Maltose transport in Escherichia coli K-12: involvement of the bacteriophage lambda receptor.

Mutants affected in lamB, the structural gene for phage lambda receptor, are unable to utilize maltose when it is present at low concentrations (less than or equal 10 muM). During growth in a chemostat at limiting maltose concentrations, the lamB mutants tested were selected against in the presence of the wild-type strain. Transport studies demonstrate that most lamB mutants have deficient maltose transport capacities at low maltose concentrations. When antibodies against purified phage lambda receptor are added to a wild-type strain, transport of maltose at low concentrations is significantly reduced. These results strongly suggest that the phage lambda receptor molecule is involved in maltose transport.     

Transmembrane permeability channels in vesicles reconstituted from single species of porins from Salmonella typhimurium.

Aggregates of the "major" outer membrane proteins, "porins," of Salmonella typhimurium form diffusion channels in reconstituted vesicle membranes. The aggregate consists of three species of porins with apparent molecular weights of 34,000, 35,000, and 36,000 when active aggregates are subjected to sodium dodecyl sulfate-acrylamide gel electrophoresis after heating in the presence of sodium dodecyl sulfate (Nakae, J. Biol. Chem. 251:2176-2178, 1976). Single species of porins were isolated by solubilization of membranes and subsequent gel filtration in the presence of sodium dodecyl sulfate from the mutant strains of Salmonella typhimurium that produced only single species of porin. The single species of porins of either 34,000, 35,000, or 36,000 daltons formed diffusion channels when assayed for sucrose permeability in the vesicle membranes reconstituted from porins, phospholipids, and lipopolysaccharides. The exclusion limits of the pores made of single species of porins were not distinguishable from each other and from the exclusion limits of the pores made of the porin aggregates from the wild-type strain, when the permeability of vesicle membranes to radioactive di-, tri-, and tetrasaccharides and to various sizes of radioactive polyethylene glycol was determined. Porin-deficient mutants produced residual amounts of porin amounting to 1 to 5% that produced by the parent strain. This residual porin made diffusion channels when the isolated porins were incorporated into the vesicle membrane and assayed for permeability of saccharides.     

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.     

Role of the receptor for bacteriophage lambda in the functioning of the maltose chemoreceptor of Escherichia coli.

Chemotaxis towards maltose is specifically defective in many strains of Escherichia coli carrying mutations affecting lamB, the gene coding for the outer membrane receptor for bacteriophage lambda. However, with one exception, the most extreme effect of lamB mutants on the maltose response as determined in the capillary assay is a shift to higher sugar concentrations and a reduction in the number of bacteria accumulated to about 25% of the wild-type level. The severity of the taxis defect is strongly correlated with reduced ability of the cells to take up the maltose present at 1 and 10 muM. Evidence presented here and in the accompanying paper indicates that the lambda receptor is involved in the transport of maltose at these concentrations. The effects of lamB mutations on maltose taxis can be explained by postulating that the high-affinity maltose transport system in which the lambda receptor participates transfers maltose from the surrounding medium across the outer membrane and into the periplasmic space. If the maltose chemoreceptor detects sugar present in the periplasmic space, and not molecules external to the outer membrane, then defective transport of low concentrations of maltose into the periplasm would result in the observed apparent reduction in the sensitivity of the maltose receptor. Thus, the lambda receptor protein would participate in maltose chemorecepton only indirectly through its role in maltose transport.     

A porin activity of purified lambda-receptor protein from Escherichia coli in reconstituted vesicle membranes.

The receptor protein for bacteriophage λ was purified to homogeneity from a mutant strain of $\text{Escherichia coli}$ K-12 producing reduced amounts of porin. In the reconstituted vesicle membranes the λ-receptor formed permeability channels that allowed the diffusion of maltose, lactose, sucrose, raffinose, amino acids, and nucleosides, but essentially not of stachyose. The permeability channels made of λ-receptor thus had a relatively low specificity for solute molecules. The active form of the protein seemed to be an oligomer of λ-receptor proteins.     

Transposon Tn10-dependent expression of the lamB gene in Escherichia coli.

Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains. The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain. Transport of maltose at concentrations of below 10(-6) M was reduced about threefold. When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored. We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.     

Distribution of mannosamine and mannosaminuronic acid among cell walls of Bacillus species

Some Escherichia coli K-12 lamB mutants, those producing reduced amounts of LamB protein (one-tenth the wild type amount), grow normally on dextrins but transport maltose when present at a concentration of 1 microM at about one-tenth the normal rate. lamB Dex- mutants were found as derivatives of these strains. These Dex- mutants are considerably impaired in the transport of maltose at low concentrations (below 10 microM), and they have a structurally altered LamB protein which is impaired in its interaction with phages lambda and K10 but still interacts with a lambda host range mutant lambda hh*. The Dex- mutants are double lamB mutants carrying one mutation, already present in the parental strains, that reduces LamB synthesis and a second that alters LamB structure. The secondary mutations, present in different independent Dex- mutants, are clustered in the same region of the lamB gene. Dex+ revertants were isolated and analyzed: when the altered LamB protein is made in wild-type amount, due to a reversion of the first mutation, the phenotype reverts to Dex+. However, these Dex+ revertants are still very significantly impaired in maltose transport at low concentrations (below 10 microM).     

Periplasmic maltose-binding protein confers specificity on the outer membrane maltose pore of Escherichia coli.

ompB mutants of Escherichia coli K-12 are markedly deficient in porin in their outer membrane. This results in a decreased rate of uptake for many substrates: the maltose pore (lambda receptor) can in some circumstances, in the absence of the periplasmic maltose-binding protein, compensate for the consequent defects in permeability to lactose, mannitol, glycylglycyl-L-valine, and tri-L-ornithine. It is postulated that the maltose-binding protein associates with the maltose pore and confers on it the specificity for maltose, and that the absence of the maltose-binding protein leaves the pore open and results in enhanced transmembrane diffusion of molecules other than maltose. This paper presents evidence to support this hypothesis.     

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.     

Induction of the lambda receptor is essential for effective uptake of trehalose in Escherichia coli.

Trehalose transport in Escherichia coli after growth at low osmolarity is mediated by enzyme IITre of the phosphotransferase system (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). The apparent Km (16 microM) of trehalose uptake is low. Since trehalose is a good source of carbon and the apparent affinity of the uptake system is high, it was surprising that the disaccharide trehalose [O-alpha-D-glucosyl(1-1)-alpha-D-glucoside] has no problems diffusing through the outer membrane at high enough rates to allow full growth, particularly at low substrate concentrations. Here we show that induction of the maltose regulon is required for efficient utilization of trehalose. malT mutants that lack expression of all maltose genes, as well as lamB mutants that lack only the lambda receptor (maltoporin), still grow on trehalose at the usual high (10 mM) trehalose concentrations in agar plates, but they exhibit the half-maximal rate of trehalose uptake at concentrations that are 50-fold higher than in the wild-type (malT+) strain. The maltose system is induced by trehalose to about 30% of the fully induced level reached when grown in the presence of maltose in a malT+ strain or when grown on glycerol in a maltose-constitutive strain [malT(Con)]. The 30% level of maximal expression is sufficient for maximal trehalose utilization, since there is no difference in the concentration of trehalose required for the half-maximal rate of uptake in trehalose-grown strains with the wild-type gene (malT+) or with strains constitutive for the maltose system [malT(Con)]. In contrast, when the expression of the lambda receptor is reduced to less than 20% of the maximal level, trehalose uptake becomes less efficient. Induction of the maltose system by trehalose requires metabolism of trehalose. Mutants lacking amylotrehalase, the key enzyme in trehalose utilization, accumulate trehalose but do not induce the maltose system.     

Identification of three porins in the outer membrane of Bacteroides fragilis

Abstract Four outer membrane proteins were purified to homogeneity from isolated outer membranes of Bacteroides fragilis ; three ( M _r 51000, 92000 and 125 000) had pore-forming activity in reconstituted liposomes as determined by swelling assay. Membrane vesicles containing the M _rmr 55 000 outer membrane protein showed no detectable pore-forming activity. The three B. fragilis porins formed pores that allowed the penetration of uncharged saccharides of M _r lower than 340–400, even though the efficiency of solute diffusion showed slight differences. The diffusion rates of glucose through the porins appeared to be lower than those through Escherichia coli porins.     

Effect of Long Generation Times on Growth of Bacteroides thetaiotaomicron in Carbohydrate-Limited Continuous Culture

lamB is the structural gene for the bacteriophage lambda receptor, a multifunctional protein located in the outer membrane of Escherichia coli K-12. We present a method for deletion mapping of any lamB mutations with a recognizable pheno-type. This method involves a transducing phage constructed by in vitro recombination which can also be used for complementation, deoxyribonucleic acid sequence, and in vitro protein synthesis studies with the mutated lamB gene. Using this method, we mapped 18 lamB missense mutations which confer resistance to phage lambda h+ (wild-type host range). The main results were the following. (i) None of the 18 mutations was located in the first 4 deletion intervals out of the 11 of the genetic map. (ii) These mutations were clustered according to their phenotype as follows. (a) Class I mutations, which allow growth of lambda h and lambda hh* (one-step and two-step host range mutants of lambda, respectively), were located in three regions--three in interval V, four in interval VIII-IX, and three in interval X-XI. Only the last three mutations still allowed growth of phage K10 which also uses the lambda receptor, and two of them still allowed reversible binding of lambda h+. (b) All seven class II mutations allowed only growth of lambda hh* and mapped in interval V. These results are discussed in the frame of a genetic approach to the functional topology of the lambda receptor.     

Derepression of LamB protein facilitates outer membrane permeation of carbohydrates into Escherichia coli under conditions of nutrient stress.

The level of LamB protein in the outer membrane of Escherichia coli was derepressed in the absence of a known inducer (maltodextrins) under carbohydrate-limiting conditions in chemostats. LamB protein contributed to the ability of the bacteria to remove sugar from glucose-limited chemostats, and well-characterized lamB mutants with reduced stability constants for glucose were less growth competitive under glucose limitation than those with wild-type affinity. In turn, wild-type bacteria were less growth competitive than lamB mutants with enhanced sugar affinity. In contrast to an earlier report, we found that LamB- bacteria were less able to compete in carbohydrate-limited chemostats (with glucose, lactose, arabinose, or glycerol as the carbon and energy sources) when mixed with LamB+ bacteria. The transport Km for [14C]glucose was affected by the presence or affinity of LamB, but only in chemostat-grown bacteria, with their elevated LamB levels. The pattern of expression of LamB and the advantage it confers for growth on low concentrations of carbohydrates are consistent with a wider role in sugar permeation than simply maltosaccharide transport, and hence the well-known maltoporin activity of LamB is but one facet of its role as the general glycoporin of E. coli. A corollary of these findings is that OmpF/OmpC porins, present at high levels in carbon-limited bacteria, do not provide sufficient permeability to sugars or even glycerol to support high growth rates at low concentrations. Hence, the sugar-binding site of LamB protein is an important contributor to the permeability of the outer membrane to carbohydrates in habitats with low extracellular nutrient concentrations.     

On Some Genetic Aspects of Phage λ Resistance in E. COLI K12

Most mutations rendering E. coli K12 resistant to phage lambda, map in two genetic regions malA and malB.-The malB region contains a gene lamB specifically involved in the lambda receptor synthesis. Twenty-one independent lamB mutations studied by complementation belonged to a single cistron. This makes it very likely that lamB is monocistronic. Among the lamB mutants some are still sensitive to a host range mutant of phage lambda. Mutations mapping in a proximal gene essential for maltose metabolism inactivate gene lamB by polarity confirming that both genes are part of the same operon. Because cases of intracistronic complementation have been found, the active lamB product may be an oligomeric protein.-Previously all lambda resistant mutations in the malA region have been shown to map in the malT cistron. malT is believed to be a positive regulatory gene necessary for the induction of the "maltose operons" in the malA region and in the malB region of the E. coli K12 genetic map. No trans dominant malT mutation have been found. Therefore if they exist, they occur at a frequency of less than 10(-8), or strongly reduce the growth rate of the mutants.     

Affinity engineering of maltoporin: variants with enhanced affinity for particular ligands

Affinity-chromatographic selection on immobilized starch was used to selectively enhance the affinity of the maltodextrin-specific pore protein ( maltoporin , LamB protein, or lambda receptor protein) in the outer membrane of E. coli. Selection strategies were established for rare bacteria in large populations producing maltoporin variants with enhanced affinities for both starch and maltose, for starch but not maltose and for maltose but not starch. Three classes of lamB mutants with up to eight-fold increase in affinity for particular ligands were isolated. These mutants provide a unique range of modifications in the specificity of a transport protein.     

Escherichia coli phage receptors. Minor porins and proteins participating in the specific transport as phage receptors

Except for the main porin proteins OmpC and OmpF there exist the membrane proteins participating in the transport of specific substrates: phosphates, nucleosides, iron, vitamin B12, maltose and maltodextrins, that also play the role of phage receptors. Some phages use as receptors the porins determined by the genes of lambdoid prophages. LamB protein that serves receptor for phage lambda exposes the amino acids sequence on the outer surface of membranes that participates in phage adsorption. The sequence is similar to tetrapeptide of fibronectin responsible for binding with the surface of cellular receptor in eucaryotes.     

Maltose-binding protein does not modulate the activity of maltoporin as a general porin in Escherichia coli.

Maltoporin (lambda receptor) is part of the maltose transport system in Escherichia coli and is necessary for the facilitated diffusion of maltose and maltodextrins across the outer membrane. Maltoporin also allows the diffusion of nonmaltodextrin substrates, albeit with less efficiency. The preference of maltoporin for maltodextrins in vivo is thought to be the result of an interaction of maltoporin with the maltose-binding protein, the malE gene product. In a recent report Heuzenroeder and Reeves (J. Bacteriol. 144:431-435, 1980) suggested that this interaction establishes a gating mechanism which inhibits the diffusion of nonmaltodextrin substrates, such as lactose. To reinvestigate this important conclusion, we constructed ompR malTc strains carrying either the malE+ gene, the nonpolar malE444 deletion, or the malE254 allele, which specifies an interaction-deficient maltose-binding protein. Lactose uptake was measured at different concentrations below the Km of this transport system and under conditions where transport was limited by the diffusion through maltoporin. We found no difference in the kinetics of lactose uptake irrespective of the malE allele. We conclude that the maltose-binding protein does not modulate the activity of maltoporin as a general outer membrane porin.     

Mutations affecting antigenic determinants of an outer membrane protein of Escherichia coli.

The Escherichia coli LamB protein is located in the outer membrane. It is both a component of the maltose and maltodextrin transport system, and the receptor for phages lambda and K10. It is a trimer composed of three identical polypeptide chains, each containing 421 residues. Six independent mutants have been isolated, in which the LamB protein is altered in its interaction with one or more monoclonal antibodies specific for regions of the protein that are exposed at the cell surface. Some of the mutations also altered the binding site for phage lambda. All of the mutations were clustered in the same region of the lamB gene, corresponding to residues 333-394 in the polypeptide. This and previous results strongly suggest that a rather large segment of the LamB polypeptide, extending from residue 315 to 401, is exposed at the outer face of the outer membrane. This segment would bear the epitopes for the four available anti-LamB monoclonal antibodies that react with the cell surface, and part of the binding site for phage lambda.     

malB Region in Escherichia coli K-12: Characterization of New Mutations

Phenotypic characterization and mapping of more than 50 Mal(-) mutations located in the malB region lead one to divide the site for Mal(-)lambdas mutations (formerly called gene malB) in that region, into two adjacent genetic segments malJ and malK. malJ and malK are both involved in maltose permeation. It is suggested that (i) malK and lamB, the only known gene specifically involved in phage lambda adsorption (20), constitute an operon of polarity malK lamB. (ii) malJ and malK correspond to two different genes, and (iii) a promoter for the malK lamB operon is located between malJ and malK. Since lambda receptors and maltose permease are inducible by maltose and absent in malT mutants, it is likely that the expression of the malK lamB operon is controlled by the product of gene malT, the positive regulatory gene of the maltose system.     

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.