Reconstitution of maltose transport in malB mutants of Escherichia coli through calcium-induced disruptions of the outer membrane.

The barrier function of the Escherichia coli outer membrane against low concentrations of maltose in strains missing the lambda receptor was partially overcome by treating the cells for 3 h with 25 mM Ca2+. Kinetic analysis of maltose-transport revealed a Ca2+-induced shift of the apparent Km of the system from about 100 microM in cells pretreated with Tris to about 15 microM in cells pretreated with Tris plus Ca2+. In contrast to maltose transport in untreated cells, that of Ca2+-treated lamB cells was inhibited by molecules with a high molecular weight, such as amylopectin (molecular weight, 20,000), and anti-maltose-binding protein antibodies. In addition, lysozyme was shown to attack Ca2+-treated cells in contrast to untreated cells. The Ca2+-induced permeability increase of the outer membrane allowed reconstitution of maltose transport in a mutant missing the maltose-binding protein with osmotic shock fluid containing the maltose-binding protein. Even though Ca2+-treatment allowed the entry of large molecules, the release of the periplasmic maltose-binding protein or alkaline phosphatase was negligible.     

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.

The reconstitution of active transport by the Ca2+ -induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+ - concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0 degree C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0 degree C and 250 mM Ca2+ was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 X 10(9)/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 microM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 X 10(7) cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidine-binding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.     

Methyl-alpha-maltoside and 5-thiomaltose: analogs transported by the Escherichia coli maltose transport system.

Neither methyl-alpha-maltoside nor 5-thiomaltose is utilized by Escherichia coli as a sole carbon source. Both are, however, effective competitive inhibitors of maltose transport into the bacterium (Km for maltose, 0.8 microM, Ki for methyl-alpha-maltoside, 5.5 microM; Ki for 5-thiomaltose, 0.2 microM). Both analogs are bound by the periplasmic maltose-binding protein. Methyl-alpha-[14C]maltoside and 5-[3H]thiomaltose were both accumulated inside E. coli. Methyl-alpha-maltoside was unchanged after accumulation, but 5-thiomaltose was converted to an unidentified compound that could exit from the bacterium. Both analogs were inhibitory to the growth of E. coli, but only when the bacteria were previously induced for the maltose transport system. The analogs are substrates for but poor inducers of the maltose transport system.     

The role of the Escherichia coli λ receptor in the transport of maltose and maltodextrins

The λ receptor is a peptidoglycan-associated integral protein that spans the outer membrane. Beside its function in phage λ adsorption it participates in transport. The latter function can be summarized as follows: (1) Receptor allows the nonspecific permeation of small molecules other than maltose and maltodextrins (in close analogy to a molecular sieve). Here the only criterion for selectivity is size and it has the properties of an unspecific pore. In this respect, it is similar to the outer membrane proteins Ia, Ib, and Ic, the porins. (2) It is a binding protein for maltodextrins. Binding affinity is low but increases by a factor of 500 as the chain length of the maltodextrins increases. In contrast, the affinity of the periplasmic maltose-binding protein for maltose and maltodextrins is similarly high (in the μM range). (3) In the in vitro system of liposomes, the λ receptor facilitates specifically the diffusion of maltodextrins that exceed the size limit given by its porin function. This clearly demonstrates that the λ receptor alone is able to specifically overcome the permeability barrier of the outer membrane for maltodextrins. (4) From the genetic and kinetic analysis of maltose and maltodextrin transport, it can be concluded that the λ receptor interacts with the periplasmic maltose-binding protein. (5) Electron microscopic studies indicate a location for the maltose-binding protein in the outer cell envelope. This location is dependent on the presence of the λ receptor.     

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. I. Transport of maltose

The malE gene encodes the periplasmic maltose-binding protein (MBP). Nineteen mutations that still permit synthesis of stable MBP were generated by random insertion of a BamHI octanucleotide into malE and six additional mutations by in-vitro recombinations between mutant genes. The sequence changes were determined; in most cases the linker insertion is accompanied by a small deletion (30 base-pairs on average). The mutant MBP were studied for export, growth on maltose and maltodextrins, maltose transport and binding, and maltose-induced fluorescence changes. Sixteen mutant MBP (out of 21 studied in detail) were found in the periplasmic space: 12 of them retained a high affinity for maltose, and 10 activity for growth on maltose. The results show that several regions of MBP are dispensable for stability, substrate binding and export. Three regions (residues 207 to 220, 297 to 303 and 364 to 370) may be involved in interactions with the MalF or MalG proteins. A region near the C-terminal end is important for maltose binding. Two regions of the mature protein (residues 18 to 42 and 280 to 296) are required for export to, or solubility in, the periplasm.     

Aspartate and maltose-binding protein interact with adjacent sites in the Tar chemotactic signal transducer of Escherichia coli.

The Tar protein of Escherichia coli is a chemotactic signal transducer that spans the cytoplasmic membrane and mediates responses to the attractants aspartate and maltose. Aspartate binds directly to Tar, whereas maltose binds to the periplasmic maltose-binding protein, which then interacts with Tar. The Arg-64, Arg-69, and Arg-73 residues of Tar have previously been shown to be involved in aspartate sensing. When lysine residues are introduced at these positions by site-directed mutagenesis, aspartate taxis is disrupted most by substitution at position 64, and maltose taxis is disrupted most by substitution at position 73. To explore the spatial distribution of ligand recognition sites on Tar further, we performed doped-primer mutagenesis in selected regions of the tar gene. A number of mutations that interfere specifically with aspartate taxis (Asp-), maltose taxis (Mal-), or both were identified. Mutations affecting residues 64 to 73 or 149 to 154 in the periplasmic domain of Tar are associated with an Asp- phenotype, whereas mutations affecting residues 73 to 83 or 141 to 150 are associated with a Mal- phenotype. We conclude that aspartate and maltose-binding protein interact with adjacent and partially overlapping regions in the periplasmic domain of Tar to initiate attractant signalling.     

Pole cap formation in Escherichia coli following induction of the maltose-binding protein

After induction with maltose, 30–40% of the total protein in the osmotic shock fluid consist of maltose-binding protein while the induction ratio (maltose versus glycerol grown cells) for the amount of binding protein synthesized as well as for maltose transport is in the order of 10. Induction of maltose transport does not occur during all times of the cell cycle, but only shortly before cell division. Electronmicroscopic analysis of cells grown logarithmically on glycerol or maltose revealed in the latter the formation of large pole caps. These pole caps arise from an enlargement of the periplasmic space. Small cells contain one pole cap, large cells contain two. Pulse label studies with strain BUG-6, a mutant that is temperature sensitive for cell division reveal the following: Growth at the non-permissive temperature prevents maltose-binding protein synthesis and formation of new transport capacity.After shifting to the permissive temperature the cells regain both functions. Simultaneously, the newly formed cells exhibit pole caps.We conclude that the induction of maltose-binding protein is responsible for the formation of pole caps. In addition, beside the presence of inducer, cell cycle events occuring during division are necessary for the synthesis of maltose-binding protein.Non Standard Abbreviations GLPT periplasmic protein, related to transport of glycerolphosphate in Escherichia coli (Silhavy et al., 1976b)     

Residues in the alpha helix 7 of the bacterial maltose binding protein which are important in interactions with the Mal FGK2 complex.

The periplasmic maltose binding protein, MalE, is a major element in maltose transport and in chemotaxis towards this sugar. Previous genetic analysis of the MalE protein revealed functional domains involved in transport and chemotactic functions. Among them the surface located alpha helix 7, which is part of the C-lobe, one of the two lobes forming the three dimensional structure of MalE. Small deletions in this region abolished maltose transport, although maintaining wild-type affinity and specificity as well as a normal chemoreceptor function. It was suggested that alpha helix 7 may be implicated in interactions between the maltose binding protein and the membrane-bound protein complex (Duplay P, Szmelcman S. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. II. Chemotaxis towards maltose. J Mol Biol 194:675-678: Duplay P, Szmelcman S, Bedouelle H, Hofnung M. 1987. Silent and functional changes in the periplasmic maltose binding protein of Escherichia coli K12. I: Transport of maltose. J Mol Biol 194:663-673). In this study, we submitted a region of 14 residues--Asp 207 to Gly 220--encompassing alpha helix 7, to genetic analysis by oligonucleotide mediated random mutagenesis. Out of 127 identified mutations, twelve single and five double mutants with normal affinities towards maltose were selected for further investigation. Two types of mutations were characterized, silent mutations that did not affect maltose transport and mutations that heavily impaired transport kinetics, even thought the maltose binding capacity of the mutant proteins remained normal. Three substitutions at Tyr 210 (Y210S, Y210L, Y210N) drastically reduced maltose transport. One substitution at Ala 213 (A213I) and one substitution at Glu 214 (E214K) also impaired transport. These three identified residues, Tyr 210, Ala 213, and Glu 214, which are constituents of alpha helix 7, therefore seem to play some important role in maltose transport, most probably in a productive interaction between the MalE protein and the membrane bound MalFGK2 complex.     

Lateral diffusion of proteins in the periplasm of Escherichia coli.

We have introduced biologically active, fluorescently labeled maltose-binding protein into the periplasmic space of Escherichia coli and measured its lateral diffusion coefficient by the fluorescence photobleaching recovery method. Diffusion of this protein in the periplasm was found to be surprisingly low (lateral diffusion coefficient, 0.9 X 10(-10) cm2 s-1), about 1,000-fold lower than would be expected for diffusion in aqueous medium and almost 100-fold lower than for an equivalent-size protein in the cytoplasm. Galactose-binding protein, myoglobin, and cytochrome c were also introduced into the periplasm and had diffusion coefficients identical to that determined for the maltose-binding protein. For all proteins nearly 100% recovery of fluorescence was obtained after photobleaching, indicating that the periplasm is a single contiguous compartment surrounding the cell. These data have considerable implications for periplasmic structure and for the role of periplasmic proteins in transport and chemotaxis.     

Silent and functional changes in the periplasmic maltose-binding protein of Escherichia coli K12. II. Chemotaxis towards maltose

We examined the chemotactic behavior of ten Escherichia coli mutants able to synthesize a modified periplasmic maltose-binding protein (MBP) retaining high affinity for maltose. Eight were able to grow on maltose (Mal+), two were not (Mal-). In the capillary assay six out of eight of the Mal+ strains showed an optimal response at the same concentration of maltose as the wild-type strain; the amplitude of the response was strongly reduced in two Mal+ mutants and partially affected in one. The amplitude of the chemotactic response of the two Mal- strains was at least equal to that of the wild type, so that the chemotactic and transport functions of MBP were dissociated in these two cases. We define two regions of the protein (residues 297 to 303 and 364 to 369), that are important both for the chemotactic response and for transport, and one region (residues 207 to 220) that is essential for transport but dispensable for chemotaxis. Interestingly, some regions that were found to be inessential for transport are also dispensable for chemotaxis.     

Dependence of maltose transport and chemotaxis on the amount of maltose-binding protein

Maltose-binding protein (MBP) is essential for maltose transport and chemotaxis in Escherichia coli. To perform these functions it must interact with two sets of cytoplasmic membrane proteins, the MalFGK transport complex and the chemotactic signal transducer Tar. MBP is present at high concentrations, on the order of 1 mM, in the periplasm of maltose-induced or malTc constitutive cells. To determine how the amount of MBP affects transport and taxis, we utilized a series of malE signal-sequence mutations that interfere with export of MBP. The MBP content in shock fluid from cells carrying the various mutations ranged from 4 to 23% of the malE+ level. The apparent Km for maltose transport varied by less than a factor of 2 among malE+ and mutant strains. At a saturating maltose concentration 9% (approximately 90 microM) of the malE+ amount of MBP was required for half-maximal uptake rates. Transport exhibited a sigmoidal dependence on the amount of periplasmic MBP, indicating that MBP may be involved in a cooperative interaction at some stage of the transport process. The chemotactic response to a saturating maltose stimulus exhibited a first-order dependence on the amount of periplasmic MBP. Thus, interaction of a single substrate-bound MBP with Tar appears sufficient to initiate a chemotactic signal from the transducer. A half-maximal chemotactic response occurred at 25% of the malE+ MBP level, suggesting that in vivo the KD for binding of maltose-loaded MBP to Tar is quite high (approximately 250 microM).     

Facilitated diffusion of p-nitrophenyl-alpha-D-maltohexaoside through the outer membrane of Escherichia coli. Characterization of LamB as a specific and saturable channel for maltooligosaccharides

LamB, an outer membrane protein of Escherichia coli, is a component of the maltose-maltooligosaccharide transport system. We used p-nitrophenyl-alpha-D-maltohexaoside, a chromogenic analog of maltohexaose, and a periplasmic amylase that hydrolyzes this compound to study the LamB-mediated diffusion of p-nitrophenyl-alpha-D-maltohexaoside into the periplasm. Using this approach, we were able to characterize LamB in vivo as a saturable channel for maltooligosaccharides. Permeation through LamB follows Michaelis-Menten kinetics, with a Km of 0.13 mM and a Vmax of 3.3 nmol/min/10(9) cells. Previous studies suggested that maltose-binding protein increases the rate of maltooligosaccharide diffusion through LamB. We show here that, at least in strains that are unable to transport maltooligosaccharides into the cytoplasm, maltose-binding protein does not influence the rate of substrate diffusion. The periplasmic amylase had been previously described as being of the alpha-type. We have now purified this protein and analyzed its mode of action using chromogenic maltooligosaccharides of varying length. Analysis of the hydrolytic products revealed that the enzyme recognizes its substrate from the nonreducing that the enzyme recognizes its substrate from the nonreducing end and preferentially liberates maltohexaose, in contrast to the behavior of classical alpha-amylases that are endohydrolases. Using p-nitrophenyl-alpha-D-maltohexaoside as a substrate, we determined a Km of 3 microM and a Vmax of 0.14 mumol/min/mg of protein.     

Crystal structure of a defective folding protein

Maltose-binding protein (MBP or MalE) of Escherichia coli is the periplasmic receptor of the maltose transport system. MalE31, a defective folding mutant of MalE carrying sequence changes Gly 32-->Asp and Ile 33-->Pro, is either degraded or forms inclusion bodies following its export to the periplasmic compartment. We have shown previously that overexpression of FkpA, a heat-shock periplasmic peptidyl-prolyl isomerase with chaperone activity, suppresses MalE31 misfolding. Here, we have exploited this property to characterize the maltose transport activity of MalE31 in whole cells. MalE31 displays defective transport behavior, even though it retains maltose-binding activity comparable with that of the wild-type protein. Because the mutated residues are in a region on the surface of MalE not identified previously as important for maltose transport, we have solved the crystal structure of MalE31 in the maltose-bound state in order to characterize the effects of these changes. The structure was determined by molecular replacement methods and refined to 1.85 A resolution. The conformation of MalE31 closely resembles that of wild-type MalE, with very small displacements of the mutated residues located in the loop connecting the first alpha-helix to the first beta-strand. The structural and functional characterization provides experimental evidence that MalE31 can attain a wild-type folded conformation, and suggest that the mutated sites are probably involved in the interactions with the membrane components of the maltose transport system.     

Engineering and rapid selection of a low-affinity glucose/galactose-binding protein for a glucose biosensor

Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.2 microM and mediates the transport of glucose within the periplasm of E. coli. The protein undergoes a large conformational change on binding glucose and, when labeled with an environmentally sensitive fluorophore, GGBP can relay glucose concentrations, making it of potential interest as a biosensor for diabetics. This use necessitates altering the glucose affinity of GGBP, bringing it into the physiologically relevant range for monitoring glucose in humans (1.7-33 mM). To accomplish this a focused library was constructed using structure-based site-saturation mutagenesis to randomize amino acids in the binding pocket of GGBP at or near direct H-bonding sites and screening the library within the bacterial periplasm. After selection, equilibrium dissociation constants were confirmed by glucose titration and fluorescence monitoring of purified mutants labeled site-specifically at E149C with the fluorophore IANBD (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene-diamine). The screening identified a single mutation A213R that lowers GGBP glucose affinity 5000-fold to 1 mM. Computational modeling suggested the large decrease in affinity was accomplished by the arginine side chain perturbing H-bonding and increasing the entropic barrier to the closed conformation. Overall, these experiments demonstrate the ability of structure-based site-saturation mutagenesis and periplasmic expression screening to discover low-affinity GGBP mutants having potential utility for measuring glucose in humans.     

Substrate transport activation is mediated through second periplasmic loop of transmembrane protein MalF in maltose transport complex of Escherichia coli

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.     

Maltose chemoreceptor of Escherichia coli.

Strains carrying mutations in the maltose system of Escherichia coli were assayed for maltose taxis, maltose uptake at 1 and 10 muM maltose, and maltose-binding activity released by osmotic shock. An earlier conclusion that the metabolism of maltose is not necessary for chemoreception is extended to include the functioning of maltodextrin phosphorylase, the product of malP, and the genetic control of the maltose receptor by the product of malT is confirmed. Mutants in malF and malK are defective in maltose transport at low concentrations as well as high concentrations, as previously shown, but are essentially normal in maltose taxis. The product of malE has been previously shown to be the maltose-binding protein and was implicated in maltose transport. Most malE mutants are defective in maltose taxis, and all those tested are defective in maltose transport at low concentrations. Thus, as previously suggested, the maltose-binding protein probably serves as the recognition component of the maltose receptor, as well as a component of the transport system. tsome malE mutants release maltose-binding activity and are tactic toward maltose, although defective in maltose transport, implying that the binding protein has separate sites for interaction with the chemotaxis and transport systems. Some mutations in lamB, whose product is the receptor for the bacteriophage lamba, cause defects in maltose taxis, indicating some involvement of that product in maltose reception.     

Maltose-binding protein interacts simultaneously and asymmetrically with both subunits of the Tar chemoreceptor

The Tar chemotactic signal transducer of Escherichia coli mediates attractant responses to L-aspartate and to maltose. Aspartate binds across the subunit interface of the periplasmic receptor domain of a Tar homodimer. Maltose, in contrast, first binds to the periplasmic maltose-binding protein (MBP), which in its ligand-stabilized closed form then interacts with Tar. Intragenic complementation was used to determine the MBP-binding site on the Tar dimer. Mutations causing certain substitutions at residues Tyr-143, Asn-145, Gly-147, Tyr-149, and Phe-150 of Tar lead to severe defects in maltose chemotaxis, as do certain mutations affecting residues Arg-73, Met-76, Asp-77, and Ser-83. These two sets of mutations defined two complementation groups when the defective proteins were co-expressed at equal levels from compatible plasmids. We conclude that MBP contacts both subunits of the Tar dimer simultaneously and asymmetrically. Mutations affecting Met-75 could not be complemented, suggesting that this residue is important for association of MBP with each subunit of the Tar dimer. When the residues involved in interaction with MBP were mapped onto the crystal structure of the Tar periplasmic domain, they localized to a groove at the membrane-distal apex of the domain and also extended onto one shoulder of the apical region.     

Photoaffinity labeling with a neuroactive steroid analogue. 6-azi-pregnanolone labels voltage-dependent anion channel-1 in rat brain

Neuroactive steroids modulate the function of gamma-aminobutyric acid, type A (GABA(A)) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3 alpha,5 beta-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABA(A) receptors as evidenced by its ability to inhibit binding of [(35)S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABA(A) receptor subunits (alpha(1)beta(2)gamma(2)). [(3)H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of approximately 35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [(3)H]6-AziP concentration of 1.9 microM, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [(3)H]6-AziP. The beta(2) and beta(3) subunits of the GABA(A) receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABA(A) receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABA(A) receptor may be mediated by binding to an accessory protein, VDAC-1.     

Linker mutagenesis in the gene encoding the periplasmic maltose-binding protein of E. coli

A plasmid carrying the malE gene, coding for the periplasmic maltose-binding protein of E. coli, was submitted to random mutagenesis by the insertion of a BamHI linker. About 25% of the clones recovered had acquired a BamHI site in the gene malE. Most of the linker insertions were accompanied by small deletions with an average size of 30 base pairs. Among 21 mutants synthesizing a stable maltose binding protein, 8 were still able to grow on maltose. A preliminary analysis of these mutants indicates that certain regions of the protein may not be essential for maltose transport.