A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Redundancy in periplasmic binding protein-dependent transport systems for trehalose,sucrose, and maltose in Sinorhizobium meliloti

We have identified a cluster of six genes involved in trehalose transport and utilization (thu) in Sinorhizobium meliloti. Four of these genes, thuE, -F, -G, and -K, were found to encode components of a binding protein-dependent trehalose/maltose/sucrose ABC transporter. Their deduced gene products comprise a trehalose/maltose-binding protein (ThuE), two integral membrane proteins (ThuF and ThuG), and an ATP-binding protein (ThuK). In addition, a putative regulatory protein (ThuR) was found divergently transcribed from the thuEFGK operon. When the thuE locus was inactivated by gene replacement, the resulting S. meliloti strain was impaired in its ability to grow on trehalose, and a significant retardation in growth was seen on maltose as well. The wild type and the thuE mutant were indistinguishable for growth on glucose and sucrose. This suggested a possible overlap in function of the thuEFGK operon with the aglEFGAK operon, which was identified as a binding protein-dependent ATP-binding transport system for sucrose, maltose, and trehalose. The K(m)s for trehalose transport were 8 +/- 1 nM and 55 +/- 5 nM in the uninduced and induced cultures, respectively. Transport and growth experiments using mutants impaired in either or both of these transport systems show that these systems form the major transport systems for trehalose, maltose, and sucrose. By using a thuE'-lacZ fusion, we show that thuE is induced only by trehalose and not by cellobiose, glucose, maltopentaose, maltose, mannitol, or sucrose, suggesting that the thuEFGK system is primarily targeted toward trehalose. The aglEFGAK operon, on the other hand, is induced primarily by sucrose and to a lesser extent by trehalose. Tests for root colonization, nodulation, and nitrogen fixation suggest that uptake of disaccharides can be critical for colonization of alfalfa roots but is not important for nodulation and nitrogen fixation per se.     

MsmE, a lipoprotein involved in sugar transport in Streptococcus mutans.

Metabolic labelling by [14C]palmitic acid showed that growth of Streptococcus mutans LT11 in raffinose, an inducer of the msm operon, resulted in increased production of a 45-kDa lipoprotein corresponding to MsmE, which is believed to be a sugar-binding protein. MsmE was also labelled when an msmE clone was expressed in Escherichia coli. The presence of a lipid anchor on MsmE provides a likely explanation of how the sugar-binding protein component of the msm binding protein-dependent multiple sugar transport system is retained at the cell surface.     

Sugar transport in Sulfolobus solfataricus is mediated by two families of binding protein-dependent ABC transporters

The extreme thermoacidophilic archaeon Sulfolobus solfataricus grows optimally at 80 degrees C and pH 3 and uses a variety of sugars as sole carbon and energy source. Glucose transport in this organism is mediated by a high-affinity binding protein-dependent ATP-binding cassette (ABC) transporter. Sugar-binding studies revealed the presence of four additional membrane-bound binding proteins for arabinose, cellobiose, maltose and trehalose. These glycosylated binding proteins are subunits of ABC transporters that fall into two distinct groups: (i) monosaccharide transporters that are homologous to the sugar transport family containing a single ATPase and a periplasmic-binding protein that is processed at an unusual site at its amino-terminus; (ii) di- and oligosaccharide transporters, which are homologous to the family of oligo/dipeptide transporters that contain two different ATPases, and a binding protein that is synthesized with a typical bacterial signal sequence. The latter family has not been implicated in sugar transport before. These data indicate that binding protein-dependent transport is the predominant mechanism of transport for sugars in S. solfataricus.     

Interaction of glucosyltransferase from Streptococcus mutans with various glucans

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.     

Influence of the 20-kDa protein from Bacillus thuringiensis ssp. israelensis on the rate of production of truncated Cry1C proteins

Abstract The multiple-sugar metabolism ( msm ) locus of Streptococcus mutans constitutes a non-PTS sugar uptake system responsible for the transport and utilization of raffinose, melibiose and isomaltotrioses. While previous studies have used polar mutations to suggest that these genes are co-transcribed, there has not been any direct evidence to support this. In this report we present direct evidence that the msm genes can be transcribed as a single operon.     

The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system.

Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes. A second site mutation in malI abolishes the constitutive expression. The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J. Reidl, K. R?misch, M. Ehrmann, and W. Boos, J. Bacteriol. 171:4888-4899, 1989). We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY. MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE). When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose. Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX. MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion. We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system. The second gene in the operon, malY, encodes a protein of 43,500 daltons. Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences. The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant. The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose. This is not the case in a malT(Con) strain that expresses the mal genes constitutively. We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis.     

Functional exchangeability of the ABC proteins of the periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli.

The periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli transport sn-glycerol-3-phosphate and maltose, respectively. The UgpC and MalK proteins of these transport systems, which couple energy to the transport process by ATP-hydrolysis, are highly homologous, suggesting that they might be functionally exchangeable. Complementation experiments showed that UgpC expression could restore growth of a malK mutant on maltose as a carbon source, provided that it was expressed at a sufficiently high level in the absence of the integral inner membrane components UgpA and/or UgpE of the Ugp system. Conversely, MalK expression could complement ugpC mutants and restore the utilization of sn-glycerol-3-phosphate as a phosphate source. The hybrid transporters appeared to be less efficient than the wild-type systems. The complementation of ugpC mutations by MalK was strongly inhibited by the presence of glucose or alpha-methylglucoside, which are substrates of the phosphotransferase system. This inhibition is probably due to hypersensitivity of the hybrid UgpBAE-MalK transporter to inducer exclusion. UgpC expression did not complement the regulatory function of MalK in mal gene expression. The exchangeability of UgpC and MalK indicates that these proteins do not contribute to a substrate-binding site conferring substrate specificity to the transporter. These are the first examples of functional, hybrid periplasmic permeases in which the energy-coupling components could be functionally exchanged.     

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.     

Biochemical evidence for the presence of two alpha-glucoside ABC-transport systems in the hyperthermophilic archaeon Pyrococcus furiosus

The hyperthermophilic archaeon Pyrococcus furiosus can utilize different carbohydrates, such as starch, maltose and trehalose. Uptake of alpha-glucosides is mediated by two different, binding protein-dependent, ATP-binding cassette (ABC)-type transport systems. The maltose transporter also transports trehalose, whereas the maltodextrin transport system mediates the uptake of maltotriose and higher malto-oligosaccharides, but not maltose. Both transport systems are induced during growth on their respective substrates.     

Evidence for catabolite inhibition in regulation of pentose utilization and transport in the ruminal bacterium Selenomonas ruminantium.

Pentose sugars can be an important energy source for ruminal bacteria, but there has been relatively little study regarding the regulation of pentose utilization and transport by these organisms. Selenomonas ruminantium, a prevalent ruminal bacterium, actively metabolizes xylose and arabinose. When strain D was incubated with a combination of glucose and xylose or arabinose, the hexose was preferentially utilized over pentoses, and similar preferences were observed for sucrose and maltose. However, there was simultaneous utilization of cellobiose and pentoses. Continuous-culture studies indicated that at a low dilution rate (0.10 h-1) the organism was able to co-utilize glucose and xylose. This co-utilization was associated with growth rate-dependent decreases in glucose phosphotransferase activity, and it appeared that inhibition of pentose utilization was due to catabolite inhibition by the glucose phosphotransferase transport system. Xylose transport activity in strain D required induction, while arabinose permease synthesis did not require inducer but was subject to repression by glucose. Since an electrical potential or a chemical gradient of protons drove xylose and arabinose uptake, pentose-proton symport systems apparently contributed to transport.     

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

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

Nucleotide sequence of the ugp genes of Escherichia coli K-12: homology to the maltose system

The nucleotide sequence of the ugp genes of Escherichia coli K-12, which encode a phosphate-limitation inducible uptake system for sn-glycerol-3-phosphate and glycerophosphoryl diesters, was determined. The genetic organization of the operon differed from previously published results. A single promoter, containing a putative pho box, was detected by S1-nuclease mapping. The promoter is followed by four open reading frames, designated ugpB, A, E and C, which encode a periplasmic binding protein, two hydrophobic membrane proteins and a protein that is likely to couple energy to the transport system, respectively. The sequences of the proteins contain the characteristics of several other binding protein-dependent transport systems, but they seem to be particularly closely related to the maltose system.