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Journal of Bacteriology, June 2006, p. 3911-3922, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.00213-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Maltose and Maltodextrin Utilization by Bacillus subtilis

Stefan Schönert,^* Sabine Seitz, Holger Krafft, Eva-Anne Feuerbaum, Iris Andernach, Gabriele Witz, and Michael K. Dahl

Department of Biology, University of Konstanz, D-78457 Konstanz, Germany

Received 8 February 2006/ Accepted 15 March 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 µM and a V_max of 91 nmol · min^–1 · (10¹⁰ 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 µM and a V_max of 4.7 nmol · min^–1 · (10¹⁰ CFU)^–1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The gram-positive soil bacterium Bacillus subtilis can utilize glycogen, starch, and amylose as carbon sources. Prior to transport through the cell membrane, these polysaccharides are hydrolyzed by the extracellular -amylase AmyE into smaller maltodextrins (15). The resulting disaccharides, maltose and isomaltose, as well as maltodextrins, are secondary metabolites that can serve as sole carbon and energy sources in B. subtilis (11, 34, 35, 44).

In contrast to the very well-known maltose and maltodextrin uptake of the Escherichia coli maltose ABC transporter (reviewed in reference 3), the uptake of these carbohydrates in B. subtilis is not very well understood. Recently, uptake of maltose via the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was postulated based on the identification of an NAD(H)-dependent phospho--1,4-glucosidase (MalA; synonym, GlvA) (46). In general, substrates that are taken up into the cell by the PTS become phosphorylated when they enter the cell. During this process, the required phosphoryl group is transferred from phosphoenolpyruvate via the general cytoplasmic proteins enzyme I and HPr to a substrate-specific membrane bound enzyme II and finally to the transported substrate (for detailed information, see references 26 and 32).

The malA gene is located in an operon composed of three genes (Fig. 1): (i) malA (synonym, glvA) (46); (ii) glvR (synonym, yfiA), encoding the potential activator of the operon (47); and (iii) malP (synonym, glvC), encoding the putative enzyme IICB (EIICB^Mal) specific for maltose (29). Inactivation of the putative EIICB^Mal resulted in a sevenfold-longer generation time on maltose minimal medium than that of the wild type (29).

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FIG. 1. The mal operon and the yvdE-pgcM region of B. subtilis. The numbers indicate the locations of the first bases of malA and yvdE and the last bases of malP and pgcM on the B. subtilis chromosome. Putative terminators are shown as stem loops. Encoded proteins and predicted proteins, based on similarities of the derived amino acid sequences, are indicated below. ?, no similarities to proteins with known functions.

However, these results contradict previous reports that concluded that uncouplers negatively affect maltose uptake in B. subtilis (44). This observation led to the conclusion that maltose transport in B. subtilis is proton motive force dependent and does not occur via the PTS, but is regulated by the PTS (44).

In addition, several genes encoding ATP binding cassette (ABC) transporters can be found on the B. subtilis chromosome, which might encode maltose and/or maltodextrin uptake systems (41). ABC transporters in general consist of four domains. Two of these domains are located in the cytoplasmic membrane and form a canal. The other two domains are ATPases that energize the transport of the substrate through the canal. In bacteria, ABC importers can be distinguished from exporters in that importers possess a high specific substrate binding protein that delivers the substrate to the transmembrane domains. These substrate binding proteins are soluble in the periplasm of gram-negative bacteria (2) and are anchored to the membrane by lipid modifications in gram-positive bacteria (42). ABC transporters do not phosphorylate or otherwise modify their substrates during transport (8, 14).

In addition to the potential maltose system mentioned above, B. subtilis contains a maltose-inducible -glucosidase activity that is associated with MalL (34). MalL activity is also induced by exogenous amylose, starch, and glycogen (35). The malL gene is located in a gene cluster consisting of nine genes (41) (Fig. 1). The last gene of this cluster, pgcM, encodes a ß-phosphoglucomutase/glucose-1-phosphosphate phosphodismutase acting on phosphorylated glucose molecules presumably resulting from the degradation of glucose oligomers (30). Based on similarities of the amino acid sequences deduced from the open reading frames, the other genes encode a transcriptional regulator (yvdE), a substrate binding protein (mdxE; formerly yvdG), and membrane-spanning components (mdxF and mdxG; formerly yvdH and yvdI, respectively) of an ABC transporter, a cytoplasmic maltogenic amylase or neopullulanase (yvdF) (5), and a maltose phosphorylase (yvdK), respectively (41). No prediction of the function of YvdJ could be made (41). Since uptake and utilization systems in bacteria are often encoded by genes located in the same operon, the region around malL is a prime candidate to encode a maltose and/or maltodextrin utilization system. However, based on computer-aided analysis, other potential ABC transporters can be found in B. subtilis that might encode potential maltose and maltodextrin uptake systems (28, 41). As in 11 of the 78 ABC transporter-encoding gene clusters of B. subtilis, the ABC-encoding open reading frame is missing in the above-described gene cluster (28, 41). However, the B. subtilis genome encodes three potential ATPases presumably involved in substrate import, namely, ylmA, yusV, and msmX, which are probably organized in monocystronic operons (28, 41). This raises the possibility that one ATP binding cassette serves different kinds of ABC transporters (28).

The aim of this study was to answer the question of how maltose or maltodextrins are taken up by B. subtilis. We show that in contrast to the gram-negative bacterium Escherichia coli, maltose is taken up by the maltose-specific enzyme IICB (MalP) of the PTS in B. subtilis and that maltotriose and presumably maltodextrins up to at least maltoheptaose are taken up by a specific ABC transporter. The latter consists of the maltodextrin-binding protein MdxE, with high affinities for maltodextrins and a low affinity for maltose, as well as the membrane components MdxF and MdxG. Furthermore, our data show that transport via this transporter is energized by MsmX as the cognate ABC domain.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents and enzymes. Restriction enzymes, Taq DNA polymerase, and T4 ligase were used as recommended by the manufacturers. Maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, para-nitrophenyl--D-glucopyranoside, and all other sugars used were purchased from Sigma (Munich, Germany). [¹⁴C]maltose (610 mCi/mmol; 200 µCi/ml) was obtained from Amersham, Braunschweig, Germany; [¹⁴C]maltotriose (800 mCi/mmol; 100 µCi/ml) was from Biotrend Chemikalien GmbH, Köln, Germany. [¹⁴C]-maltotriose was contaminated with a significant amount of a radioactive substrate that showed the same behavior as maltose on thin-Layer chromatography (data not shown). All other reagents were of analytical grade.

Bacterial strains, plasmids, media, and selection of recombinants. The plasmids and strains used in this study are listed in Table 1 and Table 2, respectively. Standard procedures were used to transform E. coli, extract plasmids, and manipulate DNA (33). PCRs (23) were performed with Taq polymerase (Boehringer Mannheim GmbH, Germany). Plasmids carrying fragments that were made by PCR were verified by sequencing.

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TABLE 1. Plasmids used in this work

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TABLE 2. Bacterial strains used in this work

Plasmid pMBP was constructed by ligating an XbaI/EcoRI-restricted 1,227-bp DNA fragment into XbaI/EcoRI-restricted pBluescript II SK(–). The fragment was obtained by PCR using primers EcoRI-yvdG (5'-GGA ATT CTG CTC AAG TTC AAA AAA TCC AGC-3') and XbaI-yvdG (5'-CCC TCT AGA CTT CCG GAC GCT ATC-3'), introducing an EcoRI site 5' to mdxE and an XbaI site 3' to the gene. Chromosomal DNA of wild-type B. subtilis 168 was used as a template. Plasmid pMBPHis6x was constructed by ligating a fragment harboring mdxE without the leader peptide-encoding sequence with vector pQE-9. Both the fragment and pQE-9 were restricted with BamHI and PstI prior to ligation. The fragment was amplified by PCR using chromosomal DNA of B. subtilis 168 as a template and the primers yvdG-BamHI (5'-CGG GAT CCT GCT CAA GTT CAA AAA ATC C-3') and yvdG-PstI (5'-CCT CTA TTC TTC CGC TGC AGA TCC C-3'), introducing a 5' BamHI site and a 3' PstI site of the truncated mdxE gene.

Plasmids pMBPK1 and pMBPK2 were obtained by inserting a 1,497-bp SmaI/StuI DNA fragment of plasmid pDG792 carrying the aphA3 kanamycin resistance cassette (13) into HpaI-restricted pMBP. Transformants of E. coli strain TG1 (12) were selected on Luria-Bertani plates supplemented with ampicillin (100 µg/ml) and kanamycin (25 µg/ml). The direction of the aphA3 gene with respect to mdxE was determined by restriction of the plasmids with HindIII.

Plasmid pBlueMBPkurz was constructed by ligation of the 3,320-bp XbaI/BglII-restricted fragment of pMBP with an XbaI/BglII-restricted 453-bp fragment obtained by PCR using primer malE-BglII (5'-GAA ATA TAC AAA AGA TCT CGA GCT GG-3') and a reverse primer (5'-AAC AGC TAT GAC CAT G-3') and plasmid pMBP as a template.

Plasmid pMalEkurzts was achieved by ligation of the 768-bp fragment resulting from the EagI/SalI restriction of pBlueMBPkurz with the 6,991-bp fragment from the EagI/SalI cleavage of pWH1509C (31).

Plasmid pBlueHI was constructed by ligation of the 2,241-bp fragment resulting from the ClaI/EheI restriction of an mdxF-mdxG-carrying PCR product (primers, a, yvdH-5' [5'-GCA AGG AAG AAA GCC GAT GAG CA-3'], and b, yvdH-3' [5'-CCC GCC TGT TAG TTT TCC GTT CCT-3']; template, chromosomal DNA of B. subtilis 168) with the 2,927-bp fragment of a ClaI/SmaI restriction of pBluescript II SK(–).

Plasmids pBlueHIK1 and pBlueHIK2 were created following a strategy similar to that described for pMBPK1 and pMBPK2 (see above). pBlueHI was restricted with Tth111I and Eco47III, followed by a fill-in modification of the Tth111I, resulting in sticky ends with T4 polymerase. The direction of the aphA3 resistance cassette with respect to the mdxF and mdxG genes was proven by BlpI/SacII restriction.

Plasmid pBluemsmX was constructed so that a 1,123-bp fragment resulting from a BamHI/SalI restriction of the PCR product obtained with primers msmX-BamHI (5'-GGG AGG ATC CAT GGC TGA ATT GCG GAT G-3') and msmX-SalI (5'-CAT GTC GAC ATG TCC GGT TTT TTT GAT CTT ATC G-3') and chromosomal DNA of wild-type B. subtilis as a template was ligated into BamHI/SalI-restricted pBluescript II SK(–).

Plasmid pmsmXK was obtained by inserting a 1,537-bp NruI/EcoRI DNA fragment of plasmid pDG792 carrying the aphA3 kanamycin resistance cassette (13) into NruI/EcoRI-restricted pBluemsmX.

B. subtilis wild-type strain 168 (1A1) was obtained from the Bacillus Genetic Stock Center (Ohio State University). For B. subtilis strain constructions (Table 2), transformation was carried out using a one-step procedure (19). Strain MD215 was constructed by transforming B. subtilis 168 with linearized plasmid pMBPK1, followed by selection on kanamycin (25 µg/ml). To obtain a nonpolar deletion of the mdxE gene, MD215 was transformed at 30°C with the circular plasmid pMalEkurzts, followed by selection on kanamycin (25 µg/ml) and chloramphenicol (5 µg/ml). The resulting mutant was used to inoculate 4 ml LB/chloramphenicol (5 µg/ml). The culture was grown at 42°C overnight; 1 ml of that culture was again transferred to 4 ml LB/chloramphenicol (5 µg/ml). Growth was allowed at 42°C for an additional 16 h. One hundred microliters of the culture was plated on LB/chloramphenicol (5 µg/ml) and incubated at 42°C overnight. The resulting colonies were used to inoculate 4 ml minimal medium M9 (22 mM potassium dihydrogen phosphate, 16.8 mM disodium hydrogen phosphate, 7.6 mM ammonium sulfate, 8.6 mM sodium chloride, 1 mM magnesium sulfate, 0.05% [wt/vol] Casamino Acids, 0.05% [wt/vol] yeast extract, pH 7.4) without antibiotics. The culture was incubated at 28°C for 2 days and diluted to 10^–6. Aliquots of 100 µl were plated on LB. About 20,000 colonies were screened for kanamycin- and chloramphenicol-sensitive mutants by replica plating, resulting in one positive clone (strain MD235).

Mutants in malP were constructed by transformation with ScaI-linearized plasmid pGP109 (29) and selection on spectinomycin (100 µg/ml). Mutants of the mdxF-mdxG region were constructed by transformation with plasmid pBlueHIK1 that was linearized with DraIII, followed by selection on kanamycin (25 µg/ml). The amyE gene was disrupted by transformation of B. subtilis with SapI-linearized plasmid pAC6 (40), followed by selection on chloramphenicol (5 µg/ml). Mutants in msmX were constructed by transformation with FspI-linearized plasmid pmsmXK (29) and selection on kanamycin (25 µg/ml).

To verify correct mutagenesis of the malP, msmX, mdxE, and mdxF-mdxG genes, chromosomal DNA of the mutants was isolated (25) and used as a template for PCR (for the primer sets, see above). The resulting DNA fragments were compared to that received using chromosomal DNA of the parental strain as a template DNA under the same conditions. Correct amyE mutants were checked for loss of amylase activity on starch plates (36).

Computer analysis. For sequence analysis, we used programs from the University of Wisconsin Genetics Computer Group (9) and the BLAST server (45) of the National Center for Biotechnology Information at the National Institutes of Health, Bethesda, Maryland (http://www.ncbi.nlm.nih.gov), as well as Clone Manager 5 (Scientific and Educational Software, Durham, NC) and Lasergene (DNAstar, Inc., Madison, WI) software.

Overproduction and purification of the maltodextrin-binding protein. Overproduction of the mdxE gene product was achieved with plasmid pMBPHis6x carrying the His tag coding region of plasmid pQE-9 fused in frame to mdxE lacking the first 23 codons of the open reading frame, leading to expression under the control of the IPTG (isopropyl-ß-D-thiogalactopyranoside)-inducible T5 promoter in E. coli strain RB791 (4, 27).

A 1-liter culture of E. coli strain RB791/pMBPHis6x was grown at 37°C in Luria-Bertani broth (33) containing ampicillin (100 µg/ml). Expression was induced by the addition of 2 mM IPTG as the culture reached an A₆₀₀ of 0.5. Growth was allowed to proceed for another 4 h after induction before the cells were harvested by centrifugation at 5,000 x g. The resulting cell pellet was washed once in lysis buffer (10 mM imidazole, 50 mM potassium dihydrogen phosphate, 50 mM potassium chloride, pH 7.0), resuspended in 10 ml of the same buffer, and used immediately. Cell extracts were prepared as described previously (24), with minor modifications. The cells were sonicated eight times for 30 s each time at 40 W with 0.9-s pulse intervals, using a Labsonic U sonicator (B. Braun, Melsungen, Germany). After centrifugation for 50 min at 30,000 x g, the supernatant was collected. Maltodextrin-binding protein was purified with an Ni²⁺-loaded 1-ml HiTrap Chelating column and a Pharmacia kta Purifier apparatus following the instructions of the manufacturer (Pharmacia, Freiburg, Germany). The elution buffer was 50 mM NaH₂PO₄, 50 mM NaCl, 500 mM imidazole, pH 7.

For further purification and to remove imidazole, the eluate was passed over a size fractionation column (HiLoad Superdex 75; buffer, 50 mM NaH₂PO₄, 50 mM NaCl, pH 7) following the instructions of the manufacturer (Pharmacia, Freiburg, Germany). Protein-containing fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE. Crude extracts of E. coli RB791/pMBPHis6x cells induced with 2 mM IPTG and lysed by SDS were prepared according to the method of Silhavy et al. (37). Aliquots of fractions from protein purification were mixed with loading buffer prior to SDS-PAGE on 10% gels (20), using a minigel system (Bio-Rad Laboratories, Richmond, CA.). The molecular size reference marker 70L was obtained from Sigma (Munich, Germany). Proteins were visualized with Coomassie blue R250.

Surface plasmon resonance spectroscopy. Interaction of the purified MdxE protein with potential substrates was analyzed using surface plasmon resonance performed on a BIAcoreX (Pharmacia Biosensor AB, Uppsala, Sweden) (21, 39). Research grade Ni²⁺-loaded nitrilotriacetic acid chips were coated with purified His₆-tagged MdxE to about 3,000 to 4,000 resonance units. The same amount of His₆-tagged GlcK (38) or Ni²⁺-loaded nitrilotriacetic acid chips without protein was used in the reference flow cell. Reaction temperatures were set to 25°C. Eluent buffer (10 mM HEPES, 150 mM NaCl, 50 µM EDTA, 0.005% Surfactant P20, pH 7.4) was used as a running buffer (Pharmacia). For further analysis, sugars (as mentioned above) were diluted in eluent buffer and passed over the experimental and reference flow cells at a flow rate of 5 µl/min. To determine the interactions between the coupled protein and the different substrates, signals obtained from the reference flow cell were subtracted from signals obtained from the experimental flow cell. To determine substrate K_d values, concentrations of 1 µM, 5 µM, 15 µM, 50 µM, 100 µM, 500 µM, 1 mM, 5 mM, and 10 mM of the indicated sugars were used. The values obtained were used to calculate the substrate affinities from Scatchard plots.

Transport assays. B. subtilis strains were grown in Luria-Bertani broth supplemented with the sugars indicated. Cells were harvested by centrifugation for 5 min at 5,000 x g, washed three times in transport buffer (50 mM Tris-HCl, 20 mM MgCl₂, pH 7.2), and resuspended in transport buffer to an A₆₀₀ of 1. Under the conditions used, a 1-ml cell suspension with an A₆₀₀ of 1 led to 2.2 x 10⁸ CFU. All washing steps were carried out at room temperature.

To determine transport activity, 500 µl of cell suspension was preincubated at 37°C for 5 min. After preincubation, ¹⁴C-labeled sugars (final concentration, 1 µM) were added. The cells were further incubated at 37°C. After 15, 30, 45, 60, and 120 seconds, 70-µl aliquots were taken, filtered through 0.45-µm-pore-size NC 45 filters (diameter, 25 mm; Schleicher & Schuell GmbH, Dassel, Germany), and washed three times with 5 ml transport buffer. The radioactivity retained on the filters was determined in a scintillation counter (LS 1801; Beckmann, Munich, Germany). Uptake rates were calculated based on the maximal gradients of the resulting curves when retained radioactivity was plot over time.

In the case of titration experiments, 490-µl preincubated cells (see above) were placed in a 10-µl mixture of ¹⁴C (final concentration, 1 µM) and ¹²C sugars (for the sugars and final concentrations, see Results). Uptake rates were determined as described above.

For kinetic studies, the 10-µl sugar mixture was composed of 2.3 pmol [¹⁴C]maltose (1.4 nCi) or 7.8 pmol [¹⁴C]maltotriose (7 nCi) and different amounts of the appropriate ¹²C sugar, leading to final overall sugar concentrations from 0.1 µM to 50 µM.

Growth experiments. To study the phenotypes of different mutants for growth on maltodextrins, we used minimal medium (44 mM potassium dihydrogen phosphate, 60 mM dipotassium hydrogen phosphate, 2.9 mM trisodium citrate, 15 mM ammonium sulfate, 245 µM tryptophan, 33 µM iron(III) citrate, 2 mM magnesium sulfate, 1 mM calcium chloride, 17 mM potassium L-glutamate) containing 0.1% maltodextrins (ICN). Cells from overnight cultures (grown in LB containing the relevant antibiotics) were washed three times with minimal medium before they were used to inoculate fresh minimal medium. Absorption was monitored at 600 nm.

-Glucosidase assays (MalL activity assay). MalL activity in crude cell extract of B. subtilis was measured according to the ß-galactosidase method of Miller (22) with slight modifications. Instead of o-nitrophenyl-ß-D-galactopyranoside, p-nitrophenyl--D-glucopyranoside was used as a substrate. Cells grown in the indicated media were harvested by centrifugation for 5 min at 5,000 x g. To prepare crude cell extracts, cells were resuspended in Z buffer (22) and treated with lysozyme/DNase I (final concentrations, lysozyme, 40 µg/ml, and DNase I, 6 µg/ml) at 37°C until lysis of the cells occurred. Cell debris was removed by centrifugation for 5 min at 10,000 x g and 4°C. The resulting supernatant was used further. Protein concentrations of the cell extracts were determined using the Bio-Rad protein assay (Bio-Rad, Munich, Germany). For the assay, 200 µl cell extract or less (when high activity was expected) was brought to 800 µl with Z buffer. The mixture was incubated at 28°C for 5 min. The reaction at 28°C was started by the addition of 200 µl p-nitrophenyl--D-glucopyranoside (4 mg/ml dissolved in 0.1 M phosphate buffer, pH 7.0) (22). After the mixture became slightly yellow, the reaction was stopped by the addition of 0.5 ml 1 M Na₂CO₃. The resulting p-nitrophenol was measured at 420 nm. As a reference, 800 µl Z buffer treated in the same way was used. Under these conditions, 1 nmol p-nitrophenol has an optical density at 420 nm of 0.0097. The specific activity is given in nmol · min^–1 · mg crude cell extract^–1.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Overproduction and purification of the His₆-tagged MdxE protein. The primary sequence deduced from the mdxE nucleotide sequence exhibits similarities to maltose-binding proteins of enteric bacteria (7, 10) and to several other binding proteins in B. subtilis (28). Several attempts to clone the complete mdxE gene in high-copy-number expression vectors replicating in E. coli failed (data not shown). Therefore, we constructed a variant in which the signal sequence (the first 22 amino acids [7, 28, 10]) was exchanged for a His₆ tag, followed by Cys23 of the native protein. This protein has a 12-residue N-terminal extension that includes the affinity tag (underlined): Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Ser-Cys (Cys23 of the native protein). The modified protein was overproduced by E. coli strain RB791/pMBPHis6x and purified to homogeneity by Ni²⁺ affinity gel chromatography and gel filtration (data not shown).

Characterization of MdxE substrate specificity. To investigate the substrate binding properties of the purified protein, substrate interactions were analyzed by surface plasmon resonance spectroscopy at sugar concentrations of 1 mM. Maltose and maltodextrins up to maltoheptaose were able to trigger response signals (Fig. 2). As expected, the number of response units increased with the size of the interacting substrate. No signals were observed under the same experimental conditions using isomaltose, isomaltotriose, isomaltotetraose, lactose, palatinose, sucrose, or trehalose (some of the controls are shown in Fig. 2). To determine the dissociation constants, we varied the substrate concentrations in the range between 1 µM and 1 mM (maximum, 10 mM for maltose) (Table 3). These data show that MdxE functions as a maltodextrin-binding protein that exhibits micromolar affinities for maltodextrins (e.g., maltohexaose; K_d, 3 µM) but only a low affinity for maltose (K_d, 1 mM). We therefore propose that the yvdG gene be renamed mdxE and its product MdxE.

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FIG. 2. Overlay of several sensorgrams derived from the interactions of MdxE with different carbohydrates. The sugars used are listed on the right. The order of the sugars corresponds to the order of the responses. Responses are given in resonance units (RU) on the ordinate, and time is given on the abscissa. The start of injection of the sugar solutions is set to 0 s. Time of injection, 360 s.

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TABLE 3. Substrate binding affinities of Bacillus subtilis MdxEa

Determination of -glucosidase (MalL) activity. To test whether mutations reduce the expression of downstream genes in the yvdE-pgcM gene cluster, we measured MalL-mediated -glucosidase activity in cell extracts obtained from cultures grown in Luria-Bertani broth supplemented with the indicated sugars at 1%. Insertion of the aphA3 resistance cassette (which carries its own constitutive promoter) in mdxE and in the same orientation as mdxE resulted in constitutive MalL activity. In this mutant, MalL activity is independent of an exogenous inducer. In a mutant which carried the mdxE483 deletion, MalL activity was comparable to that of the wild type (Fig. 3). These data showed that the mdxE483 mutation is not polar on the distal genes.

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FIG. 3. Cytoplasmic -glucosidase activities in different B. subtilis mutants compared to the wild type. All strains were grown in LB (uninduced), LB containing 1% maltose (induced), or LB containing 1% maltose and 1% glucose (repressed). The error bars represent root mean square errors.

On the other hand the mdxF-mdxG insertion showed almost no activity and is therefore polar on malL.

Maltose and maltotriose uptake. To test whether B. subtilis MdxE and the mdxF and mdxG gene-encoded products play a significant role in transport, we measured [¹⁴C]maltose and [¹⁴C]maltotriose uptake in several mutants and compared it to that of the wild type after growth in Luria-Bertani broth in the presence and absence of 1% maltose, as well as 1% maltose and 1% glucose, respectively. As seen in Fig. 4, maltose uptake was induced by maltose, but induction was reduced by glucose. No significant differences in maltose uptake rates between the wild type and the mdxE or mdxF-mdxG mutant were detectable. In contrast, in a malP single mutant or in a malP mdxE or a malP mdxF-mdxG double mutant, no maltose uptake was detectable after growth in Luria-Bertani broth in the presence of 1% maltose. These data demonstrate that maltose uptake is MalP-dependent (as indicated by Reizer et al. [29]) and independent of MdxE, MdxF, and MdxG. We also measured maltose transport in a ptsG mutant to test whether the required phosphoryl group for transport is delivered by enzyme II^Glc (PtsG). Since maltose transport in this strain is elevated even compared to the wild type, PtsG is not required for maltose transport, confirming previous observations by Reizer et al. (29), but might have influence on maltose transport regulation.

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FIG. 4. [14C]maltose transport in different B. subtilis mutants. Uptake rates are given in nmol maltose taken up per minute by 1010 CFU. All mutant strains were grown in LB containing 1% maltose (gray columns). The wild type (wt) was grown in LB containing 1% maltose (induced; gray column), LB containing 1% maltose and 1% glucose (repressed; white column) or LB (uninduced; black column). The relevant genotypes are indicated below. The error bars represent standard deviations.

To further characterize maltose transport, we analyzed [¹⁴C]maltose uptake in the mdxF-mdxG mutant in the presence of different amounts (10 µM, 100 µM, and 1 mM) of several unlabeled sugars. The mdxF-mdxG mutant was used to prevent possible interference between the maltodextrin ABC transporter and [¹⁴C]maltose uptake when maltodextrins were used in the competition experiment. As shown in Fig. 5, unlabeled maltose exhibited the most significant effect on [¹⁴C]maltose uptake. Weaker effects were obtained with glucose and trehalose. Maltotriose resulted only in slight effects at high concentrations. Nearly no effects were detected when maltotetraose, maltopentaose, maltohexaose, or maltoheptaose was used (data for maltohexaose and maltoheptaose not shown). With the exception of maltose, none of the sugars at 1 mM concentration blocked transport completely. Whether the interference of glucose and trehalose with maltose transport is direct or indirect (e.g., by competing for phosphorylated PTS proteins) cannot be determined based on these data.

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FIG. 5. Uptake of [14C]maltose by B. subtilis strain MD274 (mdxF-mdxG::aphA3) in the presence of different concentrations of 12C sugars. The 12C sugars used are indicated below the columns. For comparison, uptake of [14C]maltose (1 µM) without additional 12C sugar is shown as a positive control (hatched column). Uptake rates are given in nmol [14C]maltose taken up per minute by 1010 CFU. The error bars represent root mean square errors.

We also determined the kinetics for maltose uptake in an amyE and an mdxE mutant in order to avoid problems with extracellular hydrolytic activity or interference with the potential maltodextrin system (Table 4). An apparent K_m of 5 µM and a V_max of 91 nmol · min^–1 · (10¹⁰ CFU)^–1 for the amyE mutant, as well as an apparent K_m of 7.5 µM and a V_max of 114 nmol · min^–1 · (10¹⁰ CFU)^–1 for the mdxE mutant, was determined.

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TABLE 4. Transport affinitiesa of Bacillus subtilis

Maltotriose uptake in the wild type is induced by maltose and reduced by glucose (Fig. 6). No uptake of maltotriose was detectable in malP mdxE, malP mdxF-mdxG, or malP msmX double mutants (Fig. 6), leading to the conclusion that maltotriose uptake is mediated by the ABC transporter composed of MdxE, MdxF, MdxG, and MsmX. The observation that maltotriose uptake was only slightly or not at all affected in the corresponding single mutants (Fig. 6) was most likely caused by the contamination of [¹⁴C]maltotriose by [¹⁴C]maltose (data not shown).

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FIG. 6. [14C]maltotriose transport in different B. subtilis mutants. Uptake rates are given in nmol maltose taken up per minute by 1010 CFU. All mutant strains were grown in LB containing 1% maltose (gray columns). The wild type (wt) was grown in LB containing 1% maltose (induced; gray column), LB containing 1% maltose and 1% glucose (repressed; white column), or LB (uninduced; black column). The relevant genotypes are indicated below. The error bars represent standard deviations.

Since the purity of the [¹⁴C]maltotriose used was not satisfactory, we chose the malP mutant for further analysis of maltotriose uptake. As shown in Fig. 7, the presence of 10 µM maltotriose, maltotetraose, maltopentaose, maltohexaose, or maltoheptaose was able to reduce maltotriose uptake in this strain significantly. Glucose, maltose, and lactose lower maltotriose uptake, whereas trehalose shows no effect on it. We also determined the transport kinetics for maltotriose (Table 4), revealing that the mdxE-, mdxF-, mdxG-, and msmX-encoded ABC transporter is a high-affinity transporter with an apparent K_m of 2.2 µM and a V_max of 3.2 nmol · min^–1 · (10¹⁰ CFU)^–1. The same analysis in a malP amyE double mutant yielded an apparent K_m of 1.4 µM and a V_max of 4.7 nmol · min^–1 · (10¹⁰ CFU)^–1 for maltotriose uptake.

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FIG. 7. Uptake of [14C]maltotriose by B. subtilis strain GP110 (malP::spec) in the presence of different 12C sugars [10 µM]. The 12C sugars used are indicated below the columns. For comparison, uptake of [14C]maltotriose (1 µM) without additional 12C sugar is shown as a positive control. Uptake rates are given in nmol [14C]maltotriose taken up per minute by 1010 CFU. The error bars represent standard deviations.

These data demonstrate that maltose is taken up by MalP and that maltotriose, and probably maltodextrin, transport is mediated via the mdxE-, mdxF-, mdxG-, and msmX-encoded ABC transporter.

Growth in minimal medium with maltodextrins or maltose. To get more information about utilization of maltodextrins by B. subtilis, we analyzed the phenotypes of several mutants grown in minimal medium with potassium glutamate as a carbon source in the presence of maltose or maltodextrins. Wild-type cells grown in minimal medium with potassium glutamate as a sole carbon source served as a control.

When grown in minimal medium containing 0.1% maltose, all mutants defective in malP showed prolonged doubling times (about 74 min) and reached a final optical density (A₆₀₀) of about 1.0 to 1.4 (Table 5). This growth behavior was comparable to that of the wild type when grown in minimal medium lacking an additional carbon source (doubling time, 84 min; final absorption, 1.3). All mutants defective in mdxF-mdxG but carrying wild-type malP showed doubling times like that of the wild type grown in minimal medium containing 0.1% maltose but reached a final absorption at 600 nm of about 2.5. All other mutants showed growth behavior like that of the wild type when grown in minimal medium containing 0.1% maltose (doubling time, 59 min; final absorption, 3.4) (Table 5). These data show that neither extracellular amylase AmyE nor the maltodextrin ABC transporter is required for maltose uptake.

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TABLE 5. Phenotype analysis of Bacillus subtilis strains for growth behavior on minimal mediuma plus maltose

When the cells were grown in minimal medium containing 0.1% maltodextrins, the mdxE, mdxF-mdxG, and amyE single mutants showed doubling times comparable to that of the wild type (Table 6). However, the mdxF-mdxG mutant reached an optical density at 600 nm of only 3.0. When the amyE mutation was combined with either mdxE or mdxF-mdxG, the mutants showed prolonged doubling times and reached final absorptions of about 2.7. The malP single and malP amyE, malP mdxE, and malP mdxF-mdxG double mutants showed extremely prolonged doubling times but reached optical densities of about 2. When triple mutants (malP amyE in combination with either mdxE or mdxF-mdxG) were tested, they showed growth behavior like that of the wild type when grown in minimal medium without an additional carbon source (Table 6). Thus, maltodextrins can either be transported inside the cell by the maltodextrin ABC transporter or, after degradation to maltose by extracellular amylase AmyE, transported as maltose by MalP.

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TABLE 6. Phenotype analysis of Bacillus subtilis strains for growth behavior on minimal mediuma plus maltodextrins

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacillus subtilis contains at least 37 open reading frames encoding putative solute-binding proteins (18, 28, 41), but the specificities of many of these transporter systems are not yet known. Three of them showed significant homology to maltose/maltodextrin-binding proteins, namely, YesO, YvfK, and MdxE (YvdG) (18, 41). Recently, it was shown that YvfK interacts with linear and cyclic maltodextrins (16). The mdxE (yvdG) gene is located in a cluster of nine genes. One of the other genes, malL, encodes an -glucosidase. Its activity is inducible by maltose (34). This raised the question of whether this putative transport system that mdxE belongs to is responsible for maltose uptake.

To analyze the function of MdxE, we purified a lipidless, N-terminally His₆-tagged variant of the putative maltose binding protein. Using surface plasmon resonance spectroscopy, maltose (K_d, 1 mM) and longer maltodextrins (K_d, 3 to 6 µM) were found to bind to MdxE (Table 3). Based on the low affinity for maltose, it is not clear whether maltose is an actual substrate. Comparison of the affinities of B. subtilis MdxE with maltose binding proteins of E. coli (K_d, 1 µM for maltose) or Aeromonas hydrophila (K_d, 1.6 µM for maltose) or the affinity of glycine/betaine binding protein of B. subtilis for glycine/betaine (K_d, 6 µM) (17) suggests that B. subtilis MdxE is a maltodextrin-binding protein rather than a maltose binding protein.

To test whether the in vitro results obtained can be confirmed by in vivo experiments, we measured maltose and maltotriose transport. The data obtained show that maltose transport is inducible by maltose and underlies carbon catabolite repression mediated by glucose. Comparing the wild type grown in Luria-Bertani medium (supplemented with maltose) with the different mutants, all mutants defective in malP lost maltose transport. These data are in agreement with previous data, which demonstrated that a malP mutant showed sevenfold-increased doubling times on maltose minimal medium (29). The mdxE or mdxF-mdxG (yvdH-yvdI) single mutations showed no effect on maltose transport (Fig. 4). These data demonstrate that maltose uptake occurs only via the malP-encoded specific EIICB^Mal and not via another transport mechanism. A ptsG mutant showed even higher maltose transport rates than wild-type cells. Thus, PtsG is not the phosphoryl group donating enzyme II, as is known to be the case in sucrose or trehalose uptake (6, 43).

When a ¹⁴C-labeled maltose/maltotriose mixture was used as a substrate in transport assays, it could be shown that the malP mutant transported the maltotriose portion, whereas the mdxE, malF-malG, or msmX mutant transported the maltose portion of the labeled substrate mixture used (Fig. 6). In combination with the data obtained from competition experiments, these data show that maltotriose is taken up by the mdxE-, mdxF-, mdxG-, and msmX-encoded transport system, and they indicate that larger maltodextrins are also taken up by this system. The mechanism by which glucose and lactose inhibit maltotriose transport is unclear. The competition achieved by maltose might occur at the binding-protein level, since MdxE exhibits a low affinity for maltose (Table 3). Interestingly, maltotriose uptake in the wild type and in the malP mutant can be induced by exogenous maltose. Like maltose transport, maltotriose transport underlies carbon catabolite repression mediated by glucose (Fig. 6).

The described transport data were corroborated by growth analysis with wild-type and mutant strains. The mdxE and mdxF-mdxG single mutants showed no significant phenotype for growth on maltose. These data show that maltose is taken up by the PTS-dependent EIICB^Mal (MalP), as was suggested by Reizer et al. (29), and that inactivation of extracellular -amylase AmyE has no influence on the utilization of maltose.

In contrast to growth on maltose, utilization of maltodextrins showed an additional intermediate phenotype. Only when mutations of the ABC transporter were combined with mutations in amyE, or when amyE mutations were combined with the mutation in malP, were doubling times prolonged and the absorptions finally reached reduced. When mutations of the ABC transporter were combined with the mutation in amyE and the mutation in malP, the strains showed growth behavior similar to that of the wild type in minimal medium without an additional carbon source.

These data led to our current model of starch or glycogen utilization by B. subtilis (Fig. 8). First, these polysaccharides are hydrolyzed extracellularly by AmyE, resulting in maltose and maltodextrins (15). Maltose is then taken up by the PTS and becomes phosphorylated. Cytoplasmic maltose-P is hydrolyzed by MalA, resulting in glucose and glucose-6-P (46). Maltodextrins are taken up by the maltodextrin-specific ABC transporter composed of MdxE, MdxF, MdxG, and MsmX without phosphorylation. After they enter the cell, they are degraded in the concerted action of cytoplasmic maltogenic amylase or neopullulanase YvdF (5), maltose phosphorylase YvdK, and -glucosidase MalL (34, 35), resulting in glucose and glucose-1-P. Glucose-1-P is than converted to glucose-6-P by PgcM (30). Free glucose resulting from maltodextrin degradation, as well as from maltose-P hydrolysis, is finally phosphorylated by glucose kinase GlcK, leading to glucose-6-P (38). By this concerted action, exogenous starch and glycogen are converted to glucose-6-P that can enter glycolysis. Transcription of the mal operon is regulated by GlvR (47), whereas transcription of the maltodextrin operon remains unclear but could be regulated by YvdE. The EIIA domain that delivers the phosphoryl group to EIICB^Mal remains unknown, but it is clearly not the EIIA domain of PtsG, as in sucrose or trehalose uptake (6, 43).

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FIG. 8. Model of starch and glycogen utilization in B. subtilis. The proteins involved are shown as circles. Protein names are indicated. Starch and glycogen are hydrolyzed by AmyE extracellularly (outside) in maltodextrins and maltose. The resulting oligosaccharides are than transported into the cytoplasm (inside) over the membrane by the maltodextrin ABC transporter or the maltose-specific EIICB. Further degradation of the sugars finally leads to glucose-6-P, which can enter glycolysis. The phosphoryl moiety delivering EIIA is unknown. For further explanation, see the text.

 
We thank Winfried Boos, in whose laboratory work was done.

Financial support was from the Deutsche Forschungsgemeinschaft (TR-SFB11) and the BMBF Knoll AG.

Dedicated in loving memory to Michael K. Dahl, who died unexpectedly on 4 May 2003.

 
* Corresponding author. Mailing address: Lehrstuhl für Mikrobiologie, Fachbereich Biologie der Universität Konstanz, Universitätsstrasse 10, M605, D-78457 Konstanz, Germany. Phone: (49-7531) 882041. Fax: (49-7531) 883356. E-mail: stefan.schoenert{at}gmx.de.

Present address: Lehrstuhl für Mikrobiologie, Fachbereich Biologie/Chemie der Universität Osnabrück, D-49069 Osnabrück, Germany.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, June 2006, p. 3911-3922, Vol. 188, No. 11
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