The Bacillus subtilis ywjI (glpX) Gene Encodes a Class II Fructose-1,6-Bisphosphatase,Functionally Equivalent to the Class III Fbp Enzyme

We present here experimental evidence that the Bacillus subtilis ywjI gene encodes a class II fructose-1,6-bisphosphatase, functionally equivalent to the fbp-encoded class III enzyme, and constitutes with the upstream gene, murAB, an operon transcribed at the same level under glycolytic or gluconeogenic conditions.Under glycolytic growth conditions, unidirectional phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate is catalyzed by the 6-phosphofructokinase (EC 2.7.1.11). Under gluconeogenic growth conditions, the opposite reaction is catalyzed by the fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) and is required for the synthesis of fructose-6-phosphate and derived metabolites, such as cell wall precursors. Escherichia coli possesses two FBPases: the class I FBPase, encoded by fbp, is highly similar to eukaryotic enzymes, and the class II FBPase (GlpX) (3) has homologues in nearly all prokaryotic genera but in only a few eukaryotes (a green alga, an amoeba, and a moss) and a few archaean species (of the Methanosarcina genus). Biochemical, physiological, and genetic studies allowed the characterization of a Bacillus subtilis enzyme which defined a new class of bacterial FBPases (class III) not structurally related to those previously described and found mainly in Firmicutes (5-7). The gene encoding this activity was identified and, although structurally unrelated to the E. coli class I FBPase gene, was also named fbp (8). In E. coli, the major FBPase is the class I Fbp, whereas the class II GlpX seems to play a minor role (3). In other organisms, the major or even the only FBPase belongs to the class II GlpX family: Bacillus cereus possesses two glpX-like genes and no class I or class III FBPase-encoding gene (26); in Mycobacterium tuberculosis, FBPase activity is encoded only by a glpX-like gene, which has been shown to complement an E. coli mutant lacking such activity (18); in Corynebacterium glutamicum, the only FBPase, essential for growth on gluconeogenic carbon sources, belongs to class II (19). It has been shown that a B. subtilis fbp mutant was still able to grow on substrates such as d-fructose, glycerol, or l-malate as the sole carbon source, which indicated that this mutant could bypass the FBPase reaction during gluconeogenesis (6). Random mutagenesis (ethyl methanesulfonate treatment) performed with this fbp mutant enabled the definition of a B. subtilis locus (bfd) whose additional mutation prevented growth on gluconeogenic carbon sources, but this locus had not been characterized further (7). Determination of the nucleotide sequence of the whole B. subtilis chromosome (16) led to the identification of a putative gene, ywjI, encoding a protein displaying strong homologies with GlpX family members (e.g., 54% identity and 74% similarity with GlpX from C. glutamicum). This gene has therefore been annotated glpX, encoding a class II FBPase, but such annotation has never been validated by genetic or biochemical experimental evidence. In this work, we present experimental evidence that ywjI indeed encodes a class II FBPase.