Fructose degradation in the haloarchaeon Haloferax volcanii involves a bacterial type phosphoenolpyruvate-dependent phosphotransferase system, fructose-1-phosphate kinase, and class II fructose-1,6-bisphosphate aldolase

Abstract

The halophilic archaeon Haloferax volcanii utilizes fructose as a sole carbon and energy source. Genes and enzymes involved in fructose uptake and degradation were identified by transcriptional analyses, deletion mutant experiments, and enzyme characterization. During growth on fructose, the gene cluster HVO_1495 to HVO_1499, encoding homologs of the five bacterial phosphotransferase system (PTS) components enzyme IIB (EIIB), enzyme I (EI), histidine protein (HPr), EIIA, and EIIC, was highly upregulated as a cotranscript. The in-frame deletion of HVO_1499, designated ptfC (ptf stands for phosphotransferase system for fructose) and encoding the putative fructose-specific membrane component EIIC, resulted in a loss of growth on fructose, which could be recovered by complementation in trans. Transcripts of HVO_1500 (pfkB) and HVO_1494 (fba), encoding putative fructose-1-phosphate kinase (1-PFK) and fructose-1,6-bisphosphate aldolase (FBA), respectively, as well as 1-PFK and FBA activities were specifically upregulated in fructose-grown cells. pfkB and fba knockout mutants did not grow on fructose, whereas growth on glucose was not inhibited, indicating the functional involvement of both enzymes in fructose catabolism. Recombinant 1-PFK and FBA obtained after homologous overexpression were characterized as having kinetic properties indicative of functional 1-PFK and a class II type FBA. From these data, we conclude that fructose uptake in H. volcanii involves a fructose-specific PTS generating fructose-1-phosphate, which is further converted via fructose-1,6-bisphosphate to triose phosphates by 1-PFK and FBA. This is the first report of the functional involvement of a bacterial-like PTS and of class II FBA in the sugar metabolism of archaea.

Fig 1

Genomic arrangement and Northern blot analyses of genes involved in fructose metabolism in H. volcanii. (A) Schematic representation of fructose-specific PTS cluster and neighboring genes. Open reading frames are displayed as open arrows. glpR (HVO_1501) encodes a transcription regulator (39); pfkB (HVO_1500) encodes putative 1-phosphofructokinase (1-PFK) of the PfkB family; the HVO_1499 to HVO_1495 cluster (ptfC, ptfA, ptsH1, ptsI, and ptfB) encodes all five components of the putative fructose-specific PTS (EIIC, EIIA, HPr, EI, and EIIB); fba encodes a putative class II fructose-1,6-bisphosphate aldolase (FBA). The bar indicates a length of 1,000 bp. (B) Northern blot analyses of genes involved in fructose metabolism of H. volcanii. Total RNA was isolated from glucose (G)- or fructose (F)-grown cells and analyzed with specific probes for ptfC (HVO_1499), pfkB (HVO_1500), and fba (HVO_1494). To control equal loading, the 23S and 16S rRNAs were visualized by ethidium bromide staining (lower panels).

Fig 2

Growth analyses of in-frame deletion mutants of genes involved in fructose degradation in H. volcanii. (A to C) Growth of H. volcanii ΔptfC (A), ΔpfkB (B), and Δfba (C) strains on 25 mM fructose (■) compared to the wild type (•) and complementation strains with functional genes (▲). Precultures for growth experiments were grown in complex medium containing 1% Casamino Acids (A and B) or in synthetic medium containing 25 mM glucose (C). Growth was measured by determining the optical density at 600 nm (OD₆₀₀).

Fig 3

SDS-PAGE of purified, His-tagged 1-phosphofructokinase (1-PFK) (A) and fructose-1,6-bisphosphate aldolase (FBA) (B) of H. volcanii. M, molecular mass marker.

Fig 4

Proposed pathway of fructose uptake and degradation to triose phosphates in the haloarchaeon H. volcanii. Fructose uptake by PTS is shown schematically. It involves the putative cytoplasmic components EI, HPr, EIIA, and EIIB (encoded by HVO_1496 [ptsI], HVO_1497 [ptsH1], HVO_1498 [ptfA], and HVO_1495 [ptfB], respectively) and the transmembrane component EIIC (encoded by HVO_1499 [ptfC]), forming fructose-1-phosphate. Note that the Haloferax PTS, in contrast to bacterial counterparts, consists of five separate components. ∼P indicates the phosphoryl group which is transferred from PEP to fructose; the transient phosphorylation of PTS components is not shown. Fructose-1-phosphate is phosphorylated by 1-PFK (encoded by HVO_1500 [pfkB]) to fructose-1,6-bisphosphate, which is cleaved to dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) by FBA (encoded by HVO_1494 [fba]). Proteins and genes that were proven to be functionally involved in fructose uptake and degradation are marked in boldface. CM, cytoplasmic membrane.

Fig 5

Genomic organization of fructose-specific PTS, 1-PFK, and FBA in H. volcanii compared to selected haloarchaea and bacteria. Genes are indicated as colored arrows: 1-phosphofructokinase (1-PFK; HVO_1500 [pfkB]) in green, EIIC (HVO_1499 [ptfC]) in red, EIIA (HVO_1498[ptfA]) in blue, HPr (HVO_1497 [ptsH1]) in orange, EI (HVO_1496 [ptsI]) in purple, EIIB (HVO_1495 [ptfB]) in dark blue, and fructose-1,6-bisphosphate aldolase (FBA; HVO_1494 [fba]) in yellow. EIIC and EIIB homologs in M. thermoacetica (Moth_0013) and C. saccharolyticus (Csac2439) are fused. fruB and fruA of E. coli represent fusions of EIIA-HPr and EIIB-EIIC, respectively; white areas in these genes show regions with domains that probably are nonfunctional. rrnAC0345 codes for a class I FBA in Haloarcula marismortui (yellow with dashed line).

Fig 6

Phylogenetic relationship of class II fructose-1,6-bisphosphate aldolases from haloarchaea (class II H), bacteria, and lower eukaryotes (class II A/B). The numbers at the nodes are bootstrap values according to neighbor joining (the neighbor-joining algorithm of ClustalX was used to generate the values). The tree is based upon a multiple-sequence alignment that was generated with ClustalX using the gonnet matrix (47). The GenBank accession numbers for the aldolases from the different species are shown in parentheses: class II B, Anoxybacillus gonensis (ABL75360), Bacillus subtilis (AEP88659), Caldicellulosiruptor saccharolyticus (YP_001179983), Deinococcus radiodurans R1 (NP_295312), Desulfovibrio vulgaris (YP_002434443), Giardia lamblia (3GB6_A), Halothermothrix orenii (YP_002509546), Helicobacter pylori (CBI66947), Moorella thermoacetica (YP_429482), Mycoplasma pneumoniae (BAL21593), and Xanthobacter flavus (AAA96742); class II A, Borrelia burgdorferi (AAB91507), Campylobacter jejuni (ZP_06371648), Corynebacterium glutamicum (EHE82675), Escherichia coli (BAE76989), Euglena gracilis (CAA61912), Haemophilus haemolyticus (EGT83318), Mycobacterium tuberculosis (P67475), and Saccharomyces cerevisiae (GAA24665); and class II H, Halalkalicoccus jeotgali (YP_003738319), Haloferax volcanii (YP_003535543), Halogeometricum borinquense (ADQ67306), Halorhabdus tiamatea (ZP_08561145), Halorhabdus utahensis (YP_003129569), and Haloquadratum walsbyi (YP_658423). The scale bar corresponds to 0.1 substitutions per site.