H.L. Kornberg

Pathway of fructose utilization by Escherichia coli

Esc+erichia coli grows readily on fructose as sole source of carbon. It was long assumed that the manner in which this sugar is utilized is analogous to that by which glucose enters the main metabolic pathways of the cells; this would imply the occurrence of an initial enzymatic phosphorylation by ATP of fructose to fructose-6-phosphate and ADP, followed by a second phosphorylation to fructose-l ,ddiphosphate. Enzymes catalysing both these reactions have been demonstrated to be present E. coli [ 1,2] . However, recent studies in several laboratories have cast doubt on this view, and have suggested that fructose is initially phosphorylated to fructose-l-phosphate (reaction 1) with concomitant conversion of phosphoenolpyruvate (PEP) to pyruvate. Fructose-l-phosphate is then further phosphorylated to fructose1,6-diphosphate, through the agency of an ATP-linked fructose-l-phosphate kinase (reaction 2) distinct from the action of the phosphosfructokinase that catalyses the phosphorylation of fructose-6-phosphate to fructose-l ,6-diphosphate.

Genetic control of glucose uptake byEscherichia coli

The uptake by Escherichia coli of a variety of carbohydrates necessarily involves the activity of a PEP-dependent phosphotransferase (PT)-system [l] , in which at least three components participate. Two of these a small, histidine-containing protein (HPr) and an enzyme (Enzyme I) that catalyses the transfer of phosphate from PEP to HPr are required for the utilization of all carbohydrates that are taken up via the PTsystem: mutants devoid of either component are pleiotropically impaired in the uptake of all such sugars [2, 31. However, the membrane-linked multi-component fractions that effect the transfer or phosphate from the phosphorylated HPr to sugars (collectively termed Enzyme II) are specific for individual sugars. Thus, for example, E. coli mutants devoid of an Enzyme II for fructose do not grow or take up this sugar, although such mutants are unimpaired in their ability to grow on other hexoses and on fructose land 6-phosphates [4]. The gene (prsF) specifying this Enzyme I1 is located at about 43 min [4] on the E. coli linkage map [5] . It is the main purpose of this paper to report the isolation of l?. coli mutants deficient in an Enzyme II for the uptake of glucose and of its non-catabolizable analogue, methyl-a-glucoside (aMG). The genetic marker specifying this uptake system (umg) is located at approx. 23.5 min on the linkage map and is co-transducible with the purB marker.

PtsX: a gene involved in the uptake of glucose and fructose by Escherichia coli.

The uptake by Escherichia coli of a variety of carbohydrates necessarily involves the activity of a PEP-dependent phosphotransferase (PT) system [I], containing a number of components. Two of these, a small, histidine-containing protein (HPr) and an enzyme (Enzyme I) that catalyses the transfer of phosphate from PEP to HPr, are required for the utilization of all carbohydrates that are taken up via the PT-system [2-61. In contrast, the membranelinked multicomponent fractions that effect the transfer of phosphate from the phosphorylated HPr to sugars (collectively termed Enzymes II) are generally considered to be specific for individual sugars, although the uptake of any one sugar may involve the functioning of more than one Enzyme II. Thus, for example, the uptake by E. colt’ of fructose at low concentration (< 2 mM) involves predominantly an Enzyme II, specified by the ptsF gene (located at min. 41 on the E. coZi genome) that catalyses the formation of fructose-l-phosphate, whereas at higher concentrations (> 2 mM) fructose is phosphorylated also to fructose 6-phosphate by the action of a different Enzyme II, specified by the pfsX gene (located at min. 35.5 on the E. coli genome) [7-lo]. It is the main purpose of this paper to present evidence that the Enzyme II specified by the ptsX gene is involved also in the uptake and phosphorylation of glucose. Mutants of E. coli devoid of an Enzyme II that catalyses the phosphorylation of methyl~glucoside, specified by the umg gene (located at min. 24 on the E. coli genome, [ 1 1 ] ), are markedly impaired in glucose utilization but still grow slowly on that sugar: we here show that

Isolation and properties of a regulatory mutant in the hexose phosphate transport system of Escherichia coli

Escherichia coli can utilize a variety of hexose phosphates without first hydrolysing them to the free sugars [ 11. The system that effects the transport of these esters into the cells is inducible [ 1,2] and common to them all: growth in the presence of glucose-6phosphate, or fructose-6-phosphate, or mannosedphosphate, elicits the formation of the uptake system [3], and mutants that lack the ability to take up one of these hexose phosphates also lack the ability to take up the others [3,4] I The gene specifying this uptake system, designated uhp, is cotransducible with pyrE and is thus located at about 72 min [4] on the E. cob linkage map [5] . We recently observed [6] that E. coli can grow on frucjose1 -phosphate as sole carbon source. However, rapid growth occurred only if the cells were previously exposed to hexose-6-phosphates; the rate of growth on fructose-l-phosphate by cells thus induced decreased sharply after lI%7 doublings. This suggested that fructose-l-phosphate could be transported by the hexose phosphate uptake system but was not an inducer of that system. We now confirm this interpretation, and utilize it to select mutants that grow readily on fructose-l-phosphate without prior exposure to hexose-6-phosphates, and whose rate of growth on this substrate remains constant for many doublings. Such mutants form the uptake system for hexose phosphates constitutively; their genotype is designated uhpC. Analysis by conjugation and by phage-mediated transduction, establishes uhpc to be closely linked to uhp and to be also cotransducible with p,vrE; this raises the possibility that the uptake of hexose phosphates by E. coli may be regulated as an operon [7] .

Genetic control of inducer exclusion by Escherichia coli

There are two main ways in which enteric bacteria take up carbohydrates from their external media. In ‘group translocation’, the uptake of a number of sugars is coupled to the transfer to them of phosphate from phosphoenolpyruvate (PEP) and the sugars thus appear inside the cells as the phosphate esters. The multi-component phosphotransferase system (PTS) that brings this about was discovered by S. Roseman et al. [ 1 ] ; its properties have been extensively reviewed [ 2,3]. ‘Active transport’, on the other hand, is a term applied to the energy-linked uptake of other sugars, in which no obligatory phosphate transfer occurs and in which the sugar present externally appears, chemically unchanged, as such inside the cells. This process is intimately associated with the flow of electrons through membrane carriers and may be energized by the collapse of proton and/or electrical gradients [4,5] . Sugars that are taken up by Escherichia coli or Salmonella typhimurium via phosphotransferase-mediated ‘group translocation’ include glucose and fructose; they are designated W-sugars’ for convenience. Carbohydrates that are taken up by these bacteria via ‘active transport’ include lactose, maltose, pentoses, gluconate and hexose phosphates; they can be similarly grouped under the term ‘non-PT-sugars’ [3,6] . It would be expected from this distinction that mutants impaired in a sugar-specific component of the PEP-phosphotransferase system would not grow on the appropriate PT-sugar or, if the lesion were in a common component of that system, on any PT-sugar, but that such mutants would-grow on non-PT sugars. However, many mutants of Enzyme I of the PTS @ts Z) have been described which fail to grow both on PT-sugars

The Utilization of Propionate by Micrococcus denitrificans

Surmmary Suspensions of Micrococcus denitrificans, growing with propionate as sole carbon source, incorporated 14C from [1-14C]propionate or from sodium [14C]bicarbonate, initially into succinate and then into intermediates of the tricarboxylic acid cycle and amino acids derived therefrom. In the presence of 4 mM-sodium arsenite, the oxidation of propionate and of L-malate by washed organisms proceeded only to the level of pyruvate, which accumulated: when sodium [14C]bicarbonate was also present, the pyruvate formed from propionate, but not that formed from L-malate, was highly radioactive. Cell-free extracts of propionate-grown M. denitrificans catalysed the formation of labelled methylmalonyl-coenzyme A, succinyl-coenzyme A and succinate from sodium [14C]carbonate+ ATP + either propionate and coenzyme A, or propionyl-coenzyme A. The evidence thus obtained indicates that propionate enters the tricarboxylic acid cycle of M. denitrificans, preponderantly via activation to propionyl-coenzyme A, followed by carboxylation to methyl-malonyl coenzyme A, isomerization to succinyl-coenzyme A and hydrolysis to succinate.