP-hydrolyzing subunit KdpB. The cytosolic C-terminal domain of the KdpC subunit, which is assumed to play a role in cooperative ATP binding together with KdpB, is located in close vicinity to the nucleotide binding domain of KdpB. Overall, the arrangement of subunits agrees with biochemical data and the predictions on subunit interactions.     

Solution structure of the N-terminal domain of a potential copper-translocating P-type ATPase from Bacillus subtilis in the apo and Cu(I) loaded states

A putative partner of the already characterized CopZ from Bacillus subtilis was found, both proteins being encoded by genes located in the same operon. This new protein is highly homologous to eukaryotic and prokaryotic P-type ATPases such as CopA, Ccc2 and Menkes proteins. The N-terminal region of this protein contains two soluble domains constituted by amino acid residues 1 to 72 and 73 to 147, respectively, which were expressed both separately and together. In both cases only the 73-147 domain is folded and is stable both in the copper(I)-free and in the copper(I)-bound forms. The folded and unfolded state is monitored through the chemical shift dispersion of 15N-HSQC spectra. In the absence of any structural characterization of CopA-type proteins, we determined the structure of the 73-147 domain in the 1-151 construct in the apo state through 1H, 15N and 13C NMR spectroscopies. The structure of the Cu(I)-loaded 73-147 domain has been also determined in the construct 73-151. About 1300 meaningful NOEs and 90 dihedral angles were used to obtain structures at high resolution both for the Cu(I)-bound and the Cu(I)-free states (backbone RMSD to the mean 0.35(+/-0.06) A and 0.39(+/-0.07) A, respectively). The structural assessment shows that the structures are accurate. The protein has the typical betaalpha(betabeta)alphabeta folding with a cysteine in the C-terminal part of helix alpha1 and the other cysteine in loop 1. The structures are similar to other proteins involved in copper homeostasis. Particularly, between BsCopA and BsCopZ, only the charges located around loop 1 are reversed for BsCopA and BsCopZ, thus suggesting that the two proteins could interact one with the other. The variability in conformation displayed by the N-terminal cysteine of the CXXC motif in a number of structures of copper transporting proteins suggests that this may be the cysteine which binds first to the copper(I) carried by the partner protein.     

Menkes copper-translocating P-type ATPase (ATP7A): biochemical and cell biology properties,and role in Menkes disease

The Menkes copper-translocating P-type ATPase (ATP7A; MNK) is a ubiquitous protein that regulates the absorption of copper in the gastrointestinal tract. Inside cells the protein has a dual function: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper. The latter property is achieved through copper-dependent vesicular trafficking of the Menkes protein to the plasma membrane of the cell. The trafficking mechanism and catalytic activity combine to facilitate absorption and intercellular transport of copper. The mechanism of catalysis and copper-dependent trafficking of the Menkes protein are the subjects of this review. Menkes disease, a systemic copper deficiency disorder, is caused by mutations in the gene encoding the Menkes protein. The effect of these mutations on the catalytic cycle and the cell biology of the Menkes protein, as well as predictions of the effect of particular mutant MNKs on observed Menkes disease symptoms will also be discussed.     

The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification.     

The multi-layered regulation of copper translocating P-type ATPases

The copper-translocating Menkes (ATP7A, MNK protein) and Wilson (ATP7B, WND protein) P-type ATPases are pivotal for copper (Cu) homeostasis, functioning in the biosynthetic incorporation of Cu into copper-dependent enzymes of the secretory pathway, Cu detoxification via Cu efflux, and specialized roles such as systemic Cu absorption (MNK) and Cu excretion (WND). Essential to these functions is their Cu and hormone-responsive distribution between the trans-Golgi network (TGN) and exocytic vesicles located at or proximal to the apical (WND) or basolateral (MNK) cell surface. Intriguingly, MNK and WND Cu-ATPases expressed in the same tissues perform distinct yet complementary roles. While intramolecular differences may specify their distinct roles, cellular signaling components are predicted to be critical for both differences and synergy between these enzymes. This review focuses on these mechanisms, including the cell signaling pathways that influence trafficking and bi-functionality of Cu-ATPases. Phosphorylation events are hypothesized to play a central role in Cu homeostasis, promoting multi-layered regulation and cross-talk between cuproenzymes and Cu-independent mechanisms.     

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex from Escherichia coli (Review)

P-type ATPases are ubiquitously abundant primary ion pumps, which are capable of transporting cations across the cell membrane at the expense of ATP. Since these ions comprise a large variety of vital biochemical functions, nature has developed rather sophisticated transport machineries in all kingdoms of life. Due to the importance of these enzymes, representatives of both eu- and prokaryotic as well as archaeal P-type ATPases have been studied intensively, resulting in detailed structural and functional information on their mode of action. During catalysis, P-type ATPases cycle between the so-called E1 and E2 states, each of which comprising different structural properties together with different binding affinities for both ATP and the transport substrate. Crucial for catalysis is the reversible phosphorylation of a conserved aspartate, which is the main trigger for the conformational changes within the protein. In contrast to the well-studied and closely related eukaryotic P-type ATPases, much less is known about their homologues in Bacteria. Whereas in Eukarya there is predominantly only one subunit, which builds up the transport system, in Bacteria there are multiple polypeptides involved in the formation of the active enzyme. Such a rather unusal prokaryotic P-type ATPase is the KdpFABC complex of the enterobacterium Escherichia coli, which serves as a highly specific K⁺ transporter. A unique feature of this member of P-type ATPases is that catalytic activity and substrate transport are located on two different polypeptides. This review compares generic features of P-type ATPases with the rather unique KdpFABC complex and gives a comprehensive overview of common principles of catalysis as well as of special aspects connected to distinct enzyme functions.