The ribulose monophosphate pathway substitutes for the missing pentose phosphate pathway in the archaeon Thermococcus kodakaraensis

Abstract

The ribulose monophosphate (RuMP) pathway, involving 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI), is now recognized as a widespread prokaryotic pathway for formaldehyde fixation and detoxification. Interestingly, HPS and PHI homologs are also found in a variety of archaeal strains, and recent biochemical and genome analyses have raised the possibility that the reverse reaction of formaldehyde fixation, i.e., ribulose 5-phosphate (Ru5P) synthesis from fructose 6-phosphate, may function in the biosynthesis of Ru5P in some archaeal strains whose pentose phosphate pathways are imperfect. In this study, we have taken a genetic approach to address this possibility by using the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. This strain possesses a single open reading frame (TK0475) encoding an HPS- and PHI-fused protein. The recombinant HPS-PHI-fused enzyme exhibited the expected HPS and PHI activities in both directions (formaldehyde fixing and Ru5P synthesizing). The TK0475 deletion mutant Delta hps-phi-7A did not exhibit any growth in minimal medium, while growth of the mutant strain could be recovered by the addition of nucleosides to the medium. This auxotrophic phenotype together with the catalytic properties of the HPS-PHI-fused enzyme reveal that HPS and PHI are essential for the biosynthesis of Ru5P, the precursor of nucleotides, showing that the RuMP pathway is the only relevant pathway for Ru5P biosynthesis substituting for the classical pentose phosphate pathway missing in this archaeon.

FIG. 1.

Proposed pentose phosphate synthesis through HPS and PHI in T. kodakaraensis. Arrows with broken lines display the reactions catalyzed by enzymes whose orthologs are not apparent on the T. kodakaraensis genome. Intermediates: G6P, glucose 6-phosphate; 6PGL, 6-phosphoglucono-δ-lactone; 6PG, 6-phosphogluconate; Ru5P, ribulose 5-phosphate; Xu5P, xylulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7-phosphate; GAP, glyceraldehyde 3-phosphate; E4P, erythrose 4-phosphate; F6P, fructose 6-phosphate. Enzymes: G6PDH, glucose-6-phosphate dehydrogenase; 6PGLase, 6-phosphoglucono-δ-lactonase; 6PGDH, 6-phosphogluconate dehydrogenase; RPE, ribulose-5-phosphate-3-epimerase; RPI, ribose 5-phosphate isomerase; TK, transketolase; TA, transaldolase.

FIG. 2.

Schematic diagram of targeted disruption of hps-phi in T. kodakaraensis KW128 by use of pUDhps-phi. Relevant regions of the chromosome are illustrated for strains KW128 and Δhps-phi-7A. The gray regions display the homologous regions for recombination. The positions of primer sets used for the analyses of targeted disruption of hps-phi (CHDHPSPHI-R/CHDHPSPHI-F and CHHPSPHI-R/CHHPSPHI-F) are indicated by arrows. The thick black bar indicates the region spanned by the trpE probe used in Southern blot analyses. Abbreviations: Hd, restriction site of HindIII; Hypo, hypothetical gene; P, putative pyrF promoter region.

FIG. 3.

Western blot analyses of cell extracts of T. kodakaraensis strains KOD1, KW128, and Δhps-phi-7A. Abbreviations: CFE, cell extract; S, soluble fraction; P, particulate fraction.

FIG. 4.

PCR and Southern blot analyses of T. kodakaraensis Δhps-phi-7A (ΔpyrF ΔtrpE::pyrF Δhps-phi::trpE). (A) Amplification of the locus containing hps-phi and its homologous regions in strains KOD1 (lane 1), KW128 (lane 2), and Δhps-phi-7A (lane 3), using CHDHPSPHI-R/CHDHPSPHI-F as primers. M represents the DNA size marker, HindIII-digested λ DNA. (B) Amplification of the region within the hps-phi gene in T. kodakaraensis KOD1 (lane 1), KW128 (lane 2), and Δhps-phi-7A (lane 3), using CHHPSPHI-R/CHHPSPHI-F as primers. Primers used for these analyses are displayed in Fig. 2. (C) Southern blot analysis of T. kodakaraensis Δhps-phi-7A. The probe specific to the trpE gene was used against genomic DNA of KOD1 (lane 1), KW128 (lane 2), and Δhps-phi-7A (lane 3) digested with HindIII. The bars on the left side of the panels indicate the lengths of fragments in the DNA size marker, HindIII-digested λ DNA. The region spanned by the probe used for this analysis is indicated in Fig. 2.

FIG. 5.

Growth properties of T. kodakaraensis strains KW128 and Δhps-phi-7A in minimal medium, ASW-AA (open symbols), or a medium supplemented with five nucleosides (filled symbols). The cells were cultured at 85°C in ASW-AA media without or supplemented with five nucleosides, A, dT, G, C, and U. Symbols: open circles, KW128 without nucleosides; filled circles, KW128 with nucleosides; open triangles, Δhps-phi-7A without nucleosides; filled triangles, Δhps-phi-7A with nucleosides. Error bars represent the standard deviations from three independent experiments. OD₆₆₀, optical density at 660 nm.