doi: 10.1128/JB.180.16.4309-4313.1998.
Identification and expression of the Bacillus subtilis fructose-1, 6-bisphosphatase gene (fbp)
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- PMID:9696785
- PMCID: PMC107433
- DOI: 10.1128/JB.180.16.4309-4313.1998
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Identification and expression of the Bacillus subtilis fructose-1, 6-bisphosphatase gene (fbp)
Y Fujita et al. J Bacteriol.1998 Aug.
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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.
Figures
Identification of theB. subtilis fbp gene by examination of the transformation offbp-74 tofbp+ of PCR products covering various parts of theyycR-to-gntP region. The 19 genes (yycR togntP) 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 transformingfbp-74 tofbp+ are shown by the solid black bars, whereas those possessing no transforming activity are shown by the broken bars.
Promoter region of theB. subtilis fbp gene. A map of thefbp gene and its neighboring genes is shown at the top of the figure. Pfbp and Tfbp denote the putativefbp promoter and terminator, respectively. The locations of the Cup and Cdown primers used to obtain a PCR product for the cloning offbp inE. coli are also indicated. The nucleotide sequence of the upstream region and the 5′ part of thefbp gene are shown under the map. The −10 and −35 regions of Pfbp and an SD sequence forfbp 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.
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).
Analysis of thefbp 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 putativefbp promoter (underlined); the putative transcription start nucleotide, G, is doubly underlined.
References
- Ehrlich, S. D., and V. Vagner. Personal communication.
- Fujita, Y. Unpublished data.
- Fujita Y, Freese E. Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis. J Biol Chem. 1979;254:5340–5349. - PubMed
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