In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.     

Evidence for an intermediate methyl-acceptor for chemotaxis in Bacillus subtilis

Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.     

Identification and purification of the aspartate/glutamate carrier from bovine heart mitochondria.

The aspartate/glutamate carrier from bovine heart mitochondria was solubilized with dodecyl-octaoxyethylene ether (C12E8) and purified by chromatography on hydroxyapatite and celite. On SDS gel electrophoresis, the purified aspartate/glutamate carrier consisted of a single protein band with an apparent Mr of 31,500. When reconstituted into liposomes the aspartate/glutamate carrier protein catalyzed an N-ethylmaleimide-sensitive aspartate/aspartate exchange. It was purified 620-fold with a recovery of 17.2% and a protein yield of 0.03% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e. requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the aspartate/glutamate carrier as characterized in mitochondria.     

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.     

Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli

In bacterial chemotaxis, adaptation is correlated with methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs). Each protein migrates as a characteristic set of multiple bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. The changes in MCP methylation that accompany adaptation are not the same for all bands of a set. Adaptation to a type II repellent stimulus results in an overall decrease in MCP II methylation, but also in an increase in the amount of radioactive methyl groups in the upper band of the set. We demonstrate that this increase is not due to new methylation, but rather to reduced electrophoretic mobility of previously methylated molecules that have lost some but not all of their methyl groups. We suggest that the pattern of multiple bands is a direct reflection of multiple sites for methylation on MCP molecules, and that the distribution of radiolabel among the bands is determined by the total extent of methylation. The patterns of methylated peptides produced by limited proteolysis of different MCP bands imply that methylation of the multiple sites on a molecule may occur in a specific order.     

Methylthiol:coenzyme M methyltransferase from Methanosarcina barkeri, an enzyme of methanogenesis from dimethylsulfide and methylmercaptopropionate.

During growth on acetate, Methanosarcina barkeri expresses catabolic enzymes for other methanogenic substrates such as monomethylamine. The range of substrates used by cells grown on acetate was further explored, and it was found that cells grown on acetate also converted dimethylsulfide (DMS) and methylmercaptopropionate (MMPA) to methane. Cells or extracts of cells grown on trimethylamine or methanol did not utilize either DMS or MMPA. During growth on acetate, cultures demethylated MMPA, producing methane and mercaptopropionate. Extracts of acetate-grown cells possessed DMS- and MMPA-dependent coenzyme M (CoM) methylation activities. The activity peaks of CoM methylation with either DMS or MMPA coeluted upon gel permeation chromatography of extracts of acetate-grown cells consistent with an apparent molecular mass of 470 kDa. A 480-kDa corrinoid protein, previously demonstrated to be a CoM methylase but otherwise of unknown physiological function, was found to methylate CoM with either DMS or MMPA. MMPA was demethylated by the purified 480-kDa CoM methylase, consuming 1 mol of CoM and producing 1 mol of mercaptopropionate. DMS was demethylated by the purified protein, consuming 1 mol of CoM and producing 1 mol of methanethiol. The methylthiol:CoM methyltransferase reaction could be initiated only with the enzyme-bound corrinoid in the methylated state. CoM could demethylate, and DMS and MMPA could remethylate, the corrinoid cofactor. The monomethylamine corrinoid protein and the A isozyme of methylcobamide:CoM methyltransferase (proteins homologous to the two subunits comprising the 480-kDa CoM methylase) did not catalyze CoM methylation with methylated thiols. These results indicate that the 480-kDa corrinoid protein functions as a CoM methylase during methanogenesis from DMS or MMPA.     

Multiple electrophoretic forms of methyl-accepting chemotaxis proteins generated by stimulus-elicited methylation in Escherichia coli.

The tsr and tar genetic loci of Escherichia coli determine the presence in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of methyl-accepting chemotaxis proteins (MCPs) I and II, respectively, each of which consists of a distinct group of multiple bands. Synthesis of the tsr and tar products was directed in ultraviolet-irradiated bacteria by lambda transducing phages. The addition of appropriate chemotactic stimuli to these cells resulted in the appearance of additional, faster migrating electrophoretic forms of the Tsr and Tar polypeptides which disappeared upon removal of the stimulus. The stimulus-elicited forms comigrated with component bands of the corresponding MCPs. These results indicate that methylation itself caused shifts in electrophoretic mobility and hence led to the observed MCP band patterns. The number of Tsr species suggested that there were at least three methylated sites on the Tsr polypeptide. The conclusion that methylation generates multiplicity was supported by the results of experiments in which the tsr product was synthesized in mutant bacteria defective in specific chemotaxis functions concerned with methylation or demethylation of MCPs. Thus, the presence of a cheX defect blocked the stimulus-elicited appearance of faster migrating forms of the tsr product; conversely, the presence of a cheB defect resulted in a pronounced shift toward these forms in the absence of a chemotactic stimulus.     

Ligand-specific activation of Escherichia coli chemoreceptor transmethylation

Adaptation in the chemosensory pathways of bacteria like Escherichia coli is mediated by the enzyme-catalyzed methylation (and demethylation) of glutamate residues in the signaling domains of methyl-accepting chemotaxis proteins (MCPs). MCPs can be methylated in trans, where the methyltransferase (CheR) molecule catalyzing methyl group transfer is tethered to the C terminus of a neighboring receptor. Here, it was shown that E. coli cells exhibited adaptation to attractant stimuli mediated through either engineered or naturally occurring MCPs that were unable to tether CheR as long as another MCP capable of tethering CheR was also present, e.g., either the full-length aspartate or serine receptor (Tar or Tsr). Methylation of isolated membrane samples in which engineered tethering and substrate receptors were coexpressed demonstrated that the truncated substrate receptors (trTsr) were efficiently methylated in the presence of tethering receptors (Tar with methylation sites blocked) relative to samples in which none of the MCPs had tethering sites. The effects of ligand binding on methylation were investigated, and an increase in rate was produced only with serine (the ligand specific for the substrate receptor trTsr); no significant change in rate was produced by aspartate (the ligand specific for the tethering receptor Tar). Although the overall efficiency of methylation was lower, receptor-specific effects were also observed in trTar- and trTsr-containing samples, where neither Tar nor Tsr possessed the CheR binding site at the C terminus. Altogether, the results are consistent with a ligand-induced conformational change that is limited to the methylated receptor dimer and does not spread to adjacent receptor dimers.     

The methyl-accepting chemotaxis proteins of E. coli: a repellent-stimulated, covalent modification, distinct from methylation

The methyl-accepting chemotaxis proteins (MCPs) of Escherichia coli are integral membrane proteins that have been shown to undergo reversible methylation in response to the addition of attractants. We have shown that a second, rapid modification of MCPI and MCPII occurs, which is repellent-stimulated. This modification, which is not methylation, was detected because it causes a decrease in mobility of the MCPs on 7.5% SDS-polyacrylamide gels with a high acrylamide to bisacrylamide ratio. We have designated this modification as the CheB-modification, as it is dependent on the CheB gene product. The CheB-modification causes a decrease in the isoelectric point of MCPII by one or two charge groups. The CheB-modification is not necessary for the methylation, nor does it preclude methylation of the MCPs. Both the CheB-modified form and the unmodified, unmethylated forms of the MCPs are stable to treatment with base, which results in the hydrolysis of the methylesters (demethylation) of the MCPs. The potential role of CheB-modification in chemotaxis is discussed.     

Mercury Methylation and Demethylation in Anoxic Lake Sediments and by Strictly Anaerobic Bacteria

After spiking anoxic sediment slurries of three acidic oligotrophic lakes with either HgCl₂ at 1.0 μg/ml or CH₃HgI at 0.1 μg/ml, both mercury methylation and demethylation rates were measured. High mercury methylation potentials were accompanied by high demethylation potentials in the same sediment. These high potentials correlated positively with the concentrations of organic matter and dissolved sulfate in the sediment and with mercury levels in fish. Adjustment of the acidic sediment pH to neutrality failed to influence either the methylation or the demethylation rate of mercury. The opposing methylation and demethylation processes converged to establish similar Hg²⁺-CH₃Hg⁺ equilibria in all three sediments. Because of their metabolic dominance in anoxic sediments, mercury methylation and demethylation in pure cultures of sulfidogenic, methanogenic, and acetogenic bacteria were also measured. Sulfidogens both methylated and demethylated mercury, but the methanogen tested only catalyzed demethylation and the acetogen neither methylated nor demethylated mercury.     

Functional homology of chemotactic methylesterases from Bacillus subtilis and Escherichia coli.

The methylesterase enzyme from Bacillus subtilis was compared with that from Escherichia coli. Both enzymes were able to demethylate methyl-accepting chemotaxis proteins (MCPs) from the other organism and were similarly affected by variations in glycerol, magnesium ion, or pH. When attractants were added to a mixture of B. subtilis MCPs and E. coli methylesterase, the rate of demethylation was enhanced. Conversely, when attractants were added to a mixture of E. coli MCPs and B. subtilis methylesterase, the rate of demethylation was diminished. These effects are what would be expected if, in these in vitro systems, the MCPs determined the rate of demethylation. These data suggest that, although the enzymes are from evolutionarily divergent organisms and are different in size, they have considerable functional homology.     

The methyl-accepting chemotaxis proteins of Escherichia coli. Identification of the multiple methylation sites on methyl-accepting chemotaxis protein I

The methyl-accepting chemotaxis proteins (MCPs) are integral membrane proteins that undergo reversible methylation during adaptation of bacterial cells to environmental attractants and repellents. The numerous methylated forms of each MCP are seen as a pattern of multiple bands on polyacrylamide gels. We have characterized the methylation sites in MCPI by analyzing methyl-accepting tryptic peptides. At least two different tryptic peptides accept methyl esters; one methyl-accepting peptide contains methionine and lysine and may be methylated a maximum of four times. The second methyl-accepting tryptic peptide contains arginine and may be methylated twice. Base-catalyzed demethylations of tryptic peptides and analysis of the charge differences between the different methylated forms of MCPI show that MCPI molecules may be methylated a total of six times. The two methyl esters on the methyl-accepting arginine peptide appear to be preferentially methylated in most of the forms of MCPI in attractant-stimulated cells. The ability to acquire six methylations on MCPI allows the bacterial cells to adapt to a broad range of attractant and repellent concentrations.     

腮腺炎病毒的多肽及其在感染细胞中的合成

以差异离心和蔗糖密度梯度离心祛提纯了在鸡胚尿囊腔中繁殖的腮腺炎病毒粒子。并用SDS—PAGE分析病毒粒子的结构多肽,发现其结构多肽为11种,分子量在35K到72K之间。同时还检测到HN蛋白的多聚体和F蛋白的大亚基F1。将腮腺炎病毒分别感染Hela,Vero和CE细胞,比较这三种细胞对ME株腮腺炎病毒的敏感性,发现CE细胞是ME株的敏感宿主。用[31S]蛋氨酸标记病毒感染的CE细胞,以SDS-PAGE及放射自显影法检测到腮腺炎病毒在宿主细胞中合成了至少8种多肽,分子量在26．5K到94K之间。对这些多肽在细胞中不同时期合成情况进行了研究。还用脉冲追踪(pulsechase)技术在感染细胞中发现了FO到F这一转译后加工(Postttanslational procession)现象。此外也研究了放线菌素D和高沈度氯化钠对细胞蛋白质合成的抑制作用。     

Cardiac myosin from pig heart ventricle. Purification and enzymatic properties.

A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropomyosin, troponin, the 150,000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel electrophoresis of the purified myosin showed three closely spaced bands corresponding to the 20,000 dalton, the charge-modified 20,000 dalton and the phosphorylated 20,000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55 muM and the Vmax values were 0.263 and 0.317 mumole P1/mg/min for the Ca2+-activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.     

Molecular organization of glutamate-sensitive, chemoexcitatory membranes of nerve cells. Physico-chemical characteristics of the glutamate-binding synaptic membrane proteins of the rat cerebral cortex

Some physico-chemical properties of glutamate-binding proteins solubilized from rat cerebral cortex synaptic membranes and purified by affinity chromatography were studied. Purified proteins were shown to be homogenous during SDS polyacrylamide gel electrophoresis (Mr 14000). The Scatchard plots for L-[3H]glutamate binding to the purified membrane proteins revealed the presence of one type of binding sites with Kd 800-1000 nM and Bmax 180-200 pmol/mg of protein. Ultracentrifugation of the glutamate-binding membrane protein in sucrose linear gradient demonstrated that the position of the protein peak depends on protein concentration, i.e. after dilution of the sample the protein peak is shifted from 28 000-30 000 to 12 000-15 000. The values of sedimentation coefficients decrease correspondingly to 2.1S. Presumably, these processes are due to dissociation of receptor macromolecules. The glutamate receptor is a glycoprotein-lipid complex made up of several low molecular weight subunits.     

Differential DNA methylation reprogramming of various repetitive sequences in mouse preimplantation embryos

Genome-wide changes of DNA methylation by active and passive demethylation processes are typical features during preimplantation development. Here we provide an insight that epigenetic reprogramming of DNA methylation is regulated in a region-specific manner, not a genome-wide fashion. To address this hypothesis, methylation states of three repetitive genomic regions were monitored at various developmental stages in the mouse embryos. Active demethylation was not observed in the IAP sequences whereas methylation reprogramming of the satellite sequences was regulated only by the active mechanism. Etn elements were actively demethylated after fertilization, passively demethylated by the 8-cell stage, and de novo methylated at the morular and blastocyst stages, showing dynamic epigenetic changes. Thus, our findings suggest that the specific genomic regions or sequences may spatially/temporally have their unique characteristics in the reprogramming of the DNA methylation during preimplantation development.     

DNA methylation as a possible mechanism affecting ability of natural populations to adapt to changing climate

Environmentally induced epigenetic variation has been recently recognized as a possible mechanism allowing plants to rapidly adapt to novel conditions. Despite increasing evidence on the topic, little is known on how epigenetic variation affects responses of natural populations to changing climate. We studied the effects of experimental demethylation (DNA methylation is an important mediator of heritable control of gene expression) on performance of a clonal grass, Festuca rubra, coming from localities with contrasting temperature and moisture regimes. We compared performance of demethylated and control plants from different populations under two contrasting climatic scenarios and explored whether the response to demethylation depended on genetic relatedness of the plants. Demethylation significantly affected plant performance. Its effects interacted with population of origin and partly with conditions of cultivation. The effects of demethylation also varied between distinct genotypes with more closely related genotypes showing more similar response to demethylation. For belowground biomass, demethylated plants showed signs of adaptation to drought that were not apparent in plants that were naturally methylated. The results suggest that DNA methylation may modify the response of this species to moisture. DNA methylation may thus affect the ability of clonal plants to adapt to novel climatic conditions. Whether this variation in DNA methylation may also occur under natural conditions, however, remains to be explored. Despite the significant interactions between population of origin and demethylation, our data do not provide clear evidence that DNA methylation enabled adaptation to different environments. In fact, we obtained stronger evidence of local adaptation in demethylated than in naturally‐methylated plants. As changes in DNA methylation may be quite dynamic, it is thus possible that epigenetic variation can mask plant adaptations to conditions of their origin due to pre‐cultivation of the plants under standardized conditions. This possibility should be considered in future experiments exploring plant adaptations.     

Biochemical characterisation of the Li locus,which controls the activity of the cyanogenic β-glucosidase in Trifolium repens L.

The cyanogenic -glucosidase (linamarase) was purified from white clover leaf tissue. The enzyme is a homodimer with a molecular weight of 105 300–103 400 daltons estimated from molecular exclusion chromatography. The effect of buffer ions on the pH optimum and charge properties of the enzyme are presented. A combination of molecular exclusion chromatography and CM cellulose ion exchange chromatography purified linamarase 16 fold to a single 62 000 dalton polypeptide on SDS polyacrylamide gel electrophoresis. This polypeptide represented about 5% of the total soluble leaf protein and can be seen as a prominent band in SDS polyacrylamide gel electrophoresis of crude leaf extracts from Li Li plants. Screening backcross progeny showed that extracts from li li plants, which have no linamarase activity, lack this 62 000 dalton polypeptide. Linamarase is the major glycoprotein in white clover leaf extracts which binds to Concanavalin A-Sepharose.     

北京棒状杆菌AS1.299谷氨酰胺合成酶的提纯和性质

用热变性、硫酸铵分段沉淀和DEAE纤维素柱层析部分纯化了北京棒状杆菌AS1.299(Corynebacterium pekinense)谷氨酰胺合成酶,并用垂直平板电泳对酶进一步精制。酶对谷氨酰胺的Km值为16.4mM,对羟胺的Km值为43.5mM;酶的沉降系数为21S,SDS凝胶电泳测得酶的亚基分子量为56,000。化学修饰表明,该酶活力与硫氢基、色氨酸、精氨酸残基无关;酪氨酸修饰剂的作用引起酶活力下降,初步证明,组氨酸残基是该酶活力的必需基因。     

Factors affecting the persistence of drug-induced reprogramming of the cancer methylome

Aberrant DNA methylation is a critical feature of cancer. Epigenetic therapy seeks to reverse these changes to restore normal gene expression. DNA demethylating agents, including 5-aza-2′-deoxycytidine (DAC), are currently used to treat certain leukemias, and can sensitize solid tumors to chemotherapy and immunotherapy. However, it has been difficult to pin the clinical efficacy of these agents to specific demethylation events, and the factors that contribute to the durability of response remain largely unknown. Here we examined the genome-wide kinetics of DAC-induced DNA demethylation and subsequent remethylation after drug withdrawal in breast cancer cells. We find that CpGs differ in both their susceptibility to demethylation and propensity for remethylation after drug removal. DAC-induced demethylation was most apparent at CpGs with higher initial methylation levels and further from CpG islands. Once demethylated, such sites exhibited varied remethylation potentials. The most rapidly remethylating CpGs regained >75% of their starting methylation within a month of drug withdrawal. These sites had higher pretreatment methylation levels, were enriched in gene bodies, marked by H3K36me3, and tended to be methylated in normal breast cells. In contrast, a more resistant class of CpG sites failed to regain even 20% of their initial methylation after 3 months. These sites had lower pretreatment methylation levels, were within or near CpG islands, marked by H3K79me2 or H3K4me2/3, and were overrepresented in sites that become aberrantly hypermethylated in breast cancers. Thus, whereas DAC-induced demethylation affects both endogenous and aberrantly methylated sites, tumor-specific hypermethylation is more slowly regained, even as normal methylation promptly recovers. Taken together, these data suggest that the durability of DAC response is linked to its selective ability to stably reset at least a portion of the cancer methylome.