Reconstruction of the central carbohydrate metabolism of Thermoproteus tenax by use of genomic and biochemical data

Abstract

The hyperthermophilic, facultatively heterotrophic crenarchaeum Thermoproteus tenax was analyzed using a low-coverage shotgun-sequencing approach. A total of 1.81 Mbp (representing 98.5% of the total genome), with an average gap size of 100 bp and 5.3-fold coverage, are reported, giving insights into the genome of T. tenax. Genome analysis and biochemical studies enabled us to reconstruct its central carbohydrate metabolism. T. tenax uses a variant of the reversible Embden-Meyerhof-Parnas (EMP) pathway and two different variants of the Entner-Doudoroff (ED) pathway (a nonphosphorylative variant and a semiphosphorylative variant) for carbohydrate catabolism. For the EMP pathway some new, unexpected enzymes were identified. The semiphosphorylative ED pathway, hitherto supposed to be active only in halophiles, is found in T. tenax. No evidence for a functional pentose phosphate pathway, which is essential for the generation of pentoses and NADPH for anabolic purposes in bacteria and eucarya, is found in T. tenax. Most genes involved in the reversible citric acid cycle were identified, suggesting the presence of a functional oxidative cycle under heterotrophic growth conditions and a reductive cycle for CO2 fixation under autotrophic growth conditions. Almost all genes necessary for glycogen and trehalose metabolism were identified in the T. tenax genome.

FIG. 1.

Variant of the EMP pathway in T. tenax. The enzymes corresponding to homologs identified in this study are shown in grey-shaded boxes; enzymes identified and characterized previously are shown in open boxes. Open arrows indicate irreversible reactions. For abbreviations of enzyme names, see Table 1.

FIG. 2.

Variants of the ED pathway in T. tenax. The enzymes identified in the T. tenax genome are shown in grey-shaded boxes, and the previously characterized GDH is shown in an open box. The central intermediate KDG of both variants is boxed. Closed arrows indicate the reactions of the nonphosphorylative version of the ED pathway; open arrows indicate the reactions of the semiphosphorylative version. Grey-shaded arrows mark reactions involved in both versions. For abbreviations of enzyme names, see Table 2.

FIG. 3.

(A) Organization of functionally related genes of the central carbohydrate metabolism in T. tenax. (Gene names are given in alphabetical order at the end of the legend; gene data are given in Table 1 to Table 6.) (B) Putative promoter structures of genes organized in operons. The nucleotide sequences (5′ end) and upstream regions of genes are shown. The putative crenarchaeal promoter sequences (3, 59, 60) with BRE sites [crenarchaeal consensus sequence (A/G)N(A/T)AA(A/T)] and the TATA box [crenarchaeal consensus sequence (C/T)TTTTAAA] are in light and dark grey boxes, respectively. Putative Shine-Dalgarno sequences (GAGG) (29) are underlined, the putative start codon is shown in boldface characters, and the stop codon of preceding genes is indicated with double underlining. The operon structures shown were confirmed for the EMP genes by Northern analysis. All other clusters shown represent putative operons. Gene names and corresponding enzyme names: acn, aconitase; adh, alcohol dehydrogenase; acs, acetyl-CoA synthetase; act, acetyl-CoA transferase; amyA, α-amylase; cis1, citrate synthase 1; citE, citrate lyase β chain; fba, FBPA; frdAB, fumarate reductase α and β chains; gaa, glucan-1,4-α-glucosidase; gad, GAD; gap, GAPDH; glgA, glycogen synthase; glgP, (glycogen) phosphorylase; gltX, glutamyl tRNA synthetase; gt, glycosyltransferase; hp, hypothetical protein, hxk, HK; kdgA, KDP(G)A; kdgK, KDGK; mthfs, 5-formyltetrahydrofolate cyclo-ligase; oorABCD, oxoacid:ferredoxin oxidoreductase α, β, γ, and δ chains; orfV, orfX, orfY, and orfZ, hypothetical proteins; pncB, nicotinate phosphoribosyltransferase; pfp, PP_i-PFK; pgk, PGK; ptp, putative transport protein; rfbA, glucose-1-phosphate thymidylyltransferase; rfbB, dTDP-glucose-4,6-dehydratase; rfbC, dTDP-4-dehydrorhamnose-3,5-epimerase; rfbD, dTDP-4-dehydrorhamnose reductase; sdhABCD, succinate dehydrogenase α, β, γ, and δ chains; sucDC, succinyl-CoA synthetase α and β chains; snt, sugar phosphate nucleotidyltransferase; tpi, TIM; tpsp, TPSP; treS/treP, trehalose synthase or trehalose phosphorylase.

FIG. 3.

(A) Organization of functionally related genes of the central carbohydrate metabolism in T. tenax. (Gene names are given in alphabetical order at the end of the legend; gene data are given in Table 1 to Table 6.) (B) Putative promoter structures of genes organized in operons. The nucleotide sequences (5′ end) and upstream regions of genes are shown. The putative crenarchaeal promoter sequences (3, 59, 60) with BRE sites [crenarchaeal consensus sequence (A/G)N(A/T)AA(A/T)] and the TATA box [crenarchaeal consensus sequence (C/T)TTTTAAA] are in light and dark grey boxes, respectively. Putative Shine-Dalgarno sequences (GAGG) (29) are underlined, the putative start codon is shown in boldface characters, and the stop codon of preceding genes is indicated with double underlining. The operon structures shown were confirmed for the EMP genes by Northern analysis. All other clusters shown represent putative operons. Gene names and corresponding enzyme names: acn, aconitase; adh, alcohol dehydrogenase; acs, acetyl-CoA synthetase; act, acetyl-CoA transferase; amyA, α-amylase; cis1, citrate synthase 1; citE, citrate lyase β chain; fba, FBPA; frdAB, fumarate reductase α and β chains; gaa, glucan-1,4-α-glucosidase; gad, GAD; gap, GAPDH; glgA, glycogen synthase; glgP, (glycogen) phosphorylase; gltX, glutamyl tRNA synthetase; gt, glycosyltransferase; hp, hypothetical protein, hxk, HK; kdgA, KDP(G)A; kdgK, KDGK; mthfs, 5-formyltetrahydrofolate cyclo-ligase; oorABCD, oxoacid:ferredoxin oxidoreductase α, β, γ, and δ chains; orfV, orfX, orfY, and orfZ, hypothetical proteins; pncB, nicotinate phosphoribosyltransferase; pfp, PP_i-PFK; pgk, PGK; ptp, putative transport protein; rfbA, glucose-1-phosphate thymidylyltransferase; rfbB, dTDP-glucose-4,6-dehydratase; rfbC, dTDP-4-dehydrorhamnose-3,5-epimerase; rfbD, dTDP-4-dehydrorhamnose reductase; sdhABCD, succinate dehydrogenase α, β, γ, and δ chains; sucDC, succinyl-CoA synthetase α and β chains; snt, sugar phosphate nucleotidyltransferase; tpi, TIM; tpsp, TPSP; treS/treP, trehalose synthase or trehalose phosphorylase.

FIG. 4.

The central carbohydrate metabolism in T. tenax. A selection of growth substrates is given at the top; the labels of the different pathways are marked by grey-shaded boxes.