Enzyme IIGlc Research Articles

Enzyme IIGlc contributes to trehalose metabolism in Bacillus subtilis

The tre operon of Bacillus subtilis is regulated at the transcriptional level by the Tre repressor (TreR) and by carbon catabolite repression. The molecular inducer of TreR is trehalose-6-phosphate, which is generated by transport of trehalose across the membrane by a specific Enzyme IITre (TreP) and simultaneous phosphorylation. It has been shown that TreP lacks the Enzyme IIA domain which is necessary for the phosphoryl transfer from phosphoenolpyruvate via Enzyme I and HPr. In this study, evidence is presented supporting the hypothesis of the involvement of the A domain from Enzyme IIGlc on trehalose utilization and its potential role in the regulation of the transcription of the tre operon.

Expression of a novel gene, gluP, is essential for normal Bacillus subtilis cell division and contributes to glucose export

BackgroundThe Bacillus subtilis glucokinase operon was predicted to be comprised of the genes, yqgP (now named gluP), yqgQ, and glcK. We have previously established a role for glcK in glucose metabolism. In the absence of enzymes that phosphorylate glucose, such as GlcK and/or enzyme IIGlc, accumulated cytoplasmic glucose can be transported out of the cell. Genes within the glucokinase operon were not previously known to play a role in glucose transport. Here we describe the expression of gluP and its function in glucose transport.ResultsWe found that transcription of the glucokinase operon was regulated, putatively, by two promoters: σA and σH. Putative σA and σH-recognition sites were located upstream of and within gluP, respectively. Transcriptional glucokinase operon – lacZ fusions and Northern blotting were used to analyze the expression of gluP. GluP was predicted to be an integral membrane protein. Moreover, the prediction of GluP structure revealed interesting signatures: a rhomboid domain and two tetracopeptide repeat (TPR) motifs. Microscopic analysis showed that GluP minus cells were unable to divide completely, resulting in a filamentous phenotype. The cells were grown in either rich or minimal medium. We found GluP may be involved in glucose transport. [14C]-glucose uptake by the GluP minus strain was slightly less than in the wild type. On the other hand, trehalose-derived glucose in the growth medium of the GluP minus strain was detected in very low amounts. Experimental controls comprised of single or multiple genes mutations within the glucose transporting phosphotransferase system.ConclusionsgluP seems to be regulated only by a putative σA-dependent promoter. The glucose uptake and export assays suggest that GluP is important for glucose export and may act as an exporter. This also supports the role of the glucokinase operon in glucose utilization.

The EIIGlc protein is involved in glucose-mediated activation of Escherichia coli gapA and gapB-pgk transcription.

The Escherichia coli gapB gene codes for a protein that is very similar to bacterial glyceraldehyde-3-phosphate dehydrogenases (GAPDH). In most bacteria, the gene for GAPDH is located upstream of the pgk gene encoding 3-phosphoglycerate kinase (PGK). This is the case for gapB. However, this gene is poorly expressed and encodes a protein with an erythrose 4-phosphate dehydrogenase activity (E4PDH). The active GAPDH is encoded by the gapA gene. Since we found that the nucleotide region upstream of the gapB open reading frame is responsible for part of the PGK production, we analyzed gapB promoter activity in vivo by direct measurement of the mRNA levels by reverse transcription. We showed the presence of a unique transcription promoter, gapB P0, with a cyclic AMP (cAMP) receptor protein (CRP)-cAMP binding site centered 70.5 bp upstream of the start site. Interestingly, the gapB P0 promoter activity was strongly enhanced when glucose was used as the carbon source. In these conditions, deletion of the CRP-cAMP binding site had little effect on promoter gapB P0 activity. In contrast, abolition of CRP production or of cAMP biosynthesis (crp or cya mutant strains) strongly reduced promoter gapB P0 activity. This suggests that in the presence of glucose, the CRP-cAMP complex has an indirect effect on promoter gapB P0 activity. We also showed that glucose stimulation of gapB P0 promoter activity depends on the expression of enzyme IIGlc (EIIGlc), encoded by the ptsG gene, and that the gapA P1 promoter is also activated by glucose via the EIIGlc protein. A similar glucose-mediated activation, dependent on the EIIGlc protein, was described by others for the pts operon. Altogether, this shows that when glucose is present in the growth medium expression of the E. coli genes required for its uptake (pts) and its metabolism (gapA and gapB-pgk) are coordinately activated by a mechanism dependent upon the EIIGlc protein.

Cloning and nucleotide sequence of the ptsG gene of Bacillus subtilis.

The ptsG gene of Bacillus subtilis encodes Enzyme IIGlc of the phosphoenolpyruvate: glucose phosphotransferase system. The 3' end of the gene was previously cloned and the encoded polypeptide found to resemble the Enzymes IIIGlc of Escherichia coli and Salmonella typhimurium. We report here cloning of the complete ptsG gene of B. subtilis and determination of the nucleotide sequence of the 5' end. These results, combined with the sequence of the 3' end of the gene, revealed that ptsG encodes a protein consisting of 699 amino acids and which is similar to other Enzymes II. The N-terminal domain contains two small additional fragments, which share no similarities with the closely related Enzymes IIGlc and IINag of E. coli but which are present in the IIGlc-like protein encoded by the E. coli malX gene.

Control of glucose metabolism by enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli

The quantitative effects of variations in the amount of enzyme IIGlc of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) on glucose metabolism in Escherichia coli were studied. The level of enzyme IIGlc could be adjusted in vivo to between 20 and 600% of the wild-type chromosomal level by using the expression vector pTSG11. On this plasmid, expression of the structural gene for enzyme IIGlc, ptsG, is controlled by the tac promoter. As expected, the control coefficient (i.e., the relative increase in pathway flux, divided by the relative increase in amount of enzyme) of enzyme IIGlc decreased in magnitude if a more extensive pathway was considered. Thus, at the wild-type level of enzyme IIGlc activity, the control coefficient of this enzyme on the growth rate on glucose and on the rate of glucose oxidation was low, while the control coefficient on uptake and phosphorylation of methyl alpha-glucopyranoside (an enzyme IIGlc-specific, nonmetabolizable glucose analog) was relatively high (0.55 to 0.65). The implications of our findings for PTS-mediated regulation, i.e., inhibition of growth on non-PTS compounds by glucose, are discussed.

Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052

The glucose phosphotransferase system (PTS) of Clostridium acetobutylicum was studied by using cell extracts. The system exhibited a Km for glucose of 34 microM, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose. The analogs 3-O-methylglucoside and methyl alpha-glucoside did not inhibit glucose phosphorylation significantly. Activity showed no dependence on Mg2+ ions or on pH in the range 6.0 to 8.0. The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli. In addition to a membrane-bound enzyme IIGlc, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and IIIGlc components. The HPr was found in the soluble fraction of C. acetobutylicum extracts, whereas enzyme I, and probably also IIIGlc, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell.

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.

Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc.

The glucose permease of Bacillus subtilis is a single polypeptide chain that functions to energize the sucrose permease.

Biochemical, immunological, and sequence analyses demonstrated that the glucose permease of Bacillus subtilis, the glucose-specific Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system, is a single polypeptide chain with a C-terminal Enzyme III-like domain. A flexible hydrophilic linker, similar in length and amino acid composition to linkers previously identified in other regulatory or sensory transducing proteins, functions to tether the Enzyme IIIGlc-like domain of the protein to the membrane-embedded Enzyme IIGlc. Evidence is presented demonstrating that the Enzyme IIIGlc-like domain of the glucose permease plays a dual role and functions in the transport and phosphorylation of both glucose and sucrose. The sucrose permease appears to lack a sucrose-specific Enzyme III-like domain or a separate, soluble IIIScr protein. Enzyme IIScr was capable of utilizing the IIIGlc-like domain of the glucose permease regardless of whether the IIIGlc polypeptide was provided as a purified, soluble protein, as a membrane-bound protein within the same membrane as Enzyme IIScr, or as a membrane-bound protein within membrane fragments different from those bearing Enzyme IIScr. These observations suggest that the IIIGlc-like domain is an autonomous structural unit that assumes a conformation independent of the hydrophobic, N-terminal intramembranal domain of Enzyme IIGlc. Preferential uptake and phosphorylation of glucose over sucrose has been demonstrated by both in vivo transport studies and in vitro phosphorylation assays. Addition of the purified IIIGlc-like domain strongly stimulated the phosphorylation of sucrose, but not that of glucose, in phosphorylation assays that contained the two sugars simultaneously. The results suggest that the preferential uptake of glucose over sucrose is determined by competition of the corresponding sugar-specific permeases for the common P approximately IIIGlc/Scr domain.

Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions

Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations.

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.

Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: nucleotide sequence of ptsX, ptsH and the 5’‐end of ptsl and evidence for a ptsHI operon

The nucleotide sequence of a 1689bp fragment of the Bacillus subtilis locus containing ptsX (a crr-like gene), ptsH (coding for HPr), and the 5'-end of ptsI (coding for Enzyme I) was determined. The deduced amino acid sequences of ptsH and the N-terminal part of ptsI were compared to those of Streptococcus faecalis and Escherichia coli. Transcription fusion demonstrated that ptsHI constitutes an operon. An open reading frame overlapping the main part of ptsH in the opposite sense was shown to be expressed in vivo, using protein fusions with beta-galactosidase. The deduced amino acid sequence of ptsX showed significant homology with that of Salmonella typhimurium glucose-specific Enzyme III. ptsX was preceded by an open reading frame whose amino acid sequence showed strong homology with the C-terminal part of E. coli Enzyme IIGlc.

The ptsH, ptsI, and crr genes of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system: a complex operon with several modes of transcription

The ptsH, ptsI, and crr genes, coding for three of the proteins of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) (HPr, enzyme I, and enzyme IIIGlc, respectively) have been studied by determination of their nucleotide sequence and analysis of their expression. The three genes constitute an operon, but analysis of the ptsH, ptsI, and crr transcripts by Northern (RNA) blotting revealed the existence of three major mRNA species. One encompassed the three cistrons, a second one the ptsH gene and part of the ptsI gene, and the third one only the distal gene crr. The short crr transcripts were initiated inside the ptsI open reading frame at points which were identified by S1 mapping. Expression of the genes was studied in vivo by using operon and protein fusions between the lacZ gene and the ptsH, ptsI, or crr gene on IncW low-copy-number plasmids. The present study showed that (i) the ptsH, ptsI, and crr genes exhibited high basal expression, (ii) transcription of the ptsH and ptsI genes was stimulated threefold by the cyclic AMP-cyclic AMP receptor protein complex and also by growth on glucose, but only in the presence of an active enzyme IIGlc, (iii) crr-specific expression was not sensitive to the complex or to growth on glucose, and (iv) under the growth conditions tested, the major part of crr transcription was initiated from internal promoters.

Glucose-permease of the bacterial phosphotransferase system. Gene cloning, overproduction, and amino acid sequence of enzyme IIGlc.

The glucose-permease (IIGlc) of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It also functions as a receptor for bacterial chemotaxis. The structural gene of the permease, ptsG, has been cloned on a multicopy plasmid, and transformants constitutively overproducing the protein 10-15 times over wild-type level have been isolated. Overproduction is slightly inhibited if transformants are grown in a glucose-containing medium. The complete amino acid sequence of the glucose-permease is deduced from the nucleotide sequence. It consists of 477 residues and is moderately hydrophobic. A comparison of the glucose-permease with the mannitol-permease (Lee, C. A., and Saier, M. H., Jr. (1983) J. Biol. Chem. 258, 10761-10767) does not reveal any obvious homology at the level of amino acid sequence.

Identification and properties of distinct sucrose and glucose phosphotransferase enzyme II activities in Streptococcus mutans 6715g.

We investigated phosphoenolpyruvate-dependent phosphotransferase system enzyme II activities for sucrose and glucose in Streptococcus mutans 6715g. Two integral membrane proteins, enzyme IIscr and enzyme IIglc, each specific for its sugar substrate, sucrose or glucose, were identified by their abilities to catalyze specific sugar:sugar-phosphate exchange reactions. Some of the properties of these two transport proteins are also presented.

Glucose phosphoenolpyruvate-dependent phosphotransferase system of Streptococcus mutans GS5 studied by using cell-free extracts.

The glucose phosphotransferase system (PTS) of Streptococcus mutans GS5 has been partially characterized, using fractions derived from cells treated with the muramidase mutanolysin. Membranes retained functional PTS enzymes for the phosphoenolpyruvate-dependent phosphorylation of glucose, fructose, and mannose. This was confirmed by assaying membranes directly for enzyme I (EI) and enzyme IIglc (EIIglc) by employing specific phosphoryl-exchange reactions for each factor. Membranes prepared from glucose PTS- mutants, however, were either deficient in glucose phosphorylation or reflected the "leakiness" displayed by whole cells. Mutant membranes were unable to catalyze the glucose:glucose 6-phosphate transphosphorylation reaction, indicating a defective EIIglc in these fractions. Although total cellular EI activities in the mutant clones were about the same as that measured for the wild-type strain by employing the pyruvate:phosphoenolpyruvate phosphoryl-exchange reaction, mutant membranes were found to possess less than 10% of the specific EI activity of wild-type membranes. The cytoplasmic fractions of mutants, however, displayed markedly increased specific activities for this enzyme when compared with wild-type extracts. These results strongly suggest a molecular association of EI with a normal membrane protein, perhaps EIIglc, that is absent in mutants. This would explain the absence of fructose PTS activity in glucose PTS- mutant membranes despite the fact that whole cells of these clones are normal for this transport function.

Vicinal dithiol-disulfide distribution in the Escherichia coli mannitol specific carrier enzyme IImtl.

Escherichia coli mannitol specific EII in membrane vesicles can be inhibited by the action of the oxidizable substrate-reduced phenazine methosulfate (PMS) in a manner similar to E. coli enzyme IIGlc [Robillard, G. T., & Konings, W. (1981) Biochemistry 20, 5025-5032]. The fact that reduced PMS and various oxidizing agents protect the enzyme from inactivation by the sulfhydryl reagents N-ethylmaleimide and bromopyruvate suggests that the active form possesses a dithiol which can be protected by conversion to a disulfide. The sulfhydryl-disulfide distribution has been examined in purified EIImtl by labeling studies with N-[1-14C]ethylmaleimide ( [14C]NEM). EIImtl can be alkylated at three positions per peptide chain. When alkylation takes place in 8 M urea, only two positions are labeled. The third position becomes labeled in urea only after treatment with DTT, suggesting that the native enzyme is composed of two subunits linked by a disulfide bridge. The remaining two sulfhydryl groups per peptide chain appear to undergo changes in oxidation state as indicated by the following results. (1) Treatment of the active enzyme with NEM leads to complete inactivation and incorporation of 1 mol of [14C]NEM per peptide chain. Oxidizing agents protect the activity and prevent labeling presumably by forming a disulfide. (2) Phosphorylating the enzyme (one phosphoryl group per peptide chain) fully protects the activity, but 1 mol of NEM per peptide chain is still incorporated. Subsequent dephosphorylation by adding mannitol causes a second mole of [14C]NEM to be incorporated and results in complete inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial phosphotransferase system. Solubilization and purification of the glucose-specific enzyme II from membranes of Salmonella typhimurium.

The glucose-specific enzyme II (IIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium has been purified to homogeneity. Purification included the following steps: detergent solubilization of membranes in polydisperse octyloligooxyethylene, isoelectrofocusing, chromatofocusing, and either glycerol gradient centrifugation or gel filtration, all in the presence of the same detergent. Enzymatic activity was assayed as phosphoenolpyruvate-dependent phosphorylation of methyl-alpha-D-glucopyranoside. It could be measured after detergent dilution only and required the presence of phosphatidylglycerol in a sonicated suspension. An antiserum prepared against enzyme IIGlc specifically inhibited phosphorylation of methyl-alpha-D-glucopyranoside. In the solubilized state, purified enzyme IIGlc exists as a complex of molecular weight of 105,000 and a sedimentation coefficient of 3.8 S. In polyacrylamide gels in sodium dodecyl sulfate, it has an apparent molecular weight of about 40,000.

Stereochemical course of the reactions catalyzed by the bacterial phosphoenolpyruvate:glucose phosphotransferase system.

The overall stereochemical course of the reactions leading to the phosphorylation of methyl alpha-D-glucopyranoside by the glucose-specific enzyme II (enzyme IIGlc) of the Escherichia coli phosphotransferase system has been investigated. With [(R)-16O,17O,18O]phosphoenolpyruvate as the phosphoryl donor and in the presence of enzyme I, HPr, and enzyme IIIGlc of the phosphotransferase system, membranes from E. coli containing enzyme IIGlc catalyzed the formation of methyl alpha-D-glucopyranoside 6-phosphate with overall inversion of the configuration at phosphorus (with respect to phosphoenolpyruvate). It has previously been shown that sequential covalent transfer of the phosphoryl group of phosphoenolpyruvate to enzyme I, to HPr, and to enzyme IIIGlc occurs before the final transfer from phospho-enzyme IIIGlc to the sugar, catalyzed by enzyme IIGlc. Because overall inversion of the configuration of the chiral phospho group of phosphoenolpyruvate implies an odd number of transfer steps, the phospho group has been transferred at least five times, and transfer from phospho-enzyme IIIGlc to the sugar must occur in two steps (or a multiple thereof). On the basis that no membrane protein other than enzyme IIGlc is directly involved in the final phospho transfer steps, our results imply that a covalent phospho-enzyme IIGlc is an intermediate during transport and phosphorylation of glucose by the E. coli phosphotransferase system.

Different sidedness of functionally homologous essential thiols in two membrane-bound phosphotransferase enzymes of Escherichia coli detected by permeant and nonpermeant thiol reagents.

The membrane-bound Enzyme IIbgl and IIglc are both inactivated in vivo by the sulfhydryl reagent N-ethylmaleimide. The former is also inhibited by the hydrophilic sulfhydryl reagents p-chloromercuribenzoic acid and p-mercuriphenylsulfonic acid, while the latter is resistant to these reagents. However, inhibition of this enzyme is observed after impairment, either transient or permanent, of the permeability barrier of bacterial envelopes. Since p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid are able to cross the outer membrane of Escherichia coli, their failure to inhibit in vivo Enzyme IIglc must be due to their inability to cross the inner membrane of the bacteria. It would therefore appear that sensitive thiol group(s) of Enzyme IIglc and Enzyme IIbgl, in spite of their functional similarity, exhibit opposite orientation within the cytoplasmic membrane, the first enzyme having an -SH group accessible from the outer surface of the membrane, while the second has an -SH group accessible from the inner surface of the membrane. The present results strengthen the view that these two enzymes have in asymmetric orientation within the membrane as already suggested by their vectorial function.