Exclusion of high-molecular-weight maltosaccharides by lipopolysaccharide O-antigen of Escherichia coli and Salmonella typhimurium.

The barrier properties of lipopolysaccharide were studied by testing the influence of O-antigen on the binding of ligand to maltoporin in the outer membranes of Escherichia coli and Salmonella typhimurium. Maltoporin (LamB protein) of Escherichia coli K-12 was capable of interacting with macromolecular starch polysaccharides, as was previously shown by the binding of intact bacteria to fluorescein-labeled amylopectin or to starch-Sepharose columns. In contrast, strains with complete O-antigenic lipopolysaccharide showed reduced binding to these substrates. A similar result was obtained with Salmonella typhimurium LT2, which did not bind to starch unless rfa mutations removed noncore polysaccharide. The exclusion limit of the lipopolysaccharide permeability barrier to alpha-glucans was tested by measuring the maltoporin-dependent transport of maltose and its inhibition by maltodextrins of various sizes. Only amylopectin (molecular weight, greater than 25,000) was excluded in transport experiments, whereas maltodextrins with molecular weights of up to 2,000 were not excluded by the presence of an O-polysaccharide layer.     

MalE of group A Streptococcus participates in the rapid transport of maltotriose and longer maltodextrins

Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.     

ExbBD-dependent transport of maltodextrins through the novel MalA protein across the outer membrane of Caulobacter crescentus

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe(3+) and vitamin B(12)-the substrates hitherto known to be transported by TonB-dependent transporters-the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [(14)C]maltodextrins from [(14)C]maltose to [(14)C]maltopentaose. [(14)C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K(d) of 0.2 microM, while the second transport had a K(d) of 5 microM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 microM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [(14)C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe(3+)-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe(3+)-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K(d) values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B(12) and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B(12) has been demonstrated.     

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.     

Interspecific reconstitution of maltose transport and chemotaxis in Escherichia coli with maltose-binding protein from various enteric bacteria.

In Escherichia coli, the periplasmic maltose-binding protein (MBP), the product of the malE gene, is the primary recognition component of the transport system for maltose and maltodextrins. It is also the maltose chemoreceptor, in which capacity it interacts with the signal transducer Tar (taxis to aspartate and some repellents). In studies of the maltose system in other members of the family Enterobacteriaceae, we found that MBP is produced by Salmonella typhimurium, Klebsiella pneumoniae, Enterobacter aerogenes, and Serratia marcescens. MBP from all of these species cross-reacted with antibody against the E. coli protein and had a similar molecular weight (about 40,000). The Shigella flexneri and Proteus mirabilis strains we examined did not synthesize MBP. The isoelectric points of MBP from different species varied from the acid extreme of E. coli (4.8) to the basic extreme of E. aerogenes (8.9). All species with MBP transported maltose with high affinity, although the Vmax for K. pneumoniae was severalfold lower than that for the other species. Maltose chemotaxis was observed only in E. coli and E. aerogenes. In S. typhimurium LT2, Tar was completely inactive in maltose taxis, although it signaled normally in response to aspartate. MBP isolated from all five species could be used to reconstitute maltose transport and taxis in a delta malE strain of E. coli after permeabilization of the outer membrane with calcium.     

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.     

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.     

Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli

Crystals of maltoporin (the bacteriophage lambda receptor of Escherichia coli) that diffract X-rays to 3 A resolution can be grown reproducibly. Maltoporin is an integral membrane protein, which forms a channel in the E. coli outer membrane that specifically facilitates the diffusion of maltose and maltodextrins. The crystals have a rhombic prismatic habit and belong to the orthorhombic space group C222(1) with unit cell dimensions a = 130 A, b = 213 A and c = 216 A. X-ray structure determination is underway.     

Interaction of the lamB protein with the peptidoglycan layer in Escherichia coli K12

In Escherichia coli K12 the product of gene lamB is an outer membrane protein involved in the transport of maltose and maltodextrins and serving as a receptor for several bacteriophages including lambda. About 30 to 40% of this protein can be recovered associated to peptidoglycan when the cells are dissolved in sodium dodecyl sulfate in the presence of 2 mM Mg2+ ions. The bound protein can then be quantitatively eluted from peptidoglycan by incubating the complex in Triton X-100 and EDTA, or sodium dodecyl sulfate and NaCl. The protein eluted in such ways is still totally active in its phage-neutralizing activity. Two other membrane proteins known to behave similarly to the lamB protein are proteins Ia and Ib. However the binding of these proteins to peptidoglycan appears tighter, in several respects, than that of the lamB protein. The lamB protein may span the outer membrane since it appears to interact with the peptidoglycan on the inner side of this membrane while it is known to be accessible to both phages and antibodies at the cell surface.     

Maltose and maltodextrin utilization by Bacillus subtilis

Bacillus subtilis can utilize maltose and maltodextrins that are derived from polysaccharides, like starch or glycogen. In this work, we show that maltose is taken up by a member of the phosphoenolpyruvate-dependent phosphotransferase system and maltodextrins are taken up by a maltodextrin-specific ABC transporter. Uptake of maltose by the phosphoenolpyruvate-dependent phosphotransferase system is mediated by maltose-specific enzyme IICB (MalP; synonym, GlvC), with an apparent K(m) of 5 microM and a V(max) of 91 nmol . min(-1) . (10(10) CFU)(-1). The maltodextrin-specific ABC transporter is composed of the maltodextrin binding protein MdxE (formerly YvdG), with affinities in the low micromolar range for maltodextrins, and the membrane-spanning components MdxF and MdxG (formerly YvdH and YvdI, respectively), as well as the energizing ATPase MsmX. Maltotriose transport occurs with an apparent K(m) of 1.4 microM and a V(max) of 4.7 nmol . min(-1) . (10(10) CFU)(-1).     

Sequence determinants in the lamB gene of Escherichia coli influencing the binding and pore selectivity of maltoporin

Maltoporin (LamB protein) is a malto-oligosaccharide-selective pore protein in the outer membrane of Escherichia coli. The genetic basis of binding and transport specificity was investigated through cloning, mapping and sequencing lamB genes from seven independent mutants with various changes in maltodextrin binding affinities; these mutants were unchanged in binding phage lambda. Single amino acid substitutions specifically resulting in maltodextrin affinity changes were as follows: Arg8----His in two independent mutants resulted in much reduced affinity for all ligands and a smaller pore no longer selective for maltodextrins. A Trp74----Arg substitution resulted in a lower affinity for starch, a slight increase in maltose affinity but no striking pore changes. An Arg82----Ser resulted in lowered maltodextrin affinity, but increased affinity for sucrose in both binding and pore function. A Tyr118----Phe resulted in a higher affinity for both starch and maltose, a slightly larger pore and increased transport of maltohexaose by the pores. Asp121----Gly in two independent isolates resulted in a higher affinity for large dextrins and a marginally larger pore. These results suggest that the maltodextrin-selective functions reside in the N-terminal sequence of maltoporin and are separate from the phage lambda binding domains.     

A mutant form of maltose-binding protein of Escherichia coli deficient in its interaction with the bacteriophage lambda receptor protein.

In one malE mutant known to be deficient in the transport of maltose and maltodextrins across the outer membrane, the altered MalE protein was shown to be defective in its interaction with the phage lambda receptor, or LamB protein, of the outer membrane.     

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.     

Genetic evidence that outer membrane binding of starch is required for starch utilization by Bacteroides thetaiotaomicron

Mutagenesis of Bacteroides thetaiotaomicron with the transposon Tn4351 produced five classes of mutants that were not able to grow on amylose or amylopectin. These classes of mutants differed in their ability to grow on maltoheptaose (G7) and in the level of starch-degrading enzymes produced when bacteria were grown on maltose. All of the mutants were deficient in starch binding. Since one class of mutants retained normal levels of starch-degrading enzymes, this indicates that binding of the starch molecule by a cell surface receptor is necessary for starch utilization by B. thetaiotaomicron. Analysis of a starch-negative mutant that grew on G7 indicated that B. thetaiotaomicron possessed two starch-binding components or sites. One component (site A), apparently missing in this mutant, had an absolute preference for larger starch oligomers, whereas the other component (site M) also had a high affinity for maltodextrins (G4 through G7). Mutants not able to grow on maltodextrins (greater than G4) probably lacked both of these binding components. Only one class of mutants did not grow normally on maltose, but instead had a 4- to 5-h lag on maltose and a slower growth rate than the wild type. This class of mutants did not produce any of the starch-degrading enzymes or bind starch, even when growing on maltose. Such a phenotype probably resulted from transposon inactivation of a central regulatory gene or a gene encoding an enzyme that produces the inducer. The fact that both the degradative enzymes and the starch-binding activity were affected in this mutant indicates that genes encoding the cell surface starch-binding site are under the same regulatory control as genes encoding the enzymes.     

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 and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system.

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and alpha,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (O. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate [14C]glucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-1-phosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.     

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.     

Identification of endogenous inducers of the mal regulon in Escherichia coli.

The expression of the maltose regulon in Escherichia coli is induced when maltose or maltodextrins are present in the growth medium. Mutations in malK, which codes for a component of the transport system, result in the elevated expression of the remaining mal genes. Uninduced expression in the wild type, as well as elevated expression in malK mutants, is strongly repressed at high osmolarity. In the absence of malQ-encoded amylomaltase, expression remains high at high osmolarity. We found that uninduced expression in the wild type and elevated expression in malK mutants were paralleled by the appearance of two types of endogenous carbohydrates. One, produced primarily at high osmolarity, was identified as comprising maltodextrins that are derived from glycogen or glycogen-synthesizing enzymes. The other, produced primarily at low osmolarity, consisted of an oligosaccharide that was not derived from glycogen. We isolated a mutant that no longer synthesized this oligosaccharide. The gene carrying this mutation, termed malI, was mapped at min 36 on the E. coli linkage map. A Tn10 insertion in malI also resulted in the loss of constitutivity at low osmolarity and delayed the induction of the maltose regulon by exogenous inducers.     

Characterization and expression of the structural gene for pullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae.

Some strains of Klebsiella pneumonia secrete pullulanase, a debranching enzyme which produces linear molecules (maltodextrins, amylose) from amylopectin and glycogen. pulA, the structural gene for pullulanase, was introduced into Escherichia coli, either on a multiple-copy-number plasmid or as a single copy in the chromosome. When in E. coli, pulA was controlled by malT, the positive regulatory gene of the maltose regulon. Indeed, pulA expression was undetectable in a malT-negative mutant and constitutive in a malTc strain. Furthermore, the plasmid carrying pulA titrated the MalT protein. When produced in E. coli, pullulanase was not localized in the same way as in K. pneumoniae. In the latter case it was first exported to the outer membrane, with which it remained loosely associated, and was then released into the growth medium. In E. coli the enzyme was distributed both in the inner and the outer membranes and was never released into the growth medium.