Winfried Boos

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in Δ malP (encoding maltodextrin phosphorylase), Δ malQ (encoding amylomaltase), Δ glgA (encoding glycogen synthase), and Δ glgA Δ malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the Δ malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the Δ malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The Δ malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the Δ glgA and Δ glgA Δ malP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro , MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.

MalT is the central transcriptional activator of allmalgenes inEscherichia coli. Its activity is controlled by the inducer maltotriose. It can be inhibited by the interaction with certain proteins, and its expression can be controlled. We report here a novel aspect ofmalgene regulation: the effect of cytoplasmic glucose and glucokinase (Glk) on the activity and the expression of MalT. Amylomaltase (MalQ) is essential for the metabolism of maltose. It forms maltodextrins and glucose from maltose or maltodextrins. We found that glucose above a concentration of 0.1 mM blocked the activity of the enzyme.malQmutants when grown in the absence of maltodextrins are endogenously induced by maltotriose that is derived from the degradation of glycogen. Therefore, the fact thatglk malQ⁺mutants showed elevatedmalgene expression finds its explanation in the reduced ability to remove glucose from MalQ-catalyzed maltodextrin formation and is caused by a metabolically induced MalQ⁻phenotype. However, even in mutants lacking glycogen, Glk controls endogenous induction. We found that overexpressed Glk due to its structural similarity with Mlc, the repressor ofmalT, binds to the glucose transporter (PtsG), releasing Mlc and thus increasingmalTrepression. In addition, even in mutants lacking Mlc (and glycogen), the overexpression ofglkleads to a reduction inmalgene expression. We interpret this repression by a direct interaction of Glk with MalT concomitant with MalT inhibition. This repression was dependent on the presence of either maltodextrin phosphorylase or amylomaltase and led to the inactivation of MalT.

TrmB of Pyrococcus furiosus was discovered as the trehalose/maltose-specific repressor for the genes encoding the trehalose/maltose high-affinity ABC transporter (the TM system). TrmB also represses the genes encoding the high affinity maltodextrin-specific ABC transporter (the MD system) with maltodextrin and sucrose as inducers. In addition, TrmB binds glucose leading to an increased repression of both, the TM and the MD system. Thus, TrmB recognizes different promoters and depending on the promoter it will be activated or inactivated for promoter binding by different sugar effectors. The TrmB-like protein TrmBL1 of P. furiosus is a global regulator and recognizes preferentially, but not exclusively, the TGM (for Thermococcales–glycolytic motif) sequence that is found upstream of the MD system as well as of genes encoding enzymes involved in the glycolytic and the gluconeogenic pathway. It responds to maltose and maltotriose as inducers and functions as repressor for the genes encoding the MD system and glycolytic enzymes, but as activator for genes encoding gluconeogenic enzymes. The TrmB-like protein TrmBL2 of P. furiosus lacks the sugar-binding domain that has been determined in TrmB. It recognizes the MD promoter, but not all TGM harboring promoters. It is evolutionary the most conserved among the Thermococcales. The regulatory range of TrmBL2 remains unclear.

Summary: TrmB is a transcriptional repressor of the hyperthermophilic archaeon Pyrococcus furiosus serving at least two operons. TrmB represses genes encoding an ABC transporter for trehalose and maltose (the TM system) with trehalose and maltose as inducers. TrmB also represses genes encoding another ABC transporter for maltodextrins (the MD system) with maltotriose and sucrose as inducers. Here we report that glucose which was also bound by TrmB acted as a corepressor (causing stronger repression) for both the TM and the MD system. Binding of glucose by TrmB was increased in the presence of TM promoter DNA. Maltose which acted as inducer for the TM system acted as a corepressor for the MD system intensifying repression. We propose that the differential conformational changes of TrmB in response to binding the different sugars governs the ability of TrmB to interact with the promoter region and represents a simple mechanism for selecting the usage of one carbon source over the other, reminiscent of catabolite repression in bacteria.

We report the presence of Mlc in a thermophilic bacterium. Mlc is known as a global regulator of sugar metabolism in gram-negative enteric bacteria that is controlled by sequestration to a glucose-transporting EII ^Glc of the phosphotransferase system (PTS). Since thermophilic bacteria do not possess PTS, Mlc in Thermus thermophilus must be differently controlled. DNA sequence alignments between Mlc from T. thermophilus (Mlc _Tth ) and Mlc from E. coli (Mlc _Eco ) revealed that Mlc _Tth conserved five residues of the glucose-binding motif of glucokinases. Here we show that Mlc _Tth is not a glucokinase but is indeed able to bind glucose ( K _D = 20 μM), unlike Mlc _Eco . We found that mlc of T. thermophilus is the first gene within an operon encoding an ABC transporter for glucose and mannose, including a glucose/mannose-binding protein and two permeases. malK1 , encoding the cognate ATP-hydrolyzing subunit, is located elsewhere on the chromosome. The system transports glucose at 70°C with a K _m of 0.15 μM and a V _max of 4.22 nmol per min per ml at an optical density (OD) of 1. Mlc _Tth negatively regulates itself and the entire glucose/mannose ABC transport system operon but not malK1 , with glucose acting as an inducer. MalK1 is shared with the ABC transporter for trehalose, maltose, sucrose, and palatinose (TMSP). Mutants lacking malK1 do not transport either glucose or maltose. The TMSP transporter is also able to transport glucose with a K _m of 1.4 μM and a V _max of 7.6 nmol per min per ml at an OD of 1, but it does not transport mannose.

Strains ofEscherichia colilacking MalQ (maltodextrin glucanotransferase or amylomaltase) are endogenously induced for the maltose regulon by maltotriose that is derived from the degradation of glycogen (glycogen-dependent endogenous induction). A high level of induction was dependent on the presence of MalP, maltodextrin phosphorylase, while expression was counteracted by MalZ, maltodextrin glucosidase. Glycogen-derived endogenous induction was sensitive to high osmolarity. This osmodependence was caused by MalZ.malZ, the gene encoding this enzyme, was found to be induced by high osmolarity even in the absence of MalT, the central regulator of allmalgenes. The osmodependent expression ofmalZwas neither RpoS nor OmpR dependent. In contrast, themalPQoperon, whose expression was also increased at a high osmolarity, was partially dependent on RpoS. In the absence of glycogen, residual endogenous induction of themalgenes that is sensitive to increasing osmolarity can still be observed. This glycogen-independent endogenous induction is not understood, and it is not affected by altering the expression of MalP, MalQ, and MalZ. In particular, its independence from MalZ suggests that the responsible inducer is not maltotriose.

The maltose/maltodextrin regulon ofEscherichia coliconsists of 10 genes which encode a binding protein-dependent ABC transporter and four enzymes acting on maltodextrins. Allmalgenes are controlled by MalT, a transcriptional activator that is exclusively activated by maltotriose. By the action of amylomaltase, we prepared uniformly labeled [¹⁴C]maltodextrins from maltose up to maltoheptaose with identical specific radioactivities with respect to their glucosyl residues, which made it possible to quantitatively follow the rate of transport for each maltodextrin. IsogenicmalQmutants lacking maltodextrin phosphorylase (MalP) or maltodextrin glucosidase (MalZ) or both were constructed. The resulting in vivo pattern of maltodextrin metabolism was determined by analyzing accumulated [¹⁴C]maltodextrins. MalP⁻MalZ⁺strains degraded all dextrins to maltose, whereas MalP⁺MalZ⁻strains degraded them to maltotriose. The labeled dextrins were used to measure the rate of transport in the absence of cytoplasmic metabolism. Irrespective of the length of the dextrin, the rates of transport at a submicromolar concentration were similar for the maltodextrins when the rate was calculated per glucosyl residue, suggesting a novel mode for substrate translocation. Strains lacking MalQ and maltose transacetylase were tested for their ability to accumulate maltose. At 1.8 nM external maltose, the ratio of internal to external maltose concentration under equilibrium conditions reached 10⁶to 1 but declined at higher external maltose concentrations. The maximal internal level of maltose at increasing external maltose concentrations was around 100 mM. A strain lackingmalQ,malP, andmalZas well as glycogen synthesis and in which maltodextrins are not chemically altered could be induced by external maltose as well as by all other maltodextrins, demonstrating the role of transport per se for induction.

Summary: TrmB is the transcriptional repressor for the gene cluster of the trehalose/maltose ABC transporter of the hyperthermophilic archaea Thermococcus litoralis and Pyrococcus furiosus (malE or TM operon), with maltose and trehalose acting as inducers. We found that TrmB (the protein is identical in both organisms) also regulated the transcription of genes encoding a separate maltodextrin ABC transporter in P. furiosus (mdxE or MD operon) with maltotriose, longer maltodextrins and sucrose acting as inducers, but not with maltose or trehalose. In vitro transcription of the malE and the mdxE operons was inhibited by TrmB binding to the different operator sequences. Inhibition of the TM operon was released by maltose and trehalose whereas inhibition of the MD operon was released by maltotriose and larger maltodextrins as well as by sucrose. Scanning mutagenesis of the TM operator revealed the role of the palindromic TACTNNNAGTA sequence for TrmB recognition. TrmB exhibits a broad spectrum of sugar‐binding specificity, binding maltose, sucrose, maltotriose and trehalose in decreasing order of affinity, half‐maximal binding occurring at 20, 60, 250 and 500 µM substrate concentration respectively. Of all substrates, only maltose shows sigmoidal binding characteristics with a Hill coefficient of 2. As measured by molecular sieve chromatography and cross‐linking TrmB behaved as dimer in dilute buffer solution at room temperature. We conclude that TrmB acts as a bifunctional transcriptional regulator acting on two different promoters and being differentially controlled by binding to different sugars. We believe this to represent a novel strategy of prokaryotic transcription regulation.

Close to an operon encoding an ABC transporter for maltose and trehalose, Thermococcus litoralis contains a gene whose encoded sequence showed similarity to sugar kinases. We cloned this gene, now called frk, and expressed it as a C-terminal His-tag version in Escherichia coli. We purified the recombinant protein, identified it as an ATP-dependent and fructose-6-phosphate-forming fructokinase (Frk) and determined its biochemical properties. At its optimal temperature of 80°C, the apparent K _m and V _max values of Frk were 2.3 mM and 730 U/mg protein for fructose at saturating ATP concentration, and 0.81 mM and 920 U/mg protein for ATP at saturating fructose concentration. The enzyme did not lose activity at 80°C for 4 h. Under denaturating conditions in SDS-PAGE, it exhibited a molecular mass of 35 kDa. Gel-filtration chromatography revealed a molecular mass of 58 kDa, indicating a dimer under nondenaturating, in vitro conditions.