Sugar recognition by the glucose and mannose permeases of Escherichia coli. Steady-state kinetics and inhibition studies

The glucose (EII(Glc)) and mannose (EII(Man)) permeases of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Escherichia coli belong to structurally different families of PTS transporters. The sugar recognition mechanism of the two transporters is compared using as inhibitors and pseudosubstrates all possible monodeoxy analogues, monodeoxyfluoro analogues, and epimers of D-glucose. The analogues were tested as phosphoryl acceptors in vitro and as uptake inhibitors with intact cells. Both EII have a high K(m) of phosphorylation for glucose modified at C-4 and C-6, and these analogues also are weak inhibitors of uptake. Conversely, modifications at C-1 (and also at C-2 with EII(Man)) were well tolerated. OH-3 is proposed to interact with hydrogen bond donors on EII(Glc) and EII(Man), since only substitution by fluorine was tolerated. Glucose-6-aldehydes, which exist as gem-diols in aqueous solution, are potent and highly selective inhibitors of "nonvectorial" phosphorylation by EII(Glc) (K(I) 3-250 microM). These aldehydes are comparatively weak inhibitors of transport by EII(Glc) and of phosphorylation and transport by EII(Man). Both transporters display biphasic kinetics (with glucose and some analogues) but simple Michaelis-Menten kinetics with 3-fluoroglucose (and other analogues). Kinetic simulations of the phosphorylation activities measured with different substrates and inhibitors indicate that two independent activities are present at the cytoplasmic side of the transporter. A working model that accounts for the kinetic data is presented.     

Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.     

Effect of growth conditions on levels of components of the phosphoenolpyruvate:sugar phosphotransferase system in Streptococcus mutans and Streptococcus sobrinus grown in continuous culture.

The membrane-bound, sugar-specific enzyme II (EII) component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) in Streptococcus mutans Ingbritt is repressed by growth on glucose under various conditions in continuous culture. Compared with optimal PTS conditions (i.e., glucose limitation, dilution rate [D] of 0.1 h-1, and pH 7.0), EII activity for glucose (EIIGlc) and mannose (EIIMan) in cells grown at a D of 0.4 h-1 and pH 5.5 with the same glucose concentration was reduced 24- to 27-fold. EII activity with methyl alpha-glucoside and 2-deoxyglucose was reduced 6- and 26-fold, respectively. Growth with excess glucose (i.e., nitrogen limitation) resulted in 26- to 88-fold repression of EII activity with these substrates. The above conditions of low pH, high dilution rate, and excess glucose also repressed EII activity for fructose (EIIFru), but to a lesser extent (two- to fivefold). Conversely, growth of S. mutans DR0001 at a D of 0.2 h-1 and pH 5.5 resulted in increased EIIGlc and EIIMan activity. Unlike the EII component, the HPr concentration in S. mutans Ingbritt varied only twofold (5.5 to 11.4 nmol/mg of protein) despite growth at pH 5.5 with limiting and excess glucose. The HPr concentrations in S. mutans DR0001 and the glucose-PTS-defective mutant DR0001/6 were similar. In a companion study, the soluble components of the PTS (i.e., HPr, EI, and EIIILac) in Streptococcus sobrinus grown on limiting lactose in a chemostat were not influenced significantly by growth at various pHs (7.0 and 5.0) and growth rates (D of 0.1, 0.54, and 0.8 h-1). However, growth on lactose resulted in repression of both EIIGlc and EIIFru, confirming earlier results with batch-grown cells. Thus, the glucose-PTS in some strains of S. mutans is regulated at the level of EII synthesis by certain environmental conditions.     

A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man‐family PTS transporter influencing SLS‐mediated hemolysis

The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)‐mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC‐encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. It was found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose‐specific EII locus, encoded by manLMN, was expressed as a mannose‐inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose‐specific EII also acted to prevent the early onset of SLS‐mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain‐specific needs.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

The phosphotransferase system of Corynebacterium glutamicum: features of sugar transport and carbon regulation

In this review, we describe the phosphotransferase system (PTS) of Corynebacterium glutamicum and discuss genes for putative global carbon regulation associated with the PTS. C. glutamicum ATCC 13032 has PTS genes encoding the general phosphotransferases enzyme I, HPr and four enzyme II permeases, specific for glucose, fructose, sucrose and one yet unknown substrate. C. gluamicum has a peculiar sugar transport system involving fructose efflux after hydrolyzing sucrose transported via sucrose EII. Also, in addition to their primary PTS, fructose and glucose are each transported by a second transporter, glucose EII and a non-PTS permease, respectively. Interestingly, C. glutamicum does not show any preference for glucose, and thus co-metabolizes glucose with other sugars or organic acids. Studies on PTS-mediated sugar uptake and its related regulation in C. glutamicum are important because the production yield of lysine and cell growth are dependent on the PTS sugars used as substrates for fermentation. In many bacteria, the PTS is also involved in several regulatory processes. However, the detailed molecular mechanism of global carbon regulation associated with the PTS in this organism has not yet been revealed.     

Routes for fructose utilization by Escherichia coli

There are three main routes for the utilization of fructose by Escherichia coli. One (Route A) predominates in the growth of wild-type strains. It involves the functioning of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and a fructose operon, mapping at min. 48.7, containing genes for a membrane-spanning protein (fruA), a 1-phosphofructose kinase (fruK) and a diphosphoryl transfer protein (fruB), under negative regulation by a fruR gene mapping at min. 1.9. A second route (Route B) also involves the PTS and membrane-spanning proteins that recognize a variety of sugars possessing the 3,4,5-D-arabino-hexoseconfiguration but with primary specificity for mannose(manXYZ), mannitol (mtlA) and glucitol (gutA) and which, if over-produced, can transport also fructose. A third route (Route C), functioning in mutants devoid of Routes A and B, does not involve the PTS: fructose diffuses into the cell via an isoform (PtsG-F) of the major glucose permease of the PTS and is then phosphorylated by ATP and a manno(fructo)kinase (Mak+) specified by a normally cryptic 1032 bp ORF (yajF) of hitherto unknown function (Mak-o), mapping at min. 8.8 and corresponding to a peptide of 344 amino acids. Conversion of the Mak-o to the Mak+ phenotypeinvolves an A24D mutation in a putative regulatory region.     

Genetic and biochemical characterization of the phosphoenolpyruvate:glucose/mannose phosphotransferase system of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB(Man), two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB(Man), IIC(Man), IID(Man), and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB(Man) as well as membrane fragments containing IIC(Man) and IID(Man). These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB(Man).     

Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.     

Separation of four components of the phosphoenolpyruvate: glucose phosphotransferase system in Vibrio parahaemolyticus.

Four classes of Vibrio parahaemolyticus mutants defective in the phosphoenolpyruvate: glucose phosphotransferase system (PTS) are described. They were phenotypically different, and were defective in different PTS components. The components designated tentatively as II, I, III, and H were separated by gel filtration of a wild-type extract. Component II, which was specific for glucose and found in the particulate fraction, is probably membrane-bound, glucose-specific enzyme II. Both components I and H were soluble proteins, and the latter was relatively heat-stable. Component I was required for phosphorylation of glucose, trehalose, fructose, mannose, and mannitol. Component H was also required for phosphorylating all the above sugars except fructose. These and some additional findings strongly suggest that components I and H correspond to enzyme I and HPr, respectively. Component III, a soluble heat-stable protein, may be equivalent to the sugar-specific factor III found in other organisms, although it seems to participate in phosphorylating two sugars, glucose and trehalose. There were evidences that mutants defective in components I and III were deficient in cyclic adenosine 3',5'-monophosphate synthesis under certain conditions.     

Concentration-dependent repression of the soluble and membrane components of the Streptococcus mutans phosphoenolpyruvate: sugar phosphotransferase system by glucose.

Growth of Streptococcus mutans Ingbritt in continuous culture (pH 7.0, dilution rate of 0.1 h-1) at medium glucose concentrations above 2.6 mM resulted in repression of the sugar-specific membrane components, enzyme IIGlc (EIIGlc) and EIIMan, of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In one experiment, significant repression (27-fold) was observed with 73 mM glucose when the glycolytic capacity of the cells was reduced by only 2-fold and when the culture was still glucose limited. In a more comprehensive experiment in which cells were grown in continuous culture at eight glucose concentrations from 2.6 to 304 mM, in addition to repression of specific EII activities for glucose, mannose, 2-deoxyglucose, and fructose, synthesis of the general protein, EI, was repressed at all glucose levels above 2.6 mM to a maximum of 4-fold at 304 mM glucose when the culture was growing with excess glucose (i.e., nitrogen limited). The other PTS general protein, HPr, was less sensitive to the exogenous glucose level but was nevertheless repressed fourfold under glucose-excess conditions. The Km for glucose for EIIGlc increased from 0.22 mM during growth at 3.6 mM glucose (glucose limited) to 0.48 mM at 271 mM glucose (glucose excess). The shift from heterofermentation to homofermentation during growth with increasing glucose levels suggests the involvement of glycolytic intermediates, ATP, or another high-energy phosphate metabolite in regulation of the synthesis of the PTS components in S. mutans.     

The phosphoenolpyruvate: sugar phosphotransferase system of Streptococcus salivarius. Identification of a IIIman protein

A double-spontaneous mutant resistant to the growth inhibitory effect of alpha-methylglucoside and 2-deoxyglucose was isolated from Streptococcus salivarius. This mutant strain, called alpha S3L11, did not grow on mannose and grew poorly on 5 mM fructose and 5 mM glucose. Isolated membranes of strain alpha S3L11 were unable to catalyse the phosphoenolpyruvate-dependent phosphorylation of mannose in the presence of purified enzyme I and HPr. Addition of dialysed membrane-free cellular extract of the wild-type strain to the reaction medium restored the activity. The factor that restored the phosphoenolpyruvate-mannose phosphotransferase activity to membranes of strain alpha S3L11 was called IIIman. This factor was partially purified from the wild-type strain by DEAE-cellulose chromatography, DEAE-TSK chromatography, and molecular seiving on a column of Ultrogel AcA 34. This partially purified preparation also enhanced the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and 2-deoxyglucose in strain alpha S3L11.     

ptsS: a new regulatory element of the fructose operon in Escherichia coli

Expression of catabolite sensitive operons is repressed in E. coli mutants devoid of HPr--a component of glucose transport system. The ptsH mutants do not utilize the substrates for phosphoenolpyruvate dependent phosphotransferase system (PTS) except for fructose. Besides that, the mutants are deficient in utilization of many substrates entering the bacteria via the other transport systems. The ptsS mutation mapped in the region of the fructose regulon on the 46th min of the chromosomal map restores the growth of ptsH mutants on all substrates. The accumulation and PEP-dependent phosphorylation of proteins substrates of PTS is also restored. The synthesis of the fructose specific phosphotransferase system becomes constitutive under the effect of ptsS mutation. The mutation is supposed to impair the regulatory region of the fructose regulon.     

The glucose permease of the phosphotransferase system of Bacillus subtilis: evidence for IIGlc and IIIGlc domains

Glucose is taken up in Bacillus subtilis via the phosphoenolpyruvate:glucose phosphotransferase system (glucose PTS). Two genes, orfG and ptsX, have been implied in the glucose-specific part of this PTS, encoding an Enzyme IIGlc and an Enzyme IIIGlc, respectively. We now show that the glucose permease consists of a single, membrane-bound, polypeptide with an apparent molecular weight of 80,000, encoded by a single gene which will be designated ptsG. The glucose permease contains domains that are 40-50% identical to the IIGlc and IIIGlc proteins of Escherichia coli. The B. subtilis IIIGlc domain can replace IIIGlc in E. coli crr mutants in supporting growth on glucose and transport of methyl alpha-glucoside. Mutations in the IIGlc and IIIGlc domains of the B. subtilis ptsG gene impaired growth on glucose and in some cases on sucrose. ptsG mutants lost all methyl alpha-glucoside transport but retained part of the glucose-transport capacity. Residual growth on glucose and transport of glucose in these ptsG mutants suggested that yet another uptake system for glucose existed, which is either another PT system or regulated by the PTS. The glucose PTS did not seem to be involved in the regulation of the uptake or metabolism of non-PTS compounds like glycerol. In contrast to ptsl mutants in members of the Enterobacteriaceae, the defective growth of B. subtilis ptsl mutants on glycerol was not restored by an insertion in the ptsG gene which eliminated IIGlc. Growth of B. subtilis ptsG mutants, lacking IIGlc, was not impaired on glycerol. From this we concluded that neither non-phosphorylated nor phosphorylated IIGlc was acting as an inhibitor or an activator, respectively, of glycerol uptake and metabolism.     

Genetic and Biochemical Characterization of the Phosphoenolpyruvate:Glucose/Mannose Phosphotransferase System of Streptococcus thermophilus

In most streptococci, glucose is transported by the phosphoenolpyruvate (PEP):glucose/mannose phosphotransferase system (PTS) via HPr and IIAB^Man, two proteins involved in regulatory mechanisms. While most strains of Streptococcus thermophilus do not or poorly metabolize glucose, compelling evidence suggests that S. thermophilus possesses the genes that encode the glucose/mannose general and specific PTS proteins. The purposes of this study were to determine (i) whether these PTS genes are expressed, (ii) whether the PTS proteins encoded by these genes are able to transfer a phosphate group from PEP to glucose/mannose PTS substrates, and (iii) whether these proteins catalyze sugar transport. The pts operon is made up of the genes encoding HPr (ptsH) and enzyme I (EI) (ptsI), which are transcribed into a 0.6-kb ptsH mRNA and a 2.3-kb ptsHI mRNA. The specific glucose/mannose PTS proteins, IIAB^Man, IIC^Man, IID^Man, and the ManO protein, are encoded by manL, manM, manN, and manO, respectively, which make up the man operon. The man operon is transcribed into a single 3.5-kb mRNA. To assess the phosphotransfer competence of these PTS proteins, in vitro PEP-dependent phosphorylation experiments were conducted with purified HPr, EI, and IIAB^Man as well as membrane fragments containing IIC^Man and IID^Man. These PTS components efficiently transferred a phosphate group from PEP to glucose, mannose, 2-deoxyglucose, and (to a lesser extent) fructose, which are common streptococcal glucose/mannose PTS substrates. Whole cells were unable to catalyze the uptake of mannose and 2-deoxyglucose, demonstrating the inability of the S. thermophilus PTS proteins to operate as a proficient transport system. This inability to transport mannose and 2-deoxyglucose may be due to a defective IIC domain. We propose that in S. thermophilus, the general and specific glucose/mannose PTS proteins are not involved in glucose transport but might have regulatory functions associated with the phosphotransfer properties of HPr and IIAB^Man.     

The ptsI gene encoding enzyme I of the phosphotransferase system of Corynebacterium glutamicum.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is widespread among bacteria where it mediates carbohydrate uptake and often serves in carbon control. Here we present cloning and analysis of the monocistronic ptsI gene of Corynebacterium glutamicum R, which encodes PTS Enzyme I (EI). EI catalyzes the first reaction of PTS and the reported ptsI was shown to complement the corresponding defect in Escherichia coli. The deduced 59.2-kDa EI of 564 amino acids shares more than 50% homology with EIs from Bacillus stearothermophilus, Bacillus subtilis, and Lactobacillus sake. Chromosomal inactivation of ptsI demonstrated that EI plays an indispensable role in PTS of C. glutamicum R and this system represents a dominant sugar uptake system. Cellobiose was only transported and utilized in adaptive mutants of C. glutamicum R. Cellobiose transport was also found to be PTS-dependent and repressed by PTS sugar glucose.     

Carbohydrate Utilization in Lactobacillus sake

The ability of Lactobacillus sake to use various carbon sources was investigated. For this purpose we developed a chemically defined medium allowing growth of L. sake and some related lactobacilli. This medium was used to determine growth rates on various carbohydrates and some nutritional requirements of L. sake. Mutants resistant to 2-deoxy-d-glucose (a nonmetabolizable glucose analog) were isolated. One mutant unable to grow on mannose and one mutant deficient in growth on mannose, fructose, and sucrose were studied by determining growth characteristics and carbohydrate uptake and phosphorylation rates. We show here that sucrose, fructose, mannose, N-acetylglucosamine, and glucose are transported and phosphorylated by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The PTS permease specific for mannose, enzyme II(supMan), was shown to be responsible for mannose, glucose, and N-acetylglucosamine transport. A second, non-PTS system, which was responsible for glucose transport, was demonstrated. Subsequent glucose metabolism involved an ATP-dependent phosphorylation. Ribose and gluconate were transported by PTS-independent permeases.     

Substrate‐dependent cluster density dynamics of Corynebacterium glutamicum phosphotransferase system permeases

Many bacteria take up carbohydrates by membrane‐integral sugar specific phosphoenolpyruvate‐dependent carbohydrate:phosphotransferase systems (PTS). Although the PTS is centrally involved in regulation of carbon metabolism in different bacteria, little is known about localization and putative oligomerization of the permease subunits (EII). Here, we analyzed localization of the fructose specific PtsF and the glucose specific PtsG transporters, as well as the general components EI and HPr from Corynebacterium glutamicum using widefield and single molecule localization microscopy. PtsF and PtsG form membrane embedded clusters that localize in a punctate pattern. Size, number and fluorescence of the membrane clusters change upon presence or absence of the transported substrate, and a direct influence of EI and HPr was not observed. In presence of the transport substrate, EII clusters significantly increased in size. Photo‐activated localization microscopy data revealed that, in presence of different carbon sources, the number of EII proteins per cluster remains the same, however, the density of these clusters reduces. Our work reveals a simple mechanism for efficient membrane occupancy regulation. Clusters of PTS EII transporters are densely packed in absence of a suitable substrate. In presence of a transported substrate, the EII proteins in individual clusters occupy larger membrane areas.