Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4

With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA–DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA–DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA–DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715^T). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4. Received: April 20, 1999 / Accepted: August 31, 1999     

Unremodeled and Remodeled Cardiolipin Are Functionally Indistinguishable in Yeast

After biosynthesis, an evolutionarily conserved acyl chain remodeling process generates a final highly homogeneous and yet tissue-specific molecular form of the mitochondrial lipid cardiolipin. Hence, cardiolipin molecules in different organisms, and even different tissues within the same organism, contain a distinct collection of attached acyl chains. This observation is the basis for the widely accepted paradigm that the acyl chain composition of cardiolipin is matched to the unique mitochondrial demands of a tissue. For this hypothesis to be correct, cardiolipin molecules with different acyl chain compositions should have distinct functional capacities, and cardiolipin that has been remodeled should promote cardiolipin-dependent mitochondrial processes better than its unremodeled form. However, functional disparities between different molecular forms of cardiolipin have never been established. Here, we interrogate this simple but crucial prediction utilizing the best available model to do so, Saccharomyces cerevisiae. Specifically, we compare the ability of unremodeled and remodeled cardiolipin, which differ markedly in their acyl chain composition, to support mitochondrial activities known to require cardiolipin. Surprisingly, defined changes in the acyl chain composition of cardiolipin do not alter either mitochondrial morphology or oxidative phosphorylation. Importantly, preventing cardiolipin remodeling initiation in yeast lacking TAZ1, an ortholog of the causative gene in Barth syndrome, ameliorates mitochondrial dysfunction. Thus, our data do not support the prevailing hypothesis that unremodeled cardiolipin is functionally distinct from remodeled cardiolipin, at least for the functions examined, suggesting alternative physiological roles for this conserved pathway.     

Roles of AtpI and Two YidC-Type Proteins from Alkaliphilic Bacillus pseudofirmus OF4 in ATP Synthase Assembly and Nonfermentative Growth

AtpI, a membrane protein encoded by many bacterial atp operons, is reported to be necessary for c-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of atpI that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic Bacillus pseudofirmus OF4. A strain with a chromosomal deletion of atpI grew nonfermentatively, and its purified ATP synthase had a c-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of atpI, but not atpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F₁ domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both spoIIIJ and yqjG, but not atpI or atpZ, complemented a YidC-depleted Escherichia coli strain. Consistent with such overlapping functions, single deletions of spoIIIJ or yqjG in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of yqjG was elevated at pH 7.5 relative to that at pH 10.5 and in ΔspoIIIJ strains, but it was lower than constitutive spoIIIJ expression. Deletion of atpZ caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.     

Cardiolipin and the osmotic stress responses of bacteria

Cells control their own hydration by accumulating solutes when they are exposed to high osmolality media and releasing solutes in response to osmotic down-shocks. Osmosensory transporters mediate solute accumulation and mechanosensitive channels mediate solute release. Escherichia coli serves as a paradigm for studies of cellular osmoregulation. Growth in media of high salinity alters the phospholipid headgroup and fatty acid compositions of bacterial cytoplasmic membranes, in many cases increasing the ratio of anionic to zwitterionic lipid. In E. coli, the proportion of cardiolipin (CL) increases as the proportion of phosphatidylethanolamine (PE) decreases when osmotic stress is imposed with an electrolyte or a non-electrolyte. Osmotic induction of the gene encoding CL synthase (cls) contributes to these changes. The proportion of phosphatidylglycerol (PG) increases at the expense of PE in cls⁻ bacteria and, in Bacillus subtilis, the genes encoding CL and PG synthases (clsA and pgsA) are both osmotically regulated. CL is concentrated at the poles of diverse bacterial cells. A FlAsH-tagged variant of osmosensory transporter ProP is also concentrated at E. coli cell poles. Polar concentration of ProP is CL-dependent whereas polar concentration of its paralogue LacY, a H⁺-lactose symporter, is not. The proportion of anionic lipids (CL and PG) modulates the function of ProP in vivo and in vitro. These effects suggest that the osmotic induction of CL synthesis and co-localization of ProP with CL at the cell poles adjust the osmolality range over which ProP activity is controlled by placing it in a CL-rich membrane environment. In contrast, a GFP-tagged variant of mechanosensitive channel MscL is not concentrated at the cell poles but anionic lipids bind to a specific site on each subunit of MscL and influence its function in vitro. The sub-cellular locations and lipid dependencies of other osmosensory systems are not known. Varying CL content is a key element of osmotic adaptation by bacteria but much remains to be learned about its roles in the localization and function of osmoregulatory proteins.     

Single Site Mutations in the Hetero-oligomeric Mrp Antiporter from Alkaliphilic Bacillus pseudofirmus OF4 That Affect Na+/H+ Antiport Activity,Sodium Exclusion,Individual Mrp Protein Levels,or Mrp Complex Formation

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA–MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na⁺(Li⁺)/H⁺ antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA–MrpD and MrpE–MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA–MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na⁺]_in. Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na⁺ (Li⁺)/H⁺ antiport.     

Glutaredoxin-2 Is Required to Control Oxidative Phosphorylation in Cardiac Muscle by Mediating Deglutathionylation Reactions

Glutaredoxin-2 (Grx2) modulates the activity of several mitochondrial proteins in cardiac tissue by catalyzing deglutathionylation reactions. However, it remains uncertain whether Grx2 is required to control mitochondrial ATP output in heart. Here, we report that Grx2 plays a vital role modulating mitochondrial energetics and heart physiology by mediating the deglutathionylation of mitochondrial proteins. Deletion of Grx2 (Grx2^−/−) decreased ATP production by complex I-linked substrates to half that in wild type (WT) mitochondria. Decreased respiration was associated with increased complex I glutathionylation diminishing its activity. Tissue glucose uptake was concomitantly increased. Mitochondrial ATP output and complex I activity could be recovered by restoring the redox environment to that favoring the deglutathionylated states of proteins. Grx2^−/− hearts also developed left ventricular hypertrophy and fibrosis, and mice became hypertensive. Mitochondrial energetics from Grx2 heterozygotes (Grx2^+/−) were also dysfunctional, and hearts were hypertrophic. Intriguingly, Grx2^+/− mice were far less hypertensive than Grx2^−/− mice. Thus, Grx2 plays a vital role in modulating mitochondrial metabolism in cardiac muscle, and Grx2 deficiency leads to pathology. As mitochondrial ATP production was restored by the addition of reductants, these findings may be relevant to novel redox-related therapies in cardiac disease.     

Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis

Surfactin is a cyclic lipopeptide antibiotic that disturbs the integrity of the cytoplasmic membrane. In this study, the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus subtilis ATCC 21332 was investigated. During a 1-day cultivation, the phospholipids of the cell membrane were analyzed at the selected time points, which covered both the early and late stationary phases of growth, when surfactin concentration in the medium gradually rose from 2 to 84 μmol·l^− 1. During this time period, the phospholipid composition of the surfactin producer's membrane (Sf⁺) was compared to that of its non-producing mutant (Sf⁻). Substantial modifications of the polar head group region in response to the presence of surfactin were found, while the fatty acid content remained unaffected. Simultaneously with surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was determined in the Sf⁺ membrane, whereas the proportion of phosphatidylethanolamine remained constant. At 24 h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity.     

Phosphorylation of CpgA protein enhances both its GTPase activity and its affinity for ribosome and is crucial for Bacillus subtilis growth and morphology

In Bacillus subtilis, the ribosome-associated GTPase CpgA is crucial for growth and proper morphology and was shown to be phosphorylated in vitro by the Ser/Thr protein kinase PrkC. To further understand the function of the Escherichia coli RsgA ortholog, CpgA, we first demonstrated that its GTPase activity is stimulated by its association with the 30 S ribosomal subunit. Then the role of CpgA phosphorylation was analyzed. A single phosphorylated residue, threonine 166, was identified by mass spectrometry. Phosphoablative replacement of this residue in CpgA induces a decrease of both its affinity for the 30 S ribosomal subunit and its GTPase activity, whereas a phosphomimetic replacement has opposite effects. Furthermore, cells expressing a nonphosphorylatable CpgA protein present the morphological and growth defects similar to those of a cpgA-deleted strain. Altogether, our results suggest that CpgA phosphorylation on Thr-166 could modulate its ribosome-induced GTPase activity. Given the role of PrkC in B. subtilis spore germination, we propose that CpgA phosphorylation is a key regulatory process that is essential for B. subtilis development.     

Evidence for a close genealogical relationship between Afipia (the causal organism of cat scratch disease), Bradyrhizobium japonicum and Blastobacter denitrificans

The primary structures of the small subunit rRNA of Bradyrhizobium japonicum and Blastobacter denitrificans were determined by direct sequencing of enzymatically amplified DNA. Comparative sequence analysis revealed that Bradyrhizobium japonicum and Blastobacter denitrificans are members of the alpha-2 subgroup of the Proteobacteria and show a very close phylogenetic relationship with the genus Afipia.     

Phosphatidylethanolamine Synthesis in the Parasite Mitochondrion Is Required for Efficient Growth but Dispensable for Survival of Toxoplasma gondii

Toxoplasma gondii is a highly prevalent obligate intracellular parasite of the phylum Apicomplexa, which also includes other parasites of clinical and/or veterinary importance, such as Plasmodium, Cryptosporidium, and Eimeria. Acute infection by Toxoplasma is hallmarked by rapid proliferation in its host cells and requires a significant synthesis of parasite membranes. Phosphatidylethanolamine (PtdEtn) is the second major phospholipid class in T. gondii. Here, we reveal that PtdEtn is produced in the parasite mitochondrion and parasitophorous vacuole by decarboxylation of phosphatidylserine (PtdSer) and in the endoplasmic reticulum by fusion of CDP-ethanolamine and diacylglycerol. PtdEtn in the mitochondrion is synthesized by a phosphatidylserine decarboxylase (TgPSD1mt) of the type I class. TgPSD1mt harbors a targeting peptide at its N terminus that is required for the mitochondrial localization but not for the catalytic activity. Ablation of TgPSD1mt expression caused up to 45% growth impairment in the parasite mutant. The PtdEtn content of the mutant was unaffected, however, suggesting the presence of compensatory mechanisms. Indeed, metabolic labeling revealed an increased usage of ethanolamine for PtdEtn synthesis by the mutant. Likewise, depletion of nutrients exacerbated the growth defect (∼56%), which was partially restored by ethanolamine. Besides, the survival and residual growth of the TgPSD1mt mutant in the nutrient-depleted medium also indicated additional routes of PtdEtn biogenesis, such as acquisition of host-derived lipid. Collectively, the work demonstrates a metabolic cooperativity between the parasite organelles, which ensures a sustained lipid synthesis, survival and growth of T. gondii in varying nutritional milieus.     

嗜碱芽孢杆菌Bacillus sp.F26过氧化氢酶的分离纯化及性质研究

从一株低度嗜盐、兼性嗜碱芽孢杆菌Bacillus sp．F26中纯化得到一种碱性过氧化氢酶,并对该酶进行了性质研究。纯化过程经硫酸铵沉淀、阴离子交换层析、凝胶过滤层析及疏水层析四步最终获得电泳纯的目标酶(纯化58．5倍)。该过氧化氢酶的分子量为140kD,由两个大小相同的亚基组成。天然酶分子在408nm处显示特征吸收峰(Soret band)。吡啶血色素光谱显示了酶分子以原卟啉Ⅸ(protoheme Ⅸ)作为辅基。计算获得酶的表观米氏常数为32．5mmol／L。该过氧化氢酶不受连二亚硫酸钠的还原作用影响,但被氰化物、叠氮化物和3．氨基．1,2,4-三唑(单功能过氧化氢酶的专一抑制剂)强烈抑制。以邻联茴香胺、邻苯二胺和二氨基联苯胺作为电子供体测定酶活时,该酶不显示过氧化物酶活性。同时,酶的N-端序列比对结果说明,该过氧化氢酶与单功能过氧化氢酶亚群有一定的相似性,而与双功能过氧化氢酶亚群及猛过氧化氢酶亚群均没有同源性。因此,本文将纯化的碱性过氧化氢酶定性为单功能过氧化氢酶。此外,该酶具有热敏感的特点,且酶活在pH5～9的范围内不受pH影响,此后,活性随着pH的升高而升高,并在pH 11处有明显的酶活高峰。20℃、pH 11条件下的酶活半衰期达49h。在pH 11的高碱条件下表现出最高活力和一定的稳定性,这在已报道的过氧化氢酶中还未见描述。同时,该酶也显示了良好的盐碱稳定性,0．5mol／L NaCl、pH 10．5条件下的酶活半衰期达90h。另一方面,本文所研究的过氧化氢酶是第一个来源于嗜碱微生物的同源二聚体单功能过氧化氢酶,也是第一个来源于天然碱湖的单功能过氧化氢酶,它能部分地反映出细胞抗氧化体系对相应环境的适应情况。     

假坚强芽孢杆菌四氢嘧啶羟化酶的晶体制备及X-射线衍射研究

摘要:【目的】假坚强芽孢杆菌四氢嘧啶羟化酶蛋白纯化、晶体制备及X-射线衍射研究。【方法】通过PCR从假坚强芽孢杆菌OF4中克隆获得四氢嘧啶羟化酶基因,构建原核表达载体,经过原核表达,采用Ni-NTA亲和层析法和分子排阻色谱法纯化蛋白,289 K下采用座滴法进行晶体筛选和制备,在低温100 K下通过X-射线衍射仪(Rigaku MicroMax-007 HF)收集晶体衍射数据。【结果】通过原核表达及纯化成功获得了适合晶体生长的蛋白BpEctD。通过筛选最终在蛋白浓度为6.5 mg/mL及含有0.2 mol/L MgCl2·6H2O,0.1 mol/L Bis-Tris pH6.5,25% (W/V) 聚乙二醇3,350的缓冲液中获得了理想的蛋白晶体,其大小约为360 μm×240μm×60 μm,并在100K下成功收集了衍射数据,晶体衍射分辨率为2.40,空间群为三斜晶系P1,晶胞参数为a=45.18,b=58.87,c=68.81,α=77.48°,β=86.03°,γ=66.97°,每个不对称单位中含有2 个BpEctD单体,马修斯系数为2.44 3/Da,溶剂含量约为49.53%。【结论】衍射数据的成功收集为假坚强芽孢杆菌OF4四氢嘧啶羟化酶三维结构的解析奠定了前期基础,将有助于阐明四氢嘧啶羟化酶的催化机制。     

Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis

Cytochrome c (cyt c) release upon oxidation of cardiolipin (CL) in the mitochondrial inner membrane (IM) under oxidative stress occurs early in the intrinsic apoptotic pathway. We postulated that CL oxidation mobilizes not only cyt c but also CL itself in the form of hydroperoxide (CLOOH) species. Relatively hydrophilic CLOOHs could assist in apoptotic signaling by translocating to the outer membrane (OM), thus promoting recruitment of the pro-apoptotic proteins truncated Bid (tBid) and Bax for generation of cyt c-traversable pores. Initial testing of these possibilities showed that CLOOH-containing liposomes were permeabilized more readily by tBid plus Ca(2+) than CL-containing counterparts. Moreover, CLOOH translocated more rapidly from IM-mimetic to OM-mimetic liposomes than CL and permitted more extensive OM permeabilization. We found that tBid bound more avidly to CLOOH-containing membranes than to CL counterparts, and binding increased with increasing CLOOH content. Permeabilization of CLOOH-containing liposomes in the presence of tBid could be triggered by monomeric Bax, consistent with tBid/Bax cooperation in pore formation. Using CL-null mitochondria from a yeast mutant, we found that tBid binding and cyt c release were dramatically enhanced by transfer acquisition of CLOOH. Additionally, we observed a pre-apoptotic IM-to-OM transfer of oxidized CL in cardiomyocytes treated with the Complex III blocker, antimycin A. These findings provide new mechanistic insights into the role of CL oxidation in the intrinsic pathway of oxidative apoptosis.     

PrkC-mediated Phosphorylation of Overexpressed YvcK Protein Regulates PBP1 Protein Localization in Bacillus subtilis mreB Mutant Cells

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state.     

Chloride-Dependent Uncoupling of Oxidative Phosphorylation by Triethyllead and Triethyltin Increases Cytosolic Free Calcium in Guinea Pig Cerebral Cortical Synaptosomes

Metabolically competent isolated cerebral cortical nerve terminals were used to determine the effects of triethyllead (TEL) and triethyltin (TET) on cytosolic free calcium ([Ca2+]c), on plasma and mitochondrial membrane potentials, and on oxidative metabolism. In the presence of physiological concentrations of extracellular ions, 20 microM TEL and 20 microM TET increase [Ca2+]c from 185 nM to 390 and 340 nM, respectively. A simultaneous depolarization of plasma membrane potential (delta psi p) by only 3-4 mV occurs, a drop which is insufficient to open the voltage-sensitive Ca2+ channels. In contrast, an instant and substantial depolarization of mitochondrial membrane potential (delta psi m) upon addition of TEL and TET is evident, as monitored with safranine O fluorescence. At the same concentration, TEL and TET stimulate basal respiration of synaptosomes by 45%, induce oxidation of endogenous NAD(P)H, and reduce the terminal ATP/ADP ratio by 45%. Thus, TEL and TET inhibit ATP production of intrasynaptosomal mitochondria by a mechanism consistent with uncoupling of oxidative phosphorylation. This bioenergetic effect by TEL and TET can be prevented by omitting external chloride, and a concomitant reduction of the increase in [Ca2+]c by about 60% is observed. Uncoupling of mitochondrial ATP synthesis from oxidation by TEL and TET, [corrected] a process that is dependent on external chloride, is the main mechanism by which they [corrected] increase [Ca2+]c.     

Binding and Release of Cytochrome c in Brain Mitochondria Is Influenced by Membrane Potential and Hydrophobic Interactions with Cardiolipin

Factors influencing the release and anchorage of cytochrome c to the inner membrane of brain mitochondria have been investigated. Metabolic activity of mitochondria caused a decrease in the membrane potential Δψ_m, accompanied by detachment of the protein from the inner membrane. In a model system of cytochrome c reconstituted in cardiolipin (CL) liposomes, phosphate was used to breach the hydrophilic lipid-protein interactions. About 44% cytochrome c was removable when heart CL (80% 18:2n-6) was employed, whereas the remaining protein accounted for the tightly bound conformation characterized by hydrophobic lipid-protein interactions. Cytochrome c release from brain CL liposomes was higher compared to heart CL, consistent with lower polyunsaturated fatty acid content. The release was even higher with CL extracted from metabolically stressed mitochondria, exhibiting more saturated fatty acid profile compared to control (30% vs.17%). Therefore, weakening of the hydrophobic interactions due to saturation of CL may account for the observed cytochrome c release from mitochondria following metabolic stress. Moreover, mitochondria enriched with polyunsaturated CL exhibited higher Δψ_m, compared to less unsaturated species, suggesting that CL fatty acid composition influences Δψ_m. Mitochondria incorporated exogenous cytochrome c without protease-sensitive factors or Δψ_m. The internalized protein anchored to the inner membrane without producing swelling, as monitored by forward and side light scattering, but produced Δψ_m consumption, suggesting recovery of respiratory activity. The Δψ_m decrease is ascribed to a selected mitochondrial population containing the incorporated cytochrome c.This revised version was published online in August 2005 with a corrected cover date.     

Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.     

假坚强芽胞杆菌中乙醇降解相关酶的克隆、表达及酶学特性

【目的】研究假坚强芽胞杆菌OF4中乙醇脱氢酶和乙醛脱氢酶的酶学特性。【方法】通过引物设计,采用PCR技术从嗜碱芽胞杆菌OF4的基因组DNA中扩增获得乙醇脱氢酶(adh)基因和乙醛脱氢酶(aldh)基因,构建表达载体,通过异源原核表达,Ni-NTA柱层析纯化酶蛋白,分析其酶学特性。【结果】乙醛脱氢酶的最适反应温度为35℃,最适反应pH值为8.0,酶蛋白的活力为979.6 U/mg,其稳定性在25℃和35℃下比45℃稍好;尽管由于乙醇脱氢酶的表达量低而未能纯化获得酶蛋白,但通过双基因共表达及乙醇耐受性实验发现乙醇脱氢酶也具备较高的催化活性。【结论】成功地从假坚强芽胞杆菌OF4中克隆获得了乙醇脱氢酶和乙醛脱氢酶基因,二者共同作用能够较大提高宿主对乙醇的耐受性。     

Affinity of phosphatidylglycerophosphate synthetase for the liponucleotides in Bacillus subtilis

Abstract In Bacillus subtilis , the synthesis of phosphatidylglycerol, the more reactive phospholipid, was investigated in vitro. The phosphatidylglycerophosphate synthetase was exclusively localized in the membrane fraction. Three phospholipids (phosphatidylglycerophosphate, phosphatidylglycerol and diphosphatidylglycerol) were synthesized by this fraction. Exogenous CDP-diglyceride and dCDP-diglyceride were substrates with the same K _m (0.11 mM). In coupled system with CDP-diglyceride synthetase, endogenous dCDP-diglyceride was a less effective substrate than CDP-diglyceride.