DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.

The hyperthermophilic marine archaeon Thermococcus litoralis exhibits high-affinity transport activity for maltose and trehalose at 85؇C. The K m for maltose transport was 22 nM, and that for trehalose was 17 nM. In cells that had been grown on peptone plus yeast extract, the V max for maltose uptake ranged from 3.2 to 7.5 nmol/min/mg of protein in different cell cultures. Cells grown in peptone without yeast extract did not show significant maltose or trehalose uptake. We found that the compound in yeast extract responsible for the induction of the maltose and trehalose transport system was trehalose. [14 C]maltose uptake at 100 nM was not significantly inhibited by glucose, sucrose, or maltotriose at a 100 M concentration but was completely inhibited by trehalose and maltose. The inhibitor constant, K i , of trehalose for inhibiting maltose uptake was 21 nM. In contrast, the ability of maltose to inhibit the uptake of trehalose was not equally strong. With 20 nM [ 14 C]trehalose as the substrate, a 10-fold excess of maltose was necessary to inhibit uptake to 50%. However, full inhibition was observed at 2 M maltose. The detergent-solubilized membranes of trehalose-induced cells contained a high-affinity binding protein for maltose and trehalose, with an M r of 48,000, that exhibited the same substrate specificity as the transport system found in whole cells. We conclude that maltose and trehalose are transported by the same high-affinity membrane-associated system. This represents the first report on sugar transport in any hyperthermophilic archaeon.

glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its K m values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its V max was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR ؉ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.In Escherichia coli and most other bacteria, glucose is transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as glucose-6-phosphate (53), thus eliminating the need for glucokinase in the utilization of glucose. In contrast, the utilization of glucose-containing disaccharides such as lactose, maltose, or trehalose involves the formation of glucose inside the cell and requires its phosphorylation for the effective utilization of the disaccharides. Surprisingly, the presence of a glk mutation (22) does not appear to be a disadvantage in the utilization of these disaccharides. Severe reduction in growth is observed only when, in addition to having the glk mutation, the strain also lacks the ability to phosphorylate glucose via the PTS pathway (14, 59). Two different findings might be relevant for this phenomenon. In the first, glucose and galactose, the products of intracellular ␤-galactosidase action, have been found in large amounts outside the cell after the uptake of lactose, implying that growth on lactose is mediated via the uptake of the secreted products (PTS-mediated phosphorylation in the case of glucose) (35). In the second, internal phosphorylation of glucose by the PTS has been evoked (14). The findings indicate that the enzyme II Glc is also responsible for the utilization of internal glucose.As a consequence, the interest in E. coli glucokinase has been low. Glucokinase activity in E. coli was measured as early as 1953 (21), and MM6, an E. coli mutant defective in the utilization of glucose, was shown to contain normal amounts of glucokinase (4). In that study, the K m of...

The Escherichia coli maltose system consists of a number of genes whose products are involved in the uptake and metabolism of maltose and maltodextrins. MalT is the central positive gene activator of the regulon and is, together with the cyclic AMP-catabolite gene activator protein system, necessary for the expression of the maltose genes. Expression of malY, a MalT-independent gene, leads to the repression of all MalT-dependent genes. We have purified MalY to homogeneity and found it to be a pyridoxal-5-phosphate-containing enzyme with the enzymatic activity of a ␤C-S lyase (cystathionase). MalY is a monomeric protein of 42,000 to 44,000 Da. Strains expressing MalY constitutively abolish the methionine requirement of metC mutants. The enzymatic activity of MetC, the cleavage of cystathionine to homocysteine, ammonia, and pyruvate, can be catalyzed by MalY. However, the cystathionase activity is not required for the function of MalY in repressing the maltose system. By site-directed mutagenesis, we changed the conserved lysine residue at the pyridoxal phosphate binding site (position 233) of MalY to isoleucine. This abolished ␤C-S lyase activity but not the ability of the protein to repress the maltose system. Also, the overexpression of plasmid-encoded metC did not affect mal gene expression, nor did the deduced amino acid sequence of MetC show homology to that of MalY.The maltose regulon of Escherichia coli consists of two sets of genes which encode proteins involved in the uptake and metabolism of maltose and maltodextrins (␣1,4-linked D-glucose polymers) (29). Expression of the maltose genes is dependent on the presence of the inducer, maltotriose, bound to the positive regulator, MalT (23). Mutants lacking MalT function do not express any mal genes and are phenotypically Mal Ϫ , whereas malT(Con) mutations render mal gene expression independent of (or less dependent on) the inducer, maltotriose (10, 11). The expression of malT, as well as of some of the mal genes, is dependent on activation by the cyclic AMP-catabolite gene activator protein complex (9). Several observations demonstrate that the regulation of the maltose system is not as straightforward as implied above. First, there is the phenomenon of internal induction (12). Second, MalK, the ATP-consuming subunit of the maltose transport system, acts phenotypically as a repressor of the system, affecting the function of MalT (6,18,19,26). The third regulatory circuit affecting mal gene expression is derived from the malI malX malY gene cluster. malI was discovered as a mutation which strongly reduced the high and constitutive expression of a malK-lacZ fusion (14). Molecular analysis of malI (25) and its adjacent genes revealed that malI encodes a typical repressor protein, analogous to LacI and GalR, which prevents the expression of the adjacent and divergently oriented malX malY operon. The gene product of malX is a protein homologous to the enzyme II Glc of the phosphotransferase system. malY encodes a protein with sequence homology, including the ch...

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and a,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (0. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate ['4CJglucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-lphosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.The Escherichia coli maltose system consists of a maltodextrin-specific pore (encoded by lamB) (17, 30) in the outer membrane and a binding-protein-dependent transport system in the cell envelope (encoded by malE malF malG malK) (38), as well as one periplasmic enzyme (encoded by malS) (35) and three cytoplasmic enzymes (encoded by malQ, malP, and malZ) (27,34,41). Expression of all mal genes depends on the positive regulator MalT (33).The transport system (11,12,20,24,40) can recognize and accumulate maltose and linear a,1-4-linked maltodextrins up to a chain length of seven glucose units (15). The major enzymes of the system (see Fig. 1) are the cytoplasmic amylomaltase (MalQ) (42) and maltodextrin phosphorylase (MalP) (34). Amylomaltase recognizes maltotriose and larger maltodextrins (donors), cleaving off the reducing glucose residue and transferring the remaining dextrinyl residue onto the nonreducing end of maltodextrin (acceptors), including maltose and glucose. With maltotriose, the smallest donor substrate, as well as with longer linear maltodextrins, amylomaltase thus produces glucose and longer maltodextrins (26). Maltodextrin phosphorylase subsequently releases glucose-i-phosphate from the nonreducing end of maltodextrins with a minimal chain length of five glucose residues (37). The glucose and glucose-l-phosphate are both transformed into glucose-6-phosphat...