Identification and Expression of the Bacillus subtilis Fructose-1,6-Bisphosphatase Gene (fbp)

Abstract

The Bacillus subtilis fbp gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as yydE. The fbp gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis. fbp transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly, B. subtilis FBPase exhibited no significant similarity to other FBPases in protein sequence databases.

Fructose-1,6-bisphosphatase (EC 3.1.3.11) (FBPase) is a well-known enzyme involved in gluconeogenesis. The Bacillus subtilis FBPase appears to be a constitutive enzyme and has been purified and characterized (4). The enzyme is very labile in the absence of Mn²⁺ and is inhibited by AMP, but this inhibition can be overcome by phosphoenolpyruvate. Consequently, a change in the relative intracellular concentrations of AMP and phosphoenolpyruvate in glycolysis and gluconeogenesis may control FBPase activity (4). The Bacillus licheniformis FBPase also exhibits properties similar to those of the B. subtilis enzyme (13). In contrast to enterobacteria such as Escherichia coli (2, 22), a B. subtilis lacking FBPase is able to grow on gluconeogenic carbon sources such as malate and glycerol (5, 6).

The fbp (formerly fdp) gene required for FBPase synthesis is located between gntK and purA (6, 7), but the gene itself has not been identified. Completion of the sequencing of the B. subtilis genome (10, 11, 19–21) allowed us to identify the yydE gene as fbp.

B. subtilis strains.

The B. subtilis strains used are listed in Table 1. To construct strains YF311 and YF312, we used plasmid pMutin1, which was provided by V. Vagner (Institut National de la Recherche Agronomique, Jouy-en-Josas, France). Integration of plasmid pMutin1 into the target gene through a single crossover allows the gene’s promoter to be monitored with a lacZ reporter gene and places the downstream genes under the control of the spac promoter (1). fbp sequences were amplified by PCR using DNA from strain 1A1 as a template with primer pairs designed to generate flanking HindIII and BglII sites. The resulting PCR products were cleaved with HindIII and BglII and then ligated with plasmid pMutin1, which had been doubly digested with HindIII and BamHI. The ligated DNAs were doubly digested with BamHI and BglII to avoid self-ligation and then used for the transformation of E. coli C600 (17) to ampicillin resistance (100 μg/ml) on 2× YT plates (17). The correct cloning of PCR products into plasmid pMutin1 was confirmed by DNA sequencing. The resulting plasmids were used for the transformation (6) of B. subtilis 1A1 to erythromycin resistance (0.3 μg/ml) on tryptose blood agar base (Difco) plates containing 0.18% glucose. Correct integration of a single copy of each of the plasmids into the fbp locus of the chromosome through a single crossover was confirmed by Southern blot analysis (17).

TABLE 1.

B. subtilis strains used in this study

Strain	Relevant genotype	Reference
1A1 (= 168 trpC2)a	trpC2	19
60015	trpC2 metC7	5
YF062	bfd-1 fbp-74 glp hisA1 leuA8 metB5 trpC2	6
YF311	trpC2 fbp::pMutin1	This study
YF312	trpC2 Pfbp::pMutin1 (Pspac-fbp)	This study

^aThis strain was obtained from the Bacillus Genetic Stock Center (Columbus, Ohio). 

Identification of the fbp gene.

The B. subtilis genome sequence (10, 11, 19–21) allowed us to determine the end points of the Δigf deletion (6). This deletion was 63 kb in size, affecting 58 genes from yycR to iolI, with an 18-kb chromosomal segment at 76° replacing the 63-kb region (3). The fbp gene is located within Δigf, with the gene order being fbp, gntK, and iol (6, 7). Thus, the fbp gene must be one of the genes between yycR and gntP.

As noted above, B. subtilis can bypass FBPase (5, 6), so only strains (such as YF062) carrying both fbp-74 and bfd-1, with defective FBPase and unable to bypass it (6), are unable to grow on gluconeogenic carbon sources such as Casamino Acids (Difco). To identify the fbp gene, we examined the ability of PCR products covering various parts of the yycR-to-gntP region to transform strain YF062 (fbp-74 bfd-1) to Fbp⁺ (able to grow on Casamino Acids) (Fig. 1); bfd-1 is located outside Δigf (3, 6). Fbp⁺ transformants were selected on N medium plates (6) containing 0.5% Casamino Acids, 0.1% Na₃ citrate, 0.05 mM MnCl₂, and 50 μg of tryptophan per ml. All PCR products able to transform fbp-74 to fbp⁺ contained the yydE gene, strongly suggesting that yydE might be fbp.

FIG. 1.

Identification of the B. subtilis fbp gene by examination of the transformation of fbp-74 to fbp⁺ of PCR products covering various parts of the yycR-to-gntP region. The 19 genes (yycR to gntP) are denoted by large open arrows indicating the direction of transcription. The PCR products were synthesized with various pairs of the F and R series primers. The 5′ ends of the 20-bp hybridizing sequences of primers F7, F11, F12, F13, F14, and F15 (the complementary sequences are found in the GSDB, DDBJ, EMBL, and NCBI databases under accession no. D78193 [10]) are positions 18592, 13433, 8124, 3321, 11851, and 9341, respectively, whereas the 5′ ends of the 20-bp hybridizing sequences of primers R15, R16, R17, R18, and R19 (accession no. D78193) are positions 13411, 8102, 3376, 11832, and 9322, respectively. The 5′ end of the 20-bp hybridizing sequence of primer R14 (the complementary sequence can be found under the accession no. of AB005554 or D45242 [20]) is position 2693. Transformation of strain YF062 to Fbp⁺ with various PCR products was carried out as described previously (6). The products (F7-R14, F11-R17, F7-R16, F11-R16, F14-R17, and F7-R19) exhibited Fbp⁺ transforming activities of 553, 311, 442, 252, 74, and 166 Fbp⁺ colonies per ng of DNA, respectively, whereas the other products had no transforming activity (less than 5 colonies per ng of DNA); each value is the average of two independent experiments, with two different dilutions of each culture on a total of four plates. The products transforming fbp-74 to fbp⁺ are shown by the solid black bars, whereas those possessing no transforming activity are shown by the broken bars.

Disruption of the fbp gene.

The yydE gene contains a potential Shine-Dalgarno (SD) sequence preceding an AUG translation initiation codon at nucleotide (nt) −90 (10), as well as a second potential SD sequence preceding a UUG translation initiation codon at nt +1 (Fig. 2). Thus, two PCR products carrying nt −58 to +263 and nt +78 to +418 with and without the second SD, respectively, were used for the integration of plasmid pMutin1 through a single crossover into the yydE locus of strain 1A1, resulting in strains YF312 (Pspac-fbp) and YF311 (fbp::pMutin1).

FIG. 2.

Promoter region of the B. subtilis fbp gene. A map of the fbp gene and its neighboring genes is shown at the top of the figure. Pfbp and Tfbp denote the putative fbp promoter and terminator, respectively. The locations of the Cup and Cdown primers used to obtain a PCR product for the cloning of fbp in E. coli are also indicated. The nucleotide sequence of the upstream region and the 5′ part of the fbp gene are shown under the map. The −10 and −35 regions of Pfbp and an SD sequence for fbp are indicated. The putative transcription initiation nucleotide, G, is doubly underlined. The sequence and complementary sequence for hybridization of the Cup and Pex primers are indicated by arrows.

B. subtilis FBPase is considered to be a constitutive enzyme (4, 5), but in contrast to FBPase activity in cells of the wild-type strain 1A1 (56 nmol/min per mg of protein), strain YF311 lacked FBPase (less than 1 nmol/min per mg of protein). However, FBPase synthesis was induced in cells of strain YF312 upon the addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to the medium (data not shown). The fact that pMutin1 integration into yydE affected FBPase activity clearly indicated that the yydE gene is fbp. Moreover, pMutin1 integration through the fragment from nt −58 to +263 caused the replacement of the fbp promoter with the spac promoter, whereas pMutin1 integration through a single crossover with the fragment from nt +78 to +418 disrupted fbp. The results suggested that the assignment of the second SD sequence and a UUG initiation codon for fbp is correct. Thus, the fbp gene likely comprises 1,923 nt (641 amino acids), which is 90 nt shorter than yydE is thought to be (10).

To identify the fbp-74 mutation, we determined 2,488 bp of DNA sequence (nt −445 to +2043; containing the putative fbp promoter and coding region) of strains 60015 and YF062 (fbp-74) by successive cycle sequencing of PCR products amplified from these strains, using chromosomal DNAs as templates; the Δigf region was restored by transformation with DNA from strain 60015 in the process of isolation of strain YF062 (6). The 2,488-bp sequence of strain 60015 perfectly matched the sequence reported for the B. subtilis genome (10, 11). However, that of strain YF062 carried one mismatch, a substitution of G at nt +1406 by A, causing the substitution of Asp for Gly in residue 469 of Fbp. It seems likely that this amino acid substitution renders strain YF062 to have defective FBPase.

Expression of the fbp gene in E. coli.

To determine whether the fbp gene encodes FBPase or a positive regulator of its synthesis, we attempted to clone and express this gene in E. coli and to characterize the protein synthesized. The fbp coding region (nt −237 to +2186) carrying the fbp promoter as described below was amplified by PCR using a primer pair (Cup and Cdown) (Fig. 2) and DNA from strain 1A1 as a template to produce a PCR product with flanking BamHI and PstI sites at the respective ends. The PCR product was digested with BamHI and PstI and then ligated with plasmid pUC118 or pUC119 (17), which had been doubly digested with these endonucleases. The ligated DNAs were used for the transformation of E. coli XL1-blue (17) to produce white colonies on 2× YT plates containing 50 μg of ampicillin per ml, 40 μg of 5-bromo-4-chloro-3-indolyl-β-d-galactoside per ml, and 0.1 mM IPTG. The resultant plasmids pUC118 and pUC119 carrying the fbp gene were designated plasmids pFBP118 and pFBP119, respectively.

The levels of B. subtilis FBPase in E. coli XL1-blue carrying the various plasmids were determined as described previously (4, 5). Cells carrying plasmids pFBP118 and pFBP119 synthesized similar large amounts of FBPase (5.02 and 5.46 mmol/min per mg of protein), respectively, whereas those bearing plasmid pUC118 or pUC119 synthesized no detectable FBPase (less than 1 nmol/min per mg of protein). Since the orientations of the fbp gene were the same as and opposite that of the plasmid’s lac promoter in plasmids pFBP118 and pFBP119, respectively, the results suggest that fbp is expressed from its own promoter efficiently in E. coli.

B. subtilis FBPase requires phosphoenolpyruvate and Mn²⁺ for its activation and stability, respectively (4). When lysates from E. coli strains overexpressing Fbp were prepared and assayed in the absence of phosphoenolpyruvate and Mn²⁺, the lysates showed 14 and 2.3 times less enzyme activity, respectively (data not shown). Analysis of these lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent Western blotting analysis (8) showed that the lysate of the cells bearing plasmid pFBP118 or pFBP119 contained an abundant protein with a molecular mass of 74 kDa (data not shown), which is very close to that of the Fbp protein (74,390 Da). This protein cross-reacted with anti-FBPase (data not shown), which was produced in a rabbit against purified FBPase (4, 5). These results clearly indicate that the fbp gene is the structural gene of FBPase.

Expression of the fbp gene.

FBPase is required for gluconeogenesis. However, this enzyme was synthesized even when cells were grown on glucose as the sole carbon source, although its synthesis was somewhat lower in cells growing on glucose than in cells growing on malate (5). When B. subtilis is cultivated in NSMP medium (5), cells initially carrying out glycolysis enter gluconeogenic growth in the middle of the logarithmic phase (5) when catabolic operons such as iol (18) are released from catabolite repression (16). Consequently, we monitored FBPase synthesis in cells of strain 1A1 during both logarithmic and stationary growth phases (Fig. 3). The activity of FBPase increased in the early logarithmic growth phase (up to an optical density at 600 nm [OD₆₀₀] of 0.5) and then gradually decreased slightly in the late logarithmic and stationary phases when cells were cultivated without glucose. As observed previously (16), inositol dehydrogenase was induced in the middle of the logarithmic phase after consumption of the glycolytic carbohydrates in NSMP medium and then its expression decreased in the late logarithmic and stationary phases, probably due to consumption of the myo-inositol in the medium. When glucose was added to NSMP medium, levels of FBPase synthesized were more constant, and inositol dehydrogenase was not induced due to catabolite repression. These results indicate that expression of the fbp gene is relatively constant regardless of the necessity of FBPase. However, fbp expression is significantly dependent on the growth phase of the cell, but at present, we cannot explain this regulation.

FIG. 3.

Synthesis of FBPase during glycolytic and gluconeogenic growth. Cells of strain 1A1 were grown in NSMP medium with (open symbols) and without (closed symbols) 10 mM glucose, and the OD₆₀₀ (•, ○) was monitored during growth at 37°C in a shaking water bath. At the indicated times, the cells (OD₆₀₀ units = 9.0) were harvested and lysed in the presence of phosphoenolpyruvate and Mn²⁺ by lysozyme treatment and brief sonication (5). FBPase (■, □) and inositol dehydrogenase (▴, ▵) were assayed as described previously (4, 5, 16).

To investigate fbp transcription by means of Northern blot and primer extension analyses, RNA was prepared as follows. B. subtilis cells (strain 1A1) were grown in NSMP medium with or without 10 mM glucose, and an 80-ml sample was harvested at an OD₆₀₀ of 1. The RNA was then purified by a modified version of a procedure described elsewhere (15), using glass beads for cell disruption (9). A 0.2-ml portion of TE buffer (10 mM Tris-Cl [pH 7.4] and 1 mM Na-EDTA) containing 10 mM sodium iodoacetate was added to suspend the cell pellet. The cell suspension was mixed with 0.5 ml of 0.5-mm-diameter glass beads, 0.4 ml of phenol, and 0.8 ml of detergent solution (0.6% cetyltrimethylammonium bromide, 50 mM sodium acetate, 1 mM dithiothreitol), and then the cells were lysed by vigorous shaking with a Mini-Beadbeater (Biospec Products) for 1.5 min at 65°C. Following reextraction of the aqueous phase with phenol-chloroform-isoamyl alcohol (125/24/1, by volume), crude RNA was recovered by precipitation with an equal volume of isopropanol in the presence of 0.3 M sodium acetate at 4°C for 5 min. During incubation of crude RNA in 1 M sodium acetate at −20°C for 1 h, RNA was reprecipitated, and the RNA pellet was washed thoroughly with ethanol. The final RNA was dissolved in 50 μl of water containing 10 units of ribonuclease inhibitor (Bethesda Research Laboratories).

The fbp gene is supposed to be monocistronic, because the direction of fbp transcription is opposite that of the neighboring genes (Fig. 1 and 2). To confirm this, we determined the sizes of the fbp transcripts by Northern blotting (Fig. 4A), using a ³²P-labeled PCR product (nt −58 to +263) as a DNA probe. We detected two transcripts which were 2.4 and 2.2 kb in size, with the 2.4-kb one being dominant (Fig. 4A). There was no significant difference between the amounts of the two transcripts in the cells grown with and without glucose (Fig. 4A).

FIG. 4.

Analysis of the fbp transcript. (A) Northern blot analysis. Northern blotting was performed essentially as described previously (17, 18), using the DNA probe (nt −58 to +263) and total RNA samples prepared from the cells grown in the presence (+) and absence (−) of glucose as described in the text. The positions of the RNA size markers are indicated to the left of the panel. (B) Mapping of the 5′ end of the transcript. Primer extension was carried out essentially as described previously (17, 18), using total RNAs prepared from the cells grown in the presence (+) and absence (−) of glucose and the Pex primer (Fig. 2). The dideoxy cycle sequencing ladders (lanes G, A, T, and C) were created with the same primer using the PCR product (nt −543 to +326), which had been amplified from strain 1A1 DNA, as the template. The part of the nucleotide sequence of the noncoding strand corresponding to these ladders is shown with the −35 and −10 regions of the putative fbp promoter (underlined); the putative transcription start nucleotide, G, is doubly underlined.

To determine the 5′ ends of the transcripts, we performed primer extension (Fig. 4B) using a primer corresponding to nt +12 to +31 (Pex [Fig. 2]). When ³²P-labeled cDNAs extended from the primer were subjected to urea-polyacrylamide gel electrophoresis, we detected only one clear band corresponding to G at nt −94 (Fig. 4B). Again, there was no significant difference in the density of the bands obtained using RNAs in the cells grown with and without glucose. The transcription start site at nt −94 was preceded by the −35 and −10 sequences with significant similarity to those recognized by ς^A (Fig. 2 and 4B).

The fbp gene is followed by a sequence which may well be a ρ-independent transcription terminator, and fbp is located between nt +2220 and +2263 (AAAAAAGTCCATTCTA-12 bases-TAGGGTGGACCTTTTT). fbp transcription beginning at nt −94 and ending at this putative terminator will produce an approximately 2,360-nt product, the size of which is close to the size of the dominant transcript (2.4 kb) detected in Northern blotting. Presumably, the 2.2-kb transcript detected in Northern blotting is a degradation product of the 2.4-kb transcript.

We conclude from these results that the fbp gene is monocistronic, transcription of which is most likely to be initiated from G at nt −94, and that fbp transcription is not affected by the availability of glucose in the medium.

Homology search of B. subtilis FBPase.

We searched for proteins similar to B. subtilis FBPase in a nonredundant protein sequence database (Institute for Chemical Research, Kyoto University), using the FASTA program (14). Surprisingly, this search failed to reveal any protein exhibiting significant similarity to the B. subtilis FBPase (scores of less than 125). This indicates that the B. subtilis FBPase is unique among many FBPases in the protein sequence databases, which include those of enteric bacteria, yeasts, plants, and animals.

Although the B. subtilis FBPase showed no significant similarity to the other FBPase, these enzymes catalyze the same reaction and might exhibit small regions of homology. Therefore, local similarity between B. subtilis and E. coli FBPases was searched for by use of the LFASTA program (14). The resultant best score of 55 (16.1% identity in 137-amino-acid overlap) is low, so that it is very difficult to judge whether this similarity indicates any functional relationship between the two enzymes.

B. subtilis can bypass FBPase (5, 6), suggesting that the enzyme involved in this bypassing might be an ortholog of FBPases in the databases. However, this possibility was eliminated by the failure of an ortholog search for FBPases of other organisms among the 4,100 proteins encoded by the complete B. subtilis 168 genome (11, 12). For example, the FASTA search of a B. subtilis ortholog for E. coli FBPase gave scores of less than 113. It is quite interesting that among the B. subtilis proteins which are known or supposed to be involved in glycolysis and gluconeogenesis, only FBPase has no ortholog in the protein sequence databases at present (12). The B. subtilis FBPase gene might have arisen by convergent evolution independently of other members of the FBPase family. However, orthologs of the B. subtilis FBPase might be found at least in the Bacillus genus, because the properties of the B. licheniformis enzyme are very similar to those of the B. subtilis one (4, 13).