Sulfolobus acidocaldarius Transports Pentoses via a Carbohydrate Uptake Transporter 2 (CUT2)-Type ABC Transporter and Metabolizes Them through the Aldolase-Independent Weimberg Pathway

Abstract

Sulfolobus spp. possess a great metabolic versatility and grow heterotrophically on various carbon sources, such as different sugars and peptides. Known sugar transporters in Archaea predominantly belong to ABC transport systems. Although several ABC transporters for sugar uptake have been characterized in the crenarchaeon Sulfolobus solfataricus, only one homologue of these transporters, the maltose/maltooligomer transporter, could be identified in the closely related Sulfolobus acidocaldarius Comparison of the transcriptome of S. acidocaldarius MW001 grown on peptides alone and peptides in the presence of d-xylose allowed for the identification of the ABC transporter for d-xylose and l-arabinose transport and the gaining of deeper insights into pentose catabolism under the respective growth conditions. The d-xylose/l-arabinose substrate binding protein (SBP) (Saci_2122) of the ABC transporter is unique in Archaea and shares more similarity to bacterial SBPs of the carbohydrate uptake transporter-2 (CUT2) family than to any characterized archaeal one. The identified pentose transporter is the first CUT2 family ABC transporter analyzed in the domain of Archaea Single-gene deletion mutants of the ABC transporter subunits exemplified the importance of the transport system for d-xylose and l-arabinose uptake. Next to the transporter operon, enzymes of the aldolase-independent pentose catabolism branch were found to be upregulated in N-Z-Amine and d-xylose medium. The α-ketoglutarate semialdehyde dehydrogenase (KGSADH; Saci_1938) seemed not to be essential for growth on pentoses. However, the deletion mutant of the 2-keto-3-deoxyarabinoate/xylonate dehydratase (KDXD [also known as KDAD]; Saci_1939) was no longer able to catabolize d-xylose or l-arabinose, suggesting the absence of the aldolase-dependent branch in S. acidocaldariusIMPORTANCE Thermoacidophilic microorganisms are emerging model organisms for biotechnological applications, as their optimal growth conditions resemble conditions used in certain biotechnologies such as industrial plant waste degradation. Because of its high genome stability, Sulfolobus acidocaldarius is especially suited as a platform organism for such applications. For use in (ligno)cellulose degradation, it was important to understand pentose uptake and metabolism in S. acidocaldarius This study revealed that only the aldolase-independent Weimberg pathway is required for growth of S. acidocaldarius MW001 on d-xylose and l-arabinose. Moreover, S. acidocaldarius employs a CUT2 ABC transporter for pentose uptake, which is more similar to bacterial than to archaeal ABC transporters. The identification of pentose-inducible promoters will expedite the metabolic engineering of S. acidocaldarius for its development into a platform organism for (ligno)cellulose degradation.

Keywords: ABC transporters; Sulfolobus carbon metabolism; archaea; pentose metabolism; promoters; sugar transport.

FIG 1

Growth curves of S. acidocaldarius MW001 in Brock medium containing 0.2% (wt/vol) N-Z-Amine alone (closed circles) and 0.2% (wt/vol) N-Z-Amine and 0.4% (wt/vol) d-xylose (open circles). For the experiment, six replicates were performed for each growth condition. Cells of three replicates were harvested for RNA isolation at mid-exponential growth phase (OD₆₀₀ of ∼0.5; marked with an arrow); growth of three additional replicates was further monitored.

FIG 2

Synteny of the d-xylose-induced ABC transporter-encoding gene cluster. Absynte scores represent TBLASTN scores of the 15-kb chromosome segments normalized to the BLASTP score of the query protein sequence as maximal bit score (35). Only bacterial sequences are closely related to the S. acidocaldarius genes, while archaeal hits only had a very low similarity that was below the value of relevant similarity, which is indicated by the dashed line. The numbers in the arrows indicate the respective ORF number.

FIG 3

Changes in SBP (saci_2122) transcript levels in response to the presence of different sugars in the growth medium. Differences in saci_2122 transcript amounts were determined by qRT-PCR. Bars indicate the sugar-specific transcript levels compared to transcription of cells grown only with N-Z-Amine on a log₂-fold scale.

FIG 4

Changes in the composition of glycosylated membrane proteins of S. acidocaldarius in response to the offered carbon source. NZ, N-Z-Amine; d-xyl, d-xylose; l-ara, l-arabinose.

FIG 5

Growth of S. acidocaldarius MW001 wild-type (wt) and ΔABC-TMD, ΔABC-SBP, ΔABC-NBD, ΔαKGSADH, and ΔKDXD/KDAD gene deletion strains in the presence of different carbon sources. Cultures were grown on 0.1% (wt/vol) N-Z-Amine alone (black closed circles) or with the addition of 0.1% (wt/vol) l-arabinose (orange open squares), dextrin (green open triangles), or d-xylose (blue open circles). (A) Wild-type S. acidocaldarius MW001; (B) ΔABC-TMD, ABC type transmembrane domain (saci_2121) mutant; (C) ΔABC-SBP, ABC type substrate binding domain (saci_2122) mutant; (D) ΔABC-NBD, ABC type nucleotide binding domain (saci_2120) mutant; (E) ΔαKGSADH, α-ketoglutarate semialdehyde dehydrogenase (saci_1938) mutant; (F) ΔKDXD/KDAD, 2-keto-3-deoxyarabinoate/xylonate dehydratase (saci_1939) mutant.

FIG 6

Enzymatic activities of KDG aldolase, KDXD/KDAD, and αKGSADH in crude extracts of S. acidocaldarius wild-type (MW001) and ΔKDXD/KDAD, ΔKGSADH, and ΔABC-SBP mutant strains. The columns denote the mean enzyme activity of three biological replicates, and the error bars show standard deviations. αKGSADH, α-ketoglutarate semialdehyde dehydrogenase (Saci_1938); KDXD/KDAD, 2-keto-3-deoxyarabinoate/xylonate dehydratase (Saci_1939); ABC-SBP, ABC type substrate binding domain (Saci_2122).

FIG 7

Identification of a cis-acting element in d-xylose-inducible promoter regions. Shown is WEBLogo and promoter alignment of ara box-containing promoters in the genome of S. acidocaldarius. Sequences containing the ara box motif AMCWWGTT (M = A or C; W = A or T) in the region −40 to −50 bp upstream of the transcription start site were used to generate a sequence logo by WEBLogo (http://weblogo.berkeley.edu/) (30).

FIG 8

β-Galactosidase (β-gal) activity of promoter-reporter fusions in cells grown in the presence of l-arabinose, dextrin, and d-xylose. The columns in panel A denote the mean enzyme activity of three biological replicates, and the error bars show the standard deviations. The dextrin condition represents the negative control. Sequences of the different promoters are shown in panel B.

FIG 9

Model of d-xylose and l-arabinose transport and metabolism in S. acidocaldarius MW001. The ABC transporter consists of the SBD Saci_2122 XylF (purple), the TMD Saci_2121 XylH (orange), and the NBD Saci_2120 XylG (green) and transports d-xylose and l-arabinose into the cell. An additional unknown transporter can import pentoses in the presence of peptides. Within the cells, these pentose sugars were shown to be degraded via the aldolase-independent Weimberg pathway to α-ketoglutarate, which enters the citric acid cycle. Conversely, the aldolase-dependent Dahms pathway converting the pentoses to pyruvate and glycolaldehyde (entering the citric acid cycle via glyoxylate) is not utilized under the chosen growth conditions. Enzymes transcriptionally upregulated on d-xylose are in blue, and stars mark promiscuous enzymes, which function simultaneously in hexose and pentose metabolism. Abbreviations: GDH-1, glucose dehydrogenase (isoenzyme 1); d-XAD/l-AraD, xylonate/arabinoate dehydratase; d-KDXD/l-KDAD, 2-keto-3-deoxy-xylonate/arabinoate dehydratase; α-KGSADH, α-ketoglutarate semialdehyde dehydrogenase.