Mercury resistance and accumulation in Escherichia coli with cell surface expression of fish metallothionein

Recombinant tilapia (Oreochromis mossambicus) fish metallothionein (MT) was used as a surface biosorbent for mercury removal in Escherichia coli. Fish MT conferred better resistance than did mouse or human MT. When tilapia MT (tMT) was fused with an outer-membrane protein, outer membrane protein C (OmpC), the membrane-targeted fusion protein, OmpC–tMT, gave enhanced resistance compared with cytoplasmic tMT expressed in the same host cell. The cytoplasmically expressed tMT showed high mercury adsorption (4.3 ± 0.4 mg/g cell dry weight). The cell surface that expressed E. coli showed about 25% higher adsorption ability (5.6 ± 0.4 mg/g) than the cells expressing cytoplasmic MT, attaining almost twice the level of adsorption of the control plasmid (3.0 ± 0.4 mg/g). As MTs are also known for their ability to scavenge hydroxyl-free radicals, it was also shown that tMT exhibited better radical-scavenging activities than glutathione. These results suggest that fish MT has potential for the development of a bioremediation system for mercury removal that protects the harboring E. coli host by free-radical scavenging.     

Cytoplasmic linker protein-170 enhances spreading and phagocytosis in activated macrophages by stabilizing microtubules

Activation of macrophages causes increased cell spreading, increased secretion of cytokines and matrix metalloproteinases, and enhanced phagocytosis. The intracellular mechanisms driving the up-regulation of these activities have not been completely clarified. We observe that classical activation of murine resident peritoneal or RAW 264.7 macrophages with a combination of IFN-gamma and LPS induces an increase in stabilized cytoplasmic microtubules (MTs), measured with an anti-acetylated alpha-tubulin Ab. We examined the mechanism of this MT stabilization and find that macrophage activation causes redistribution of the MT plus-end tracking protein, cytoplasmic linker protein-170 (CLIP-170). CLIP-170 is localized at the distal plus-ends of MTs in resting macrophages, but accumulates along the length of MTs in IFN-gamma/LPS-activated cells. A direct involvement of CLIP-170 in MT stabilization has not been thoroughly established. In this study, we show that expression of a mutant CLIP-170 chimeric protein (dominant-negative CLIP-170-GFP), lacking the MT-binding domain, prevents MT stabilization in activated RAW 264.7 macrophages. Furthermore, we find enhanced CLIP-170 association with MTs and MT stabilization by treating resting macrophages with okadaic acid, implicating the protein phosphatase 2A in CLIP-170 binding and MT stabilization in RAW 264.7 cells. Finally, we observed enhanced cell spreading and phagocytosis in both IFN-gamma/LPS-activated and okadaic acid-treated resting RAW 264.7 cells, which are markedly reduced in activated cells expressing dominant-negative CLIP-170-GFP. These results identify CLIP-170 as a key regulator of MT stabilization and establish a prominent role for stabilized MTs in cell spreading and phagocytosis in activated macrophages.     

Functional homologs of fungal metallothionein genes from Arabidopsis.

Metallothioneins (MTs) are cysteine-rich proteins required for heavy metal tolerance in animals and fungi. Two cDNAs encoding proteins with homology to animal and fungal MTs have been isolated from Arabidopsis. The genes represented by these cDNAs are referred to as MT1 and MT2. When expressed in an MT-deficient (cup1 delta) mutant of yeast, both MT1 and MT2 complemented the cup1 delta mutation, providing a high level of resistance to CuSO4 and moderate resistance to CdSO4. Although the MT-deficient yeast was not viable in the presence of either 300 microM CuSO4 or 5 microM CdSO4, cells expressing MT1 were able to grow in medium supplemented with 3 mM CuSO4 and 10 microM CdSO4, and those expressing MT2 grew in the presence of 3 mM CuSO4 and 100 microM CdSO4. In plants, MT1 mRNA was more abundant in roots and dark-grown seedlings than in leaves. In contrast, MT2 mRNA accumulated more in leaves than in either roots or darkgrown seedlings. MT2 mRNA was strongly induced in seedlings by CuSO4, but only slightly by CdSO4 or ZnSO4. However, MT1 mRNA was induced by CuSO4 in excised leaves that were submerged in medium. These results indicated that Arabidopsis MT genes are involved in copper tolerance. Plants also synthesized metal binding phytochelatins (poly[gamma-glutamylcysteine]glycine) when exposed to heavy metals. The results presented here argue against the hypothesis that phytochelatins are the sole molecules involved in heavy metal tolerance in plants. We conclude that Arabidopsis MT1 and MT2 are functional homologs of yeast MT.     

Tobacco microtubule-associated protein,MAP65-1c,bundles and stabilizes microtubules

Three genes that encode MAP65-1 family proteins have been identified in the Nicotiana tabacum genome. In this study, NtMAP65-1c fusion protein was shown to bind and bundle microtubules (MTs). Further in vitro investigations demonstrated that NtMAP65-1c not only alters MT assembly and nucleation, but also exhibits high MT stabilizing activity against cold or katanin-induced destabilization. Analysis of NtMAP65-1c-GFP expressing BY-2 cells clearly demonstrated that NtMAP65-1c was able to bind to MTs during specific stages of the cell cycle. Furthermore, in vivo, NtMAP65-1c-GFP-bound cortical MTs displayed an increase in resistance against the MT-disrupting drug, propyzamide, as well as against cold temperatures. Taken together, these results strongly suggest that NtMAP65-1c stabilizes MTs and is involved in the regulation of MT organization and cellular dynamics.     

Isolation of Zn-responsive genes from two accessions of the hyperaccumulator plant <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis>

Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.     

丙酮丁醇梭菌(Clostridium acetobutylicum)原生质体融合育种研究

为了提高丙酮丁醇梭菌(Clostridium acetobutylicum)的丁醇耐受能力和培养基总糖产丁醇的转化率,通过原生质体融合的方法,研究了溶菌酶浓度及其作用时间、再生培养基种类、55℃条件下菌体致死时间、不同PEG分子量以及作用时间、Ca^2+和Mg^2+不同的添加量对丙酮丁醇梭菌原生质体制备、融合、再生的影响,得到了一套比较系统的丙酮丁醇梭菌的原生质体融合条件,同时通过气相色谱检测了融合菌的产溶剂能力并计算总糖转化率。结果显示,最终得到的215I菌株的总糖转化率比原始菌株提高了34.7%,产丁醇能力比原始菌株提高了32.2%,并且发现1株融合菌能产生新物质。原生质体融合方法在丙酸丁醇梭菌育种方面有广泛的应用潜力,通过融合得到的菌株为丁醇生产奠定了基础。     

Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase

The central spindle forms between segregating chromosomes during anaphase and is required for cytokinesis. Although anaphase-specific bundling and stabilization of interpolar microtubules (MTs) contribute to formation of the central spindle, it remains largely unknown how these MTs are prepared. Using live imaging of MT plus ends and an MT depolymerization and regrowth assay, we show that de novo MT generation in the interchromosomal region during anaphase is important for central spindle formation in human cells. Generation of interchromosomal MTs and subsequent formation of the central spindle occur independently of preanaphase MTs or centrosomal MT nucleation but require augmin, a protein complex implicated in nucleation of noncentrosomal MTs during preanaphase. MTs generated in a hepatoma up-regulated protein (HURP)-dependent manner during anaphase also contribute to central spindle formation redundantly with preanaphase MTs. Based on these results, a new model for central spindle assembly is proposed.     

Centrosome fragments and microtubules are transported asymmetrically away from division plane in anaphase

Spinning disc confocal microscopy of LLCPK1 cells expressing GFP-tubulin was used to demonstrate that microtubules (MTs) rapidly elongate to the cell cortex after anaphase onset. Concurrently, individual MTs are released from the centrosome and the centrosome fragments into clusters of MTs. Using cells expressing photoactivatable GFP-tubulin to mark centrosomal MT minus ends, a sevenfold increase in MT release in anaphase is documented as compared with metaphase. Transport of both individually released MTs and clusters of MTs is directionally biased: motion is directed away from the equatorial region. Clusters of MTs retain centrosomal components at their focus and the capacity to nucleate MTs. Injection of mRNA encoding nondegradable cyclin B blocked centrosome fragmentation and the stimulation of MT release in anaphase despite allowing anaphase-like chromosome segregation. Biased MT release may provide a mechanism for MT-dependent positioning of components necessary for specifying the site of contractile ring formation.     

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.     

融和蛋白GST-SUMO-MT在大肠杆菌中的表达、纯化及其活性研究

将编码融合蛋白GST-SUMO-MT的DNA片段连接到大肠杆菌表达载体pET-28a中,构建重组表达质粒pET-GS-MT并转化到大肠杆菌Origami（DE3）中。20℃,1mM的IPTG诱导20h后,获得分子量约为43Kd的融合蛋白,表达量占菌体上清总蛋白的38.4％。利用谷胱甘肽交联琼脂糖（Glutathione Sepharose 4B）凝胶柱和Sephardex G-25分子筛联用可以得到纯度为95%以上的融合蛋白,得率约为70mg/L。该融合蛋白可与GST抗体产生阳性反应。融合蛋白GST-SUMO-MT可以显著提高宿主对Cd2+、Zn2+和Cu2+离子聚积的能力,其耐受能力比对照组分别提高4.2倍、4倍及1.6倍。此外,原子吸收光谱法测定,每分子GST-SUMO-MT可以结合2-3个Cd2+离子。     

Zn tolerance of novel Colocasia esculenta metallothionein and its domains in Escherichia coli and tobacco

Contrary to extensive researches on the roles of metallothioneins (MTs) in metal tolerance of animals, the roles of plant MTs in metal tolerance are largely under investigation. In this study, we evaluated the functional role of type 2 MT from Colocasia esculenta (CeMT2b) in Zn tolerance of tobacco and E. coli cells. Under Zn-stress conditions, transgenic tobacco overexpressing CeMT2b displayed much better seedling growth, a significant decrease in the levels of H₂O₂ and an increase in Zn accumulation compared with the wild type. Overexpression of CeMT2b in E. coli greatly enhanced Zn tolerance and Zn accumulation under Zn stresses compared with control cells. CeMT2b bound 5.38?±?0.29 atoms of Zn per protein. To identify a structural domain of CeMT2b for Zn binding, we investigated the growth of E. coli expressing each of the N-terminal, C-terminal, and central linker domains or a CNC motif deletion from the C-terminus of full-length CeMT2b. The results showed that the CNC motif is required for Zn tolerance, and the N-terminal domain is more effective in Zn tolerance than the C-terminal domain. Taken together, our results provide direct evidence for functional contributions of CeMT2b in Zn tolerance of tobacco and E. coli cells.     

Removal of MAP4 from microtubules in vivo produces no observable phenotype at the cellular level

Microtubule-associated protein 4 (MAP4) promotes MT assembly in vitro and is localized along MTs in vivo. These results and the fact that MAP4 is the major MAP in nonneuronal cells suggest that MAP4's normal functions may include the stabilization of MTs in situ. To understand MAP4 function in vivo, we produced a blocking antibody (Ab) to prevent MAP4 binding to MTs. The COOH-terminal MT binding domain of MAP4 was expressed in Escherichia coli as a glutathione transferase fusion protein and was injected into rabbits to produce an antiserum that was then affinity purified and shown to be monospecific for MAP4. This Ab blocked > 95% of MAP4 binding to MTs in an in vitro assay. Microinjection of the affinity purified Ab into human fibroblasts and monkey epithelial cells abolished MAP4 binding to MTs as assayed with a rat polyclonal antibody against the NH2-terminal projection domain of MAP4. The removal of MAP4 from MTs was accompanied by its sequestration into visible MAP4-Ab immunocomplexes. However, the MT network appeared normal. Tubulin photoactivation and nocodazole sensitivity assays indicated that MT dynamics were not altered detectably by the removal of MAP4 from the MTs. Cells progressed to mitosis with morphologically normal spindles in the absence of MAP4 binding to MTs. Depleting MAP4 from MTs also did not affect the state of posttranslational modifications of tubulin subunits. Further, no perturbations of MT- dependent organelle distribution were detected. We conclude that the association of MAP4 with MTs is not essential for MT assembly or for the MT-based functions in cultured cells that we could assay. A significant role for MAP4 is not excluded by these results, however, as MAP4 may be a component of a functionally redundant system.     

Screening and characterization of butanol‐tolerant micro‐organisms

Aims: Poor butanol tolerance of solventogenic stains directly limits their butanol production during industrial‐scale fermentation process. This study was performed to search for micro‐organisms possessing elevated tolerance to butanol. Methods and Results: Two strains, which displayed higher butanol tolerance compared to commonly used solventogenic Clostridium acetobutylicum, were isolated by evolution and screening strategies. Both strains were identified as lactic acid bacteria (LAB). On this basis, a LAB culture collection was tested for butanol tolerance, and 60% of the strains could grow at a butanol concentration of 2·5% (v/v). In addition, an isolated strain with superior butanol tolerance was transformed using a certain plasmid. Conclusions: The results indicate that many strains of LAB possessed inherent tolerance of butanol. Significance and Impact of the Study: This study suggests that LAB strains may be capable of producing butanol to elevated levels following suitable genetic manipulation.     

Phospholipase D signaling regulates microtubule organization in the fucoid alga Silvetia compressa

Recent studies in higher plants or animals have shown that phospholipase D (PLD) signaling regulates many aspects of development, including organization of microtubules (MTs), actin and the endomembrane system. PLD hydrolyzes structural phospholipids to form the second messenger phosphatidic acid (PA). To begin to understand the signaling pathways and molecules that regulate cytoskeletal and endomembrane arrays during early development in the brown alga, Silvetia compressa, we altered PLD activity by applying butyl alcohols to zygotes. 1-Butanol activates PLD and is a preferred substrate, primarily forming phosphatidyl butanol (P-butanol), which is not a signaling molecule. Treatment with 1-butanol inhibited cell division and cytokinesis but not photopolarization or germination, suggesting an MT-based effect. Immunolabeling revealed that 1-butanol treatment rapidly disrupted MT arrays and caused zygotes to arrest in metaphase. MT arrays recovered rapidly following butanol washout, but subsequent development depended on the timing of the treatment regime. Additionally, treatment with 1-butanol early in development disrupted endomembrane organization, known to require functional MTs. Interestingly, treatment with higher concentrations of 2-butanol, which also activates PLD, mimicked the effects of 1-butanol. In contrast, the control t-butanol had no effect on MTs or development. These results indicate that S. compressa zygotes utilize PLD signaling to regulate MT arrays. In contrast, PLD signaling does not appear to regulate actin arrays or endomembrane trafficking directly. This is the first report describing the signaling pathways that regulate cytoskeletal organization in the stramenopile (heterokont) lineage.     

Microtubule-associated proteins in higher plants

A variety of microtubule-associated proteins (MAPs) have been reported in higher plants. Microtubule (MT) polymerization starts from the γ-tubulin complex (γTuC), a component of the MT nucleation site. MAP200/MOR1 and katanin regulate the length of the MT by promoting the dynamic instability of MTs and cutting MTs, respectively. In construction of different MT structures, MTs are bundled or are associated with other components—actin filaments, the plasma membrane, and organelles. The MAP65 family and some of kinesin family are important in bundling MTs. MT plus-end-tracking proteins (+TIPs) including end-binding protein 1 (EB1), Arabidopsis thaliana kinesin 5 (ATK5), and SPIRAL 1 (SPR1) localize to the plus end of MTs. It has been suggested that +TIPs are involved in binding of MT to other structures. Phospholipase D (PLD) is a possible candidate responsible for binding of MTs to the plasma membrane. Many candidates have been reported as actin-binding MAPs, for example calponin-homology domain (KCH) family kinesin, kinesin-like calmodulin-binding protein (KCBP), and MAP190. RNA distribution and translation depends on MT structures, and several RNA-related MAPs have been reported. This article gives an overview of predicted roles of these MAPs in higher plants.     

Rapid assembly and collective behavior of microtubule bundles in the presence of polyamines

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-β tubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.     

Low cell surface hydrophobicity is one of the key factors for high butanol tolerance of Lactic acid bacteria

Highly butanol‐tolerant strains have always been attractive because of their potential as microbial hosts for butanol production. However, due to the amphiphilic nature of 1‐butanol as a solvent, the relationship between the cell surface hydrophobicity and butanol resistance remained ambiguous to date. In this work, the quantitatively estimated cell surface hydrophobicity of 74 Lactic acid bacteria strains were juxtaposed to their tolerance to various butanol concentrations. The obtained results revealed that the strains’ hydrophobicity was inversely proportional to their butanol tolerance. All highly butanol‐resistant strains were hydrophilic (cell surface hydrophobicity<1%), whereas the more hydrophobic the strains were, the more sensitive to butanol they were. Furthermore, cultivation at increasing butanol concentrations showed a clear tendency to decrease the level of hydrophobicity in all tested organisms, thus suggesting possible adaptation mechanisms. Purposeful reduction of cell surface hydrophobicity (by removal of S‐layer proteins from the cell envelope) also led to an increase of butanol resistance. Since the results covered 23 different Lactic acid bacteria species of seven genera, it could be concluded that regardless of the species, the lower degree of cells’ hydrophobicity clearly correlates with the higher level of butanol tolerance.     

Cytoplasmic dynein is involved in the retention of microtubules at the centrosome in interphase cells

Cytoplasmic dynein is known to be involved in the establishment of radial microtubule (MT) arrays. During mitosis, dynein activity is required for tethering of the MTs at the spindle poles. In interphase cells, dynein inhibitors induce loss of radial MT organization; however, the exact role of dynein in the maintenance of MT arrays is unclear. Here, we examined the effect of dynein inhibitors on MT distribution and the centrosome protein composition in cultured fibroblasts. We found that while these inhibitors induced rapid ( t _1/2 ∼ 20 min) loss of radial MT organization, the levels of key centrosomal proteins or the rates of MT nucleation did not change significantly in dynein-inhibited cells, suggesting that the loss of dynein activity does not affect the structural integrity of the centrosome or its capacity to nucleate MTs. Live observations of the centrosomal activity showed that dynein inhibition enhanced the detachment of MTs from the centrosome. We conclude that the primary role of dynein in the maintenance of a radial MT array in interphase cells consists of retention of MTs at the centrosome and hypothesize that dynein has a role in the MT retention, separate from the delivery to the centrosome of MT-anchoring proteins.     

Centromere protein F includes two sites that couple efficiently to depolymerizing microtubules

Firm attachments between kinetochores and dynamic spindle microtubules (MTs) are important for accurate chromosome segregation. Centromere protein F (CENP-F) has been shown to include two MT-binding domains, so it may participate in this key mitotic process. Here, we show that the N-terminal MT-binding domain of CENP-F prefers curled oligomers of tubulin relative to MT walls by approximately fivefold, suggesting that it may contribute to the firm bonds between kinetochores and the flared plus ends of dynamic MTs. A polypeptide from CENP-F’s C terminus also bound MTs, and either protein fragment diffused on a stable MT wall. They also followed the ends of dynamic MTs as they shortened. When either fragment was coupled to a microbead, the force it could transduce from a shortening MT averaged 3–5 pN but could exceed 10 pN, identifying CENP-F as a highly effective coupler to shortening MTs.