Involvement of the central loop of the lactose permease of Escherichia coli in its allosteric regulation by the glucose-specific enzyme IIA of the phosphoenolpyruvate-dependent phosphotransferase system.

Allosteric regulation of several sugar transport systems such as those specific for lactose, maltose and melibiose in Escherichia coli (inducer exclusion) is mediated by the glucose-specific enzyme IIA (IIAGlc) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Deletion mutations in the cytoplasmic N and C termini of the lactose permease protein, LacY, and replacement of all cysteine residues in LacY with other residues did not prevent IIAGlc-mediated inhibition of lactose uptake, but several point and insertional mutations in the central cytoplasmic loop of this permease abolished transport regulation and IIAGlc binding. The results substantiate the conclusion that regulation of the lactose permease in E. coli by the PTS is mediated by a primary interaction of IIAGlc with the central cytoplasmic loop of the permease.     

Interactions in vivo between IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and the glycerol and maltose uptake systems of Salmonella typhimurium

Our previous studies indicated that the ability of phosphoenolpyruvate:sugar phosphotransferase system (PTS) substrates to inhibit the uptake of glycerol or maltose in Salmonella typhimurium is dependent on the relative cellular content of the PTS-sensitive uptake system and of the PTS protein IIIGlc. Our present study confirms and extends those observations. The maltose and glycerol uptake systems are rendered (wholly or partially) insensitive to PTS inhibition by the presence of a second PTS-sensitive uptake system (respectively that for glycerol or maltose) and its substrate. Both the second PTS-sensitive uptake system and its substrate were needed for this protective effect. Galactose and the galactose permease (a PTS-insensitive transport system) did not have any effect on PTS-mediated inhibition of the maltose uptake system. The protective effect of the second PTS-sensitive uptake system and its substrate is counteracted by increasing the cellular levels of IIIGlc. Overproduction of IIIGlc in crr-plasmid-containing strains renders the glycerol and maltose uptake systems hypersensitive to inhibition by PTS substrates. We interpret our results on the basis of a stoichiometric interaction between IIIGlc and a PTS-sensitive uptake system, in which the IIIGlc--transport-system complex is inactive. Competition between two PTS-sensitive transport systems for formation of inactive complex with IIIGlc lowers the free intracellular concentration of IIIGlc resulting in a mutual protective effect against inhibition by IIIGlc.     

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.     

Physiological studies on regulation of glycerol utilization by the phosphoenolpyruvate:sugar phosphotransferase system in Enterococcus faecalis.

In vitro studies with purified glycerol kinase from Enterococcus faecalis have established that this enzyme is activated by phosphorylation of a histidyl residue in the protein, catalyzed by the phosphoenolpyruvate-dependent phosphotransferase system (PTS), but the physiological significance of this observation is not known. In the present study, the regulation of glycerol uptake was examined in a wild-type strain of E. faecalis as well as in tight and leaky ptsI mutants, altered with respect to their levels of enzyme I of the PTS. Glycerol kinase was shown to be weakly repressible by lactose and strongly repressible by glucose in the wild-type strain. Greatly reduced levels of glycerol kinase activity were also observed in the ptsI mutants. Uptake of glycerol into intact wild-type and mutant cells paralleled the glycerol kinase activities in extracts. Glycerol uptake in the leaky ptsI mutant was hypersensitive to inhibition by low concentrations of 2-deoxyglucose or glucose even though the rates and extent of 2-deoxyglucose uptake were greatly reduced. These observations provide strong support for the involvement of reversible PTS-mediated phosphorylation of glycerol kinase in the regulation of glycerol uptake in response to the presence or absence of a sugar substrate of the PTS in the medium. Glucose and 2-deoxyglucose were shown to elicit rapid efflux of cytoplasmic [14C]lactate derived from [14C]glycerol. This phenomenon was distinct from the inhibition of glycerol uptake and was due to phosphorylation of the incoming sugar by cytoplasmic phosphoenolpyruvate. Lactate appeared to be generated by sequential dephosphorylation and reduction of cytoplasmic phosphoenolpyruvate present in high concentrations in resting cells. The relevance of these findings to regulatory phenomena in other bacteria is discussed.     

Autoregulation of lactose uptake through the LacY permease by enzyme IIAGlc of the PTS in Escherichia coli K-12

Bacterial growth on one or more carbon sources requires careful control of the uptake and metabolism of these carbon sources. In Escherichia coli, the phosphorylation state of enzyme IIAGlc of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) is involved in this control in two ways. The unphosphorylated form of IIAGlc causes 'inducer exclusion', the inhibition of uptake of a number of non-PTS carbon sources, including lactose uptake by the lactose permease. The phosphorylated form of enzyme IIAGlc probably activates adenylate cyclase. In cells growing on lactose, enzyme IIAGlc was approximately 50% dephosphorylated, suggesting that lactose could inhibit its own uptake. This inhibition could be demonstrated by comparing lactose uptake rates in the wild-type strain and in a mutant in which the lactose carrier was insensitive to inducer exclusion. In this deregulated mutant strain, lactose was consumed much faster, and large amounts of glucose were excreted. It was shown that enzyme IIAGlc was dephosphorylated more strongly and that the cAMP level was lower in the mutant, most probably causing the observed decrease in lac expression level. When the lac expression level in the mutant strain was increased to that of the parent strain by adding exogenous cAMP, growth on lactose was slower, suggesting that enzyme IIAGlc-mediated inhibition of lactose uptake and downregulation of the lac expression level protected the cells against excessive lactose influx. An even stronger increase in the lac expression level in a mutant lacking enzyme IIAGlc caused complete growth arrest. We conclude that the autoregulatory mechanism that controls lactose uptake is an important mechanism for the cells in adjusting the uptake rate to their metabolic capacity.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Enterotoxin A synthesis in Staphylococcus aureus: inhibition by glycerol and maltose

Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS- mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of beta-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.     

Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system inSalmonella typhimurium LT2

InEnterobacteriaceae the nonphosphorylated form of IIA^G1c of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the Ma1K protein of the maltose transport system, respectively. In this report we show that the IIA^Glc-Iike domain of the membrane-bound II^{N-acetylglucosamine} (II^Nag) of the PTS can replace IIA^Glc in aSalmonella typhimurium crr mutant strain that lacks all soluble IIA^Glc. The inhibition was most severe in cells which were partially induced for the glycerol or maltose up take systems. TheStreptococcus thermophilus lactose transporter LacS, which also contains a IIA^Glc-like domain, could not replace IIA^Glc. Neither IINag nor LacS could replace IIA^Glc in activation of adenylate cyclase.     

Replacing the general energy-coupling proteins of the phospho-enol-pyruvate: sugar phosphotransferase system of Salmonella typhimurium with fructose-inducible counterparts results in the inability to utilize nonphosphotransferase system sugars

A Salmonella typhimurium mutant lacking Enzyme I and HPr, general proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), but producing homologues EI(Fructose) and FPr constitutively, did not grow in minimal medium supplemented with non-PTS sugars (melibiose, glycerol, and maltose) in the absence of any trace of Luria-Bertani broth; adding cyclic AMP allowed growth. On melibiose, rapid growth began only when melibiose permease activity had reached a threshold level. Wild-type cultures reached this level within about 2 h, but the mutant only after a 12-14 h lag period, and then only when cyclic AMP had been added to the medium. On a mixture of melibiose and a PTS sugar, permease was undetectable in either the wild type or mutant until the PTS sugar had been exhausted. Permease then appeared, increasing with time, but in the mutant it never reached the threshold allowing rapid growth on melibiose unless cyclic AMP had been added. On rich medium supplemented with melibiose or glycerol, the mutant produced lower (30%) levels of melibiose permease or glycerol kinase compared with the wild type. We propose that poor phosphorylation of the regulatory protein Enzyme IIA(Glucose), leading to constitutive inducer exclusion and catabolite repression in this strain, accounts for these results.     

IIAGlc inhibition of glycerol kinase: a communications network tunes protein motions at the allosteric site

Steady-state and time-resolved fluorescence anisotropy methods applied to an extrinsic fluorophore that is conjugated to non-native cysteine residues demonstrate that amino acids in an allosteric communication network within a protein subunit tune protein backbone motions at a distal site to enable allosteric binding and inhibition. The unphosphorylated form of the phosphocarrier protein IIAGlc is an allosteric inhibitor of Escherichia coli glycerol kinase, binding more than 25 A from the kinase active site. Crystal structures that showed a ligand-dependent conformational change and large temperature factors for the IIAGlc-binding site on E. coli glycerol kinase suggest that motions of the allosteric site have an important role in the inhibition. Three E. coli glycerol kinase amino acids that are located at least 15 A from the active site and the allosteric site were shown previously to be necessary for transplanting IIAGlc inhibition into the nonallosteric glycerol kinase from Haemophilus influenzae. These three amino acids are termed the coupling locus. The apparent allosteric site motions and the requirement for the distant coupling locus to transplant allosteric inhibition suggest that the coupling locus modulates the motions of the IIAGlc-binding site. To evaluate this possibility, variants of E. coli glycerol kinase and the chimeric, allosteric H. influenzae glycerol kinase were constructed with a non-native cysteine residue replacing one of the native residues in the IIAGlc-binding site. The extrinsic fluorophore Oregon Green 488 (2',7'-difluorofluorescein) was conjugated specifically to the non-native cysteine residue. Steady-state and time-resolved fluorescence anisotropy measurements show that the motions of the fluorophore reflect backbone motions of the IIAGlc-binding site and these motions are modulated by the amino acids at the coupling locus.     

A Cys-less variant of the bacterial ATP binding cassette protein MalK is functional in maltose transport and regulation

The cysteine residues of the ABC protein MalK from Salmonella typhimurium maltose transport system (C40, C350, C360) were consecutively replaced by serines. Cys-less MalK was fully functional in maltose transport in vivo. Moreover, the activity of MalK as a repressor of other maltose-regulated genes was also retained. The absence of cysteine residues in the purified protein was verified by its failure to react with fluorescein-5-maleimide. In contrast to purified wild-type MalK, the ATPase activity of the C40S variant was insensitive to inhibition by N-ethylmaleimide.     

beta-D-phosphogalactoside galactohydrolase of Streptococcus faecalis and the inhibition of its synthesis by glucose.

The lactose hydrolysing system of Streptococcus faecalis is described. It is closely related to that one of the group N streptocci as it consists of a beta-D-phosphogalactoside galactohydrolase (beta-Pgal). The uptake of methyl-beta-D-thiogalactoside (TMG), lactose, and glucose is maintained by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) but the uptake of galactose is not. The induction time is 6--7 min. Inducers are lactose and galactose but not isopropyl-beta-D-galactoside (IPTG) and TMG. In the presence of glucose, mannose, and maltose no induction of beta-Pgal occurs but pyruvate and glycerol allow induction. The competitive inhibition of uptake of TMG by glucose suggests inducer exclusion by this sugar. TMG accumulates in the cells exclusively as a derivative.     

Phosphatidylglycerol Directs Binding and Inhibitory Action of EIIAGlc Protein on the Maltose Transporter

The signal-transducing protein EIIA^Glc belongs to the phosphoenolpyruvate carbohydrate phosphotransferase system. In its dephosphorylated state, EIIA^Glc is a negative regulator for several permeases, including the maltose transporter MalFGK₂. How EIIA^Glc is targeted to the membrane, how it interacts with the transporter, and how it inhibits sugar uptake remain obscure. We show here that acidic phospholipids together with the N-terminal tail of EIIA^Glc are essential for the high affinity binding of the protein to the transporter. Using protein docking prediction and chemical cross-linking, we demonstrate that EIIA^Glc binds to the MalK dimer, interacting with both the nucleotide-binding and the C-terminal regulatory domains. Dissection of the ATPase cycle reveals that EIIA^Glc does not affect the binding of ATP but rather inhibits the capacity of MalK to cleave ATP. We propose a mechanism of maltose transport inhibition by this central amphitropic regulatory protein.     

Carbon catabolite repression by the catabolite control protein CcpA in Staphylococcus xylosus

Carbon catabolic repression (CR) by the catabolite control protein CcpA has been analyzed in Staphylococcus xylosus. Genes encoding components needed to utilize lactose, sucrose, and maltose were found to be repressed by CcpA. In addition, the ccpA gene is under negative autogenous control. Among several tested sugars, glucose caused strongest CcpA-dependent repression. Glucose can enter S. xylosus in nonphosphorylated form via the glucose uptake protein GlcU. Internal glucose is then phosphorylated by the glucose kinase GlkA. Alternatively, glucose can be transported and concomitantly phosphorylated by glucose-specific permease(s) of the phosphotransferase system (PTS). S. xylosus mutant strains deficient in GlcU or GlkA showed partial relief of glucose-specific, CcpA-dependent repression. Likewise, blocking PTS activity completely by inactivation of the gene encoding the general PTS protein enzyme I resulted in diminished glucose-mediated repression. Thus, both glucose entry routes contribute to glucose-specific CR in S. xylosus. The sugar transport activity of the PTS is not required to trigger glucose-specific repression. The phosphocarrier protein HPr however, is absolutely essential for CcpA activity. Inactivation of the HPr gene led to a complete loss of CR. Repression is also abolished upon inactivation of the HPr kinase gene or by replacing serine at position 46 of HPr by alanine. These results clearly show that HPr kinase provides the signal, seryl-phosphorylated HPr, to activate CcpA in S. xylosus.     

Effects of mutations and truncations on the kinetic behavior of IIAGlc, a phosphocarrier and regulatory protein of the phosphoenolpyruvate phosphotransferase system of Escherichia coli

IIAGlc, a component of the glucose-specific phosphoenolpyruvate:phosphotransferase system (PTS) of Escherichia coli, is important in regulating carbohydrate metabolism. In Glc uptake, the phosphotransfer sequence is: phosphoenolpyruvate --> Enzyme I --> HPr --> IIAGlc --> IICBGlc --> Glc. (HPr is the first phosphocarrier protein of the PTS.) We previously reported two classes of IIAGlc mutations that substantially decrease the P-transfer rate constants to/from IIAGlc. A mutant of His75 which adjoins the active site (His90), (H75Q), was 0.5% as active as wild-type IIAGlc in the reversible P-transfer to HPr. Two possible explanations were offered for this result: (a) the imidazole ring of His75 is required for charge delocalization and (b) H75Q disrupts the hydrogen bond network: Thr73, His75, phospho-His90. The present studies directly test the H-bond network hypothesis. Thr73 was replaced by Ser, Ala, or Val to eliminate the network. Because the rate constants for phosphotransfer to/from HPr were largely unaffected, we conclude that the H-bond network hypothesis is not correct. In the second class of mutants, proteolytic truncation of seven residues of the IIAGlc N terminus caused a 20-fold reduction in phosphotransfer to membrane-bound IICBGlc from Salmonella typhimurium. Here, we report the phosphotransfer rates between two genetically constructed N-terminal truncations of IIAGlc (Delta7 and Delta16) and the proteins IICBGlc and IIBGlc (the soluble cytoplasmic domain of IICBGlc). The truncations did not significantly affect reversible P-transfer to IIBGlc but substantially decreased the rate constants to IICBGlc in E. coli and S. typhimurium membranes. The results support the hypothesis (Wang, G., Peterkofsky, A., and Clore, G. M. (2000) J. Biol. Chem. 275, 39811-39814) that the N-terminal 18-residue domain "docks" IIAGlc to the lipid bilayer of membranes containing IICBGlc.     

Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli. Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile.

Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile. Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake. Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside. Protein synthesis did not appear to be required for these effects. The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation. Inhibition was abolished by the crr mutation. The results suggest that Enzyme I functions as a catalytic component of the regulatory system. Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system.     

Regulation of glycerol uptake by the phosphoenolpyruvate-sugar phosphotransferase system in Bacillus subtilis.

Enteric bacteria have been previously shown to regulate the uptake of certain carbohydrates (lactose, maltose, and glycerol) by an allosteric mechanism involving the catalytic activities of the phosphoenolpyruvate-sugar phosphotransferase system. In the present studies, a ptsI mutant of Bacillus subtilis, possessing a thermosensitive enzyme I of the phosphotransferase system, was used to gain evidence for a similar regulatory mechanism in a gram-positive bacterium. Thermoinactivation of enzyme I resulted in the loss of methyl alpha-glucoside uptake activity and enhanced sensitivity of glycerol uptake to inhibition by sugar substrates of the phosphotransferase system. The concentration of the inhibiting sugar which half maximally blocked glycerol uptake was directly related to residual enzyme I activity. Each sugar substrate of the phosphotransferase system inhibited glycerol uptake provided that the enzyme II specific for that sugar was induced to a sufficiently high level. The results support the conclusion that the phosphotransferase system regulates glycerol uptake in B. subtilis and perhaps in other gram-positive bacteria.     

Overproduction, solubilization, and reconstitution of the maltose transport system from Escherichia coli

Maltose is transported across the cytoplasmic membrane of Escherichia coli by a binding protein-dependent transport system. We observed a 10-fold increase in the level of transport activity in assays with membrane vesicles when the three membrane-associated components of the transport system (the MalF, MalG, and MalK proteins) were overproduced. In addition, we have successfully reconstituted maltose transport activity in proteoliposome vesicles from solubilized proteins using a detergent dilution procedure. The addition of ATP as an energy source was sufficient to obtain transport, and this activity was dependent on the presence of maltose binding protein and was not seen in proteoliposomes prepared from a strain with a deletion of the maltose genes. We determined that hydrolysis of ATP was directly coupled to maltose uptake. In the majority of these experiments, an average of 1.4 mol of ATP was hydrolyzed for each mole of maltose accumulated. However, in the remaining experiments, ATP hydrolysis was observed to be much higher and averaged 17 mol of ATP hydrolyzed per mol of maltose transported. Possible explanations for a variable stoichiometry are discussed. These results provide strong evidence that it is the hydrolysis of ATP by a component of the transport complex that provides the energy required for active maltose transport.