Facilitation of bacteriophage lambda DNA injection by inner membrane proteins of the bacterial phosphoenol-pyruvate: carbohydrate phosphotransferase system (PTS)

Infection of Escherichia coli by bacteriophage lambda depends on two membrane protein complexes: (i) maltoporin (LamB) in the outer membrane for adsorption and (ii) the IIC(Man)-IID(Man) complex of the mannose transporter in the inner membrane for DNA penetration. IIC(Man) and IID(Man) are components of the phosphoenolpyruvate: sugar phosphotransferase system (PTS) which together with the IIAB(Man) subunit mediate transport and phosphorylation of sugars. To identify structural determinants important for penetration of lambda DNA, the homologous IIC-IID complexes of E. coli, K. pneumoniae and B. subtilis, and chimeric complexes between the IIC and IID were characterized. All three complexes support sugar transport in E. coli. Only IIC-IID of E. coli and B. subtilis also support bacteriophage lambda infection. The six chimeric complexes had lost transport activity, but three containing IIC of E. coli or B. subtilis continue to support bacteriophage lambda infection. Complexes containing IIC(Man) and fusion proteins between truncated IID(Man) and alkaline phosphatase or beta-galactosidase support penetration of lambda DNA if less than 100 residues are missing from the C-terminus of IID(Man). Truncation of IIC(Man) renders the complex unstable. Taken together, these results suggest, that IIC is the major specificity determinant for lambda infection but that the IIC subunit is stably expressed only in a complex with the IID subunit. Lambda DNA in transit across the periplasmic space, but not transforming plasmid DNA, is inaccessible to the non-specific nuclease NucA of Anabaena sp. targeted to the periplasmic space either in soluble form or as a fusion protein to the C-terminus of IID(Man).     

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).     

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.     

A conserved glutamate residue, Glu-257, is important for substrate binding and transport by the Escherichia coli mannitol permease.

The mannitol permease, or D-mannitol-specific enzyme II of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) of Escherichia coli, both transports and phosphorylates its substrate. Previous analyses of the amino acid sequences of PTS permeases specific for various carbohydrates in different species of bacteria revealed several regions of similarity. The most highly conserved region includes a GIXE motif, in which the glutamate residue is completely conserved among the permeases that contain this motif. The corresponding residue in the E. coli mannitol permease is Glu-257, which is located in a large putative cytoplasmic loop of the transmembrane domain of the protein. We used site-directed mutagenesis to investigate the role of Glu-257. The properties of proteins with mutations at position 257 suggest that a carboxylate side chain at this position is essential for mannitol binding. E257A and E257Q mutant proteins did not bind mannitol detectably, while the E257D mutant could still bind this substrate. Kinetic studies with the E257D mutant protein also showed that a glutamate residue at position 257 of this permease is specifically required for efficient mannitol transport. While the E257D permease phosphorylated mannitol with kinetic parameters similar to those of the wild-type protein, the Vmax for mannitol uptake by this mutant protein is less than 5% that of the wild type. These results suggest that Glu-257 of the mannitol permease and the corresponding glutamate residues of other PTS permeases play important roles both in binding the substrate and in transporting it through the membrane.     

Genetic dissection of specificity determinants in the interaction of HPr with enzymes II of the bacterial phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli

The histidine protein (HPr) is the energy-coupling protein of the phosphoenolpyruvate (PEP)-dependent carbohydrate:phosphotransferase system (PTS), which catalyzes sugar transport in many bacteria. In its functions, HPr interacts with a number of evolutionarily unrelated proteins. Mainly, it delivers phosphoryl groups from enzyme I (EI) to the sugar-specific transporters (EIIs). HPr proteins of different bacteria exhibit almost identical structures, and, where known, they use similar surfaces to interact with their target proteins. Here we studied the in vivo effects of the replacement of HPr and EI of Escherichia coli with the homologous proteins from Bacillus subtilis, a gram-positive bacterium. This replacement resulted in severe growth defects on PTS sugars, suggesting that HPr of B. subtilis cannot efficiently phosphorylate the EIIs of E. coli. In contrast, activation of the E. coli BglG regulatory protein by HPr-catalyzed phosphorylation works well with the B. subtilis HPr protein. Random mutations were introduced into B. subtilis HPr, and a screen for improved growth on PTS sugars yielded amino acid changes in positions 12, 16, 17, 20, 24, 27, 47, and 51, located in the interaction surface of HPr. Most of the changes restore intermolecular hydrophobic interactions and salt bridges normally formed by the corresponding residues in E. coli HPr. The residues present at the targeted positions differ between HPrs of gram-positive and -negative bacteria, but within each group they are highly conserved. Therefore, they may constitute a signature motif that determines the specificity of HPr for either gram-negative or -positive EIIs.     

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.     

Solution NMR structures of productive and non-productive complexes between the A and B domains of the cytoplasmic subunit of the mannose transporter of the Escherichia coli phosphotransferase system

Solution structures of complexes between the isolated A (IIA(Man)) and B (IIB(Man)) domains of the cytoplasmic component of the mannose transporter of Escherichia coli have been solved by NMR. The complex of wild-type IIA(Man) and IIB(Man) is a mixture of two species comprising a productive, phosphoryl transfer competent complex and a non-productive complex with the two active site histidines, His-10 of IIA(Man) and His-175 of IIB(Man), separated by approximately 25A. Mutation of the active site histidine, His-10, of IIA(Man) to a glutamate, to mimic phosphorylation, results in the formation of a single productive complex. The apparent equilibrium dissociation constants for the binding of both wild-type and H10E IIA(Man) to IIB(Man) are approximately the same (K(D) approximately 0.5 mM). The productive complex can readily accommodate a transition state involving a pentacoordinate phosphoryl group with trigonal bipyramidal geometry bonded to the Nepsilon2 atom of His-10 of IIA(Man) and the Ndelta1 atom of His-175 of IIB(Man) with negligible (<0.2A) local backbone conformational changes in the immediate vicinity of the active site. The non-productive complex is related to the productive one by a approximately 90 degrees rotation and approximately 37A translation of IIB(Man) relative to IIA(Man), leaving the active site His-175 of IIB(Man) fully exposed to solvent in the non-productive complex. The interaction surface on IIA(Man) for the non-productive complex comprises a subset of residues used in the productive complex and in both cases involves both subunits of IIA(Man). The selection of the productive complex by IIA(Man)(H10E) can be attributed to neutralization of the positively charged Arg-172 of IIB(Man) at the center of the interface. The non-productive IIA(Man)-IIB(Man) complex may possibly be relevant to subsequent phosphoryl transfer from His-175 of IIB(Man) to the incoming sugar located on the transmembrane IIC(Man)-IID(Man) complex.     

Functional expression of two Bacillus subtilis chromosomal genes in Escherichia coli.

EcoRI-cleaved deoxyribonucleic acid segments carrying two genes from Bacillus subtilis, pyr and leu, have been cloned in Escherichia coli by insertion into a derivative of the E. coli bacteriophage lambda. Lysogenization of pyrimidine- and leucine-requiring auxotrophs of E. coli by the hybrid phages exhibited prototrophic phenotypes, suggesting the expression of B. subtilis genes in E. coli. Upon induction, these lysogens produced lysates capable of transducing E. coli pyr and leu auxotrophs to prototrophy with high frequency. Isolated DNAs of these bacteriophages have the ability to transform B. subtilis auxotrophs to pyr and leu independence and contain EcoRI-cleaved segments which hybridize to corresponding segments of B. subtilis.     

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.     

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.     

Secondary transporters for citrate and the Mg(2+)-citrate complex in Bacillus subtilis are homologous proteins.

Citrate uptake in Bacillus subtilis is mediated by a secondary transporter that transports the complex of citrate and divalent metal ions. The gene coding for the transporter termed CitM was cloned, sequenced, and functionally expressed in Escherichia coli. Translation of the base sequence to the primary sequence revealed a transporter that is not homologous to any known secondary transporter. However, CitM shares 60% sequence identity with the gene product of open reading frame N15CR that is on the genome of B. subtilis and for which no function is known. The hydropathy profiles of the primary sequences of CitM and the unknown gene product are very similar, and secondary structure prediction algorithms predict 12 transmembrane-spanning segments for both proteins. Open reading frame N15CR was cloned and expressed in E. coli and was shown to be a citrate transporter as well. The transporter is termed CitH. A remarkable difference between the two transporters is that citrate uptake by CitM is stimulated by the presence of Mg2+ ions, while citrate uptake by CitH is inhibited by Mg2+. It is concluded that the substrate of CitM is the Mg(2+)-citrate complex and that CitH transports the free citrate anion. Uptake experiments in right-side-out membrane vesicles derived from E. coli cells expressing either CitM or CitH showed that both transporters catalyze electrogenic proton/substrate symport.     

Enhanced metallosorption of Escherichia coli cells due to surface display of beta- and alpha-domains of mammalian metallothionein as a fusion to LamB protein

The lamB gene was inserted at with DNA fragments encoding N-terminal beta- and C-terminal alpha-domains of human metallothionein 1A (HMT1A). The hybrid LamB proteins were expressed as full-length products. Virtually whole pool of hybrid LamB proteins was found localized in the outer membrane of E. coli to and cells expressing LamB variants retained sensitivity to lambda phage, indicating their correct folding. Expression of hybrid LamB proteins increased natural ability of E. coli accumulate bivalent heavy metals ions with the highest efficiency observed for cadmium. The order of amount of cadmium accumulated is alpha-domain of HMT1A > HMT1A > beta-domain of HMT1A. This correlates with affinity for cadmium and stability of metallothionein and its individual domains. This confirms suitability of LamB vehicle for surface display of various bioactive molecules and suggests possibility of engineering of cell surface for bioremediation of heavy metals.     

The conserved cytoplasmic module of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis in Bacillus subtilis

Escherichia coli cells use two distinct sensory circuits during chemotaxis towards carbohydrates. One circuit requires the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and is independent of any specific chemoreceptor, whereas the other uses a chemoreceptor-dependent sensory mechanism analogous to that used during chemotaxis towards amino acids. Work on the carbohydrate chemotaxis sensory circuit of Bacillus subtilis reported in this article indicates that the B. subtilis circuit is different from either of those used by E. coli. Our chemotactic analysis of B. subtilis strains expressing various chimeric chemoreceptors indicates that the cytoplasmic, C-terminal module of the chemoreceptor McpC acts as a sensory-input element during carbohydrate chemotaxis. Our results also indicate that PTS-mediated carbohydrate transport, but not carbohydrate metabolism, is required for production of a chemotactic signal. We propose a model in which PTS-transport-induced chemotactic signals are transmitted to the C-terminal module of McpC for control of chemotaxis towards PTS carbohydrates.     

Isolation and characterization of ButA, a secondary glycine betaine transport system operating in Tetragenococcus halophila

Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.     

Analytical sedimentation of the IIAChb and IIBChb proteins of the Escherichia coli N,N'-diacetylchitobiose phosphotransferase system. Demonstration of a model phosphotransfer transition state complex

The phosphoenolpyruvate:glycose transferase system (PTS) is a prototypic signaling system responsible for the vectorial uptake and phosphorylation of carbohydrate substrates. The accompanying papers describe the proteins and product of the Escherichia coli N, N-diacetylchitobiose ((GlcNAc)(2)) PTS-mediated permease. Unlike most PTS transporters, the Chb system is composed of two soluble proteins, IIA(Chb) and IIB(Chb), and one transmembrane receptor (IIC(Chb)). The oligomeric states of PTS permease proteins and phosphoproteins have been difficult to determine. Using analytical ultracentrifugation, both dephospho and phosphorylated IIA(Chb) are shown to exist as stable dimers, whereas IIB(Chb), phospho-IIB(Chb) and the mutant Cys10SerIIB(Chb) are monomers. The mutant protein Cys10SerIIB(Chb) is unable to accept phosphate from phospho-IIA(Chb) but forms a stable higher order complex with phospho-IIA(Chb) (but not with dephospho-IIA(Chb)). The stoichiometry of proteins in the purified complex was determined to be 1:1, indicating that two molecules of Cys10SerIIB(Chb) are associated with one phospho-IIA(Chb) dimer in the complex. The complex appears to be a transition state analogue in the phosphotransfer reaction between the proteins. A model is presented that describes the concerted assembly and disassembly of IIA(Chb)-IIB(Chb) complexes contingent on phosphorylation-dependent conformational changes, especially of IIA(Chb).