L-Lysine transport in Corynebacterium glutamicum was investigated. The bacterium was shown to possess a highly specific, energy-dependent system of active lysine transport. The system transferred lysine into the cells and exchanged intra-and extracellular lysine. Mutations in the transport system did not lead to overproduction of the amino acid. Resting cells of the parent strain, or of its lysine-producer derivatives with a defective transport system, failed to excrete lysine into the medium. An efflux of intracellular lysine could be induced by a hyperosmotic shock, and by different membrane-active substances. It has been suggested that C. ghtamicum cells are equipped with channels (pores) for excreting lysine from the cytoplasm. These channels appeared to open in response to an increase in the intracellular lysine concentration. The channel permeability also depends on the membrane structure.

G . M . M A L I N AND G. I . BOURD. 1991. The transport system for glucose and its non-metabolizable analogue methyl-a-D-glucoside (MG) has been described in Corynebacterium glutamtcum. The initial product of the transport reaction was shown to be a phosphate ester of MG (MGP).Free M G appeared inside the cells as a result of MGP dephosphorylation. The bacteria transported MG with an apparent K,,, of 0.08 & 0.017 mmol/l and V,,, of 21 f 2.3 nmol/(min x mg dry wt). Toluenized cells and crude cell extracts catalysed phosphoenolpyruvate (PEP)-dependent phosphorylation of M G and glucose. Both the membrane and the cytoplasmic fractions of bacterial extracts were required for phosphotransferase reaction. Most of the spontaneous mutants resistant to 2-deoxyglucose (DG), xylitol and 5-thioglucose were defective both in transport and in PEP-dependent phosphorylation of MG. Some strains were defective only in glucose utilization and some were also unable to grow on a number of other sugars. The phosphotransferase activity in extracts from mutant cells was restored by the addition of either membrane or cytoplasmic fraction from wild type bacteria. It was concluded that Corynebacterium glutamicum accumulated glucose and MG by means of a PEP-dependent phosphotransferase system (PTS).

No abstract

Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsl and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cyu mutation) and synthesis of cyclic-AMP-receptor protein (crp mutation) Measurements of the differential rate of 1-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in luc', l a d , luc0" and cyu bacteria, but it was lost in lucP and crp strains.It was concluded that mutational damage to the general components of the phosphoenolpyruvatedependent phosphotransferase system diminishes activity of the lac promoter.The results obtained led to the conclusion that pts gene products (apparently phospho -HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.The phosphoenolpyruvate : carbohydrate phosphotransferase system of Escherichia coli carries out the phosphorylation of many carbohydrates [ 1 -41. This process involves the activities of several protein components. Enzyme I catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate to a small histidine-containing heat-stable protein HPr. The phospho -HPr generated serves as a donor of the phosphoryl moiety in the phosphotransferase reaction catalysed by the enzyme 11 which is specfic for a certain carbohydrate or its analogue. Enzyme I and HPr are designated the general proteins of the system since they are required for the phosphorylation of all sugar substrates of the phosphotransferase system. These two components are constitutive soluble proteins ; their synthesis is determined by theptsl (for enzyme I) and ptsH (for HPr) genes [3]. At present it is well established that the phosphotransferase system is necessary for the transport and metabolism of carbohydrates of D-gluco and u-munno configuration [2,4]. Transport of the sugar is catalyzed concomitantly with phosphorylation of the carbohydrate at the expense of phosphoenolpyruvate. Sugars that have an obligatory requirement for phosphorylation during transfer are called phosphotransferase sugars. Non-phosphotransferase substrates are those transported into bacterial cell via their specific systems [2].Mutational damage to the general proteins of the phosphotransferase system (ptsl, ptsI,H and ptsH mutations) leads to severe disturbance of transport of the phosphotransferase sugars into E. coli cells and these mutants are unable to grow in media containing these sugars as the sole source of carbon [5-1.51.Some properties of the p t s mutants indicate that the products of the p t s genes are relevant to the regulation of the synthesis of catabolite-sensitive enzymes. As was demonstrated in our laboratory, pts

1. Two spontaneous Escherichia coli K12 mutants resistant to glucose catabolite repression were isolated using minimal agar plates with methyl a-D-glucoside. Mutants grow well on glucose and mannitol.2. Glucose does not inhibit the inducible enzyme synthesis in isolated mutants: mutant cell (in contrast to parent cells) produce high levels of j-galactosidase and L-tryptophanase under the conditions of glucose catabolite repression.3. The isolated mutants are negative in methyl-a-D-glucoside transport; glucose uptake is not severely damaged. But the mutants (named tgl, transport of glucose) retained the ability to phosphorylate methyl a-D-glucoside in vitro at the expense of phosphoenolpyruvate.4. The tgf mutation is cotransduced with purB and pyrC markers, i.e. locates near 24 min of the E. coli chromosome map.5. It is thought that E. coli cells possess two glucose transport systems. The first one is represented by the glucose-specific enzyme I1 of the phosphoenolpyruvate-dependent phosphotransferase system. The second glucose transport system (coded for tgf gene) functions as permease and possesses high affinity to methyl a-D-ghcoside. The integrity of glucose permease determine the sensitivity of the cell to glucose catabolite repression.The transfer of glucose into Escherichia coli K 12 is mediated by the phosphoenofpyruvate : carbohydrate phosphotransferase system [l]. The phosphotransferase reaction coupled with the glucose transport involves the necessary activity of some proteins. Enzyme I catalyses the transfer of the phosphate group from phosphoenolpyruvate to a small histidine containing protein (Hpr) [2,3]. The phosphorylated form of Hpr (phospho -Hpr) is the phosphate donor in the second step of the phosphotransferase reaction at which the membrane-linked enzyme 11, specific for glucose, carries out phosphorylation of the carbohydrate [2,4]. At present there is some evidence that Genetic Nou~w~claturr. tgl = lack of thc glucose permease; gptA = lack of the phospho -Hpr : glucose phosphotransferase activity; um,y = lack of the methyl-a-D-glucoside uptake system. Other symbols are standard, see [9].Enzyme.s. P-Galactosidase (EC 3.2.1.23); L-tryptophanase (EC 4.2.1.20).in E. coli a cytoplasmic factor I11 may participate in the last step of the phosphotransferase reaction [4].Recently it has been reported that there exist two phospho -Hpr : glucose phosphotransferase systems in crude extracts of E. coli cells. The two systems differ in their substrate specificity [ S ] . In the preliminary communication it was mentioned that system A phosphorylates both glucose and methyl a-D-glucoside (nonmetabolizable glucose analogue) ; in the wild type of E. coli 40 % of the glucose phosphotransferase activity is due to system A. The remaining 60 % of the activity is maintained by system €3 which phosphorylates only glucose and has little affinity for methyl a-D-glucoside. Curtis and Epstein consider that system B represents the membrane-linked glucose-specific enzyme I1 but not the glucokinase or some other soluble...