Genetic evidence identifying the true gluconeogenic fructose-1,6-bisphosphatase in Thermococcus kodakaraensis and other hyperthermophiles

Abstract

Fructose-1,6-bisphosphatase (FBPase) is one of the key enzymes in gluconeogenesis. Although FBPase activity has been detected in several hyperthermophiles, no orthologs corresponding to the classical FBPases from bacteria and eukaryotes have been identified in their genomes. An inositol monophosphatase (IMPase) from Methanococcus jannaschii which displayed both FBPase and IMPase activities and a structurally novel FBPase (FbpTk) from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 have been proposed as the "missing" FBPase. For this study, using T. kodakaraensis, we took a genetic approach to elucidate which candidate is the major gluconeogenic enzyme in vivo. The IMPase/FBPase ortholog in T. kodakaraensis, ImpTk, was confirmed to possess high FBPase activity along with IMPase activity, as in the case of other orthologs. We therefore constructed Deltafbp and Deltaimp strains by applying a gene disruption system recently developed for T. kodakaraensis and investigated their phenotypes. The Deltafbp strain could not grow under gluconeogenic conditions while glycolytic growth was unimpaired, and the disruption resulted in the complete abolishment of intracellular FBPase activity. Evidently, fbpTk is an indispensable gene for gluconeogenesis and is responsible for almost all intracellular FBPase activity. In contrast, the endogenous impTk gene could not complement the defect of the fbp deletion, and its disruption did not lead to any detectable phenotypic changes under the conditions examined. These facts indicated that impTk is irrelevant to gluconeogenesis, despite the high FBPase activity of its protein product, probably due to insufficient transcription. Our results provide strong evidence that the true FBPase for gluconeogenesis in T. kodakaraensis is the FbpTk ortholog, not the IMPase/FBPase ortholog.

FIG. 1.

Schematic drawing of pUDImp (A) and pUDFbp (B) for disruption of imp and fbp in T. kodakaraensis KW128. The homologous regions between the circular DNAs and the chromosome of T. kodakaraensis are shaded. Restriction site abbreviations: A, ApaI; H, HindIII. The bold gray bar below the trpE gene indicates the region spanned by the trpE probe for Southern blot analyses. The bold black bars below the imp and fbp genes indicate the regions spanned by the imp and fbp probes used for Northern blot analyses.

FIG. 2.

(A) Amplification of imp locus from T. kodakaraensis KOD1, KW128, and Δimp-2A, with CHDIMP-R and CHDIMP-F as primers. (B) Amplification of fbp locus from T. kodakaraensis KOD1, KW128, and Δfbp-8J, with CHDFBP-R and CHDFBP-F as primers. (C) Southern blot analysis using the trpE probe with genomic DNAs of KOD1, KW128, and Δimp-2A digested with HindIII. (D) Southern blot analysis using the trpE probe with genomic DNAs of KOD1, KW128, and Δfbp-8J digested with ApaI. The region corresponding to the trpE probe is indicated in Fig. 1. M, DNA size marker (HindIII-digested λ DNA).

FIG. 3.

Growth properties of T. kodakaraensis KW128, Δimp-2A, and Δfbp-8J under glycolytic (open symbols) or gluconeogenic (closed symbols) conditions. The cells were cultured in ASW-AA medium supplemented with soluble starch or pyruvate at 85°C. Symbols: open circles, KW128 with starch; open squares, Δimp-2A with starch; open triangles, Δfbp-8J with starch; closed circles, KW128 with pyruvate; closed squares, Δimp-2A with pyruvate; closed triangles, Δfbp-8J with pyruvate. Error bars represent standard deviations for repeated independent experiments.

FIG. 4.

Northern blot analysis with imp probe (A) and fbp probe (B). Total RNAs were isolated from cells of strains KW128, Δimp-2A, and Δfbp-8J grown in ASW-YT medium supplemented with pyruvate (P) or starch (S). The regions corresponding to the respective probes are indicated in Fig. 1. Each lane contained 30 μg of total RNA. The signal intensities between the panels cannot be directly compared due to the prolonged chromogenic reaction time (>10 times longer) for panel A compared to that for panel B. Numbers on the left are lengths of RNA size markers (in bases).