Maltose and maltodextrin transport in the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius is mediated by a high-affinity transport system that includes a maltose binding protein tolerant to low pH

Abstract

We have studied the uptake of maltose in the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius, which grows best at 57 degrees C and pH 3.5. Under these conditions, accumulation of [(14)C]maltose was observed in cells grown with maltose but not in those grown with glucose. At lower temperatures or higher pH values, the transport rates substantially decreased. Uptake of radiolabeled maltose was inhibited by maltotetraose, acarbose, and cyclodextrins but not by lactose, sucrose, or trehalose. The kinetic parameters (K(m) of 0.91 +/- 0.06 microM and V(max) ranging from 0.6 to 3.7 nmol/min/mg of protein) are consistent with a binding protein-dependent ATP binding cassette (ABC) transporter. A corresponding binding protein (MalE) that interacts with maltose with high affinity (K(d) of 1.5 microM) was purified from the culture supernatant of maltose-grown cells. Immunoelectron microscopy revealed distribution of the protein throughout the cell wall. The malE gene was cloned and sequenced. Five additional open reading frames, encoding components of a maltose transport system (MalF and MalG), a putative transcriptional regulator (MalR), a cyclodextrinase (CdaA), and an alpha-glucosidase (GlcA), were identified downstream of malE. The malE gene lacking the DNA sequence that encodes the signal sequence was expressed in Escherichia coli. The purified wild-type and recombinant proteins bind maltose with high affinity over a wide pH range (2.5 to 7) and up to 80 degrees C. Recombinant MalE cross-reacted with an antiserum raised against the wild-type protein, thereby indicating that the latter is the product of the malE gene. The MalE protein might be well suited as a model to study tolerance of proteins to low pH.

FIG. 1

Maltose uptake by A. acidocaldarius cells. Cells were grown in minimal medium supplemented with maltose (10 mM) (○), glucose (10 mM) (□), or maltose and glucose (10 mM each) (●) as a carbon source. Uptake assays were performed at 57°C, pH 3.6, and 3 μM [¹⁴C]maltose.

FIG. 2

Determination of apparent K_m and V_max values of maltose transport in A. acidocaldarius cells (Lineweaver-Burk plot). Cells were grown in minimal medium in the presence of 10 mM maltose and assayed at 57°C and pH 3.6 as described in Materials and Methods. Data are from a single experiment. The K_m is 1 μM, and the V_max is 1.25 nmol/min/mg of protein.

FIG. 3

Maltose uptake by A. acidocaldarius cells (A) and maltose binding activity of MalE_Aa (B) in the presence of competing substrates. (A) Uptake was performed at 57°C, pH 3.6, and 3 μM [¹⁴C]maltose in the presence of the indicated sugars at a concentration of 1 mM each. Cells were preincubated for 2 min with nonlabeled sugars in minimal medium without a carbon source, and uptake was stopped after 1 min. (B) Binding assays were performed at 57°C, pH 3.6, and 5 μM [¹⁴C]maltose in the presence of the indicated sugars at a concentration of 1 mM each. MalE_Aa (5 μg) was preincubated for 1 min with nonlabeled sugars in buffer (pH 3.6), and the binding reaction was stopped after 1 min. Shaded bars, wild type MalE_Aa; black bars, recombinant MalE_Aa.

FIG. 4

SDS gel of purified MalE_Aa. Wild-type and recombinant MalE_Aa proteins were purified from the culture supernatant of A. acidocaldarius cells and from the cytosol of E. coli strain ED169(pAH18), respectively, by affinity chromatography through agarose-coupled amylose. Aliquots were subjected to SDS-polyacrylamide gel electrophoresis, and the gel was subsequently stained with Coomassie brilliant blue. Lane 1, wild-type MalE_Aa (2.5 μg); lane 2, recombinant MalE_Aa (5 μg); lane St, molecular weight standards (in thousands). The observed difference in molecular weight is due to exposure of the wild-type protein to an extracellular protease that clips 23 amino acids from the N terminus (24).

FIG. 5

Scatchard plot of maltose binding by MalE_Aa. The affinity of maltose binding was determined by a precipitation assay as described in Materials and Methods. The line fitted to a K_d of 1.5 μM is indicated.

FIG. 6

Electron micrographs of thin sections of A. acidocaldarius cells reacted with monospecific antiserum to purified wild-type MalE_Aa. (A) Sections reacted with monospecific rabbit anti-MalE_Aa and protein A-gold conjugate; (B) sections reacted with irrelevant rabbit serum and protein A-gold conjugate. Representative pictures are shown.

FIG. 7

Synthesis of MalE_Aa in the presence of different sugars. Wild-type A. acidocaldarius was grown in minimal salt medium supplemented with the following sugars (10 mM each): glucose (lane 1), maltose (lane 2), starch (0.2%, wt/vol) (lane 3), maltotetraose (lane 4), α-cyclodextrin (lane 5), β-cyclodextrin (lane 6), and maltose plus glucose (lane 7). Proteins from whole-cell extracts were separated by SDS-polyacrylamide gel electrophoresis and subsequently subjected to immunoblot analysis.

FIG. 8

Organization of the genomic region around malE_Aa. ORFs were named according their homologs identified by database searches using BLAST. The sequence at the 3′ end of the glcA gene, including the predicted transcriptional termination structure (underlined), is also shown. Arrows indicate the predicted direction of transcription. Genes encoding maltose transport components are shaded. p, putative promoter region upstream of amyA (32).

FIG. 9

Temperature (A) and pH (B) dependence of maltose binding to purified wild-type (▴) and recombinant (●) MalE_Aa. Binding assays were performed at the indicated temperatures in Na-acetate buffer (pH 3.6) and in the presence of 5 μM [¹⁴C]maltose. (B) Binding assays were performed at 57°C in citrate-phosphate buffer of the indicated pH. MalE of S. enterica serovar Typhimurium (■) was assayed at pH 7.4 (A) and 37°C (B). MBP, maltose binding protein.