Scientific Publications - Work Done by Microbiology Reader Bioscreen C

J. Biochem. 126, 333-339 (1999)

Regulation of the  Bacillus subtilis Phosphotransacetylase  Gene1

Byung-Sik Shin, Soo-Keun Choi, and Seung-Hwan Park²

Bacterial Molecular Genetics R.U., Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea 305-333

Received May 6, 1999; accepted May 28, 1999

ABSTRACT

The enzyme, phosphotransacetylase (Pta), catalyzes the conversion of acetyl coenzyme A to acetyl phosphate. The putative pta gene of Bacillus subtilis, which had been sequenced as part of the Genome Project, was cloned and overexpressed in Escherichia coli. We confirmed that the gene encodes Pta by measuring the enzymatic activity of the purified protein. Insertional mutagenesis of the pta gene resulted in complete loss of the Pta activity, indicating that B. subtilis contains only one kind of pta gene. Expression of a pta-lacZ fusion was induced in the presence of excess glucose in the growth medium, and the intact ccpA gene was required for this activation. The transcriptional start site of the pta gene was located at 37 nucleotides upstream of the pta start codon, and a cre (catabolite responsive element) sequence, a cis-acting element that is responsible for the catabolite repression of a number of carbon utilization genes in B. subtilis, was identified upstream of the tentative promoter site. Experiments involving oligonucleotide-directed mutagenesis showed that the cre sequence is involved in glucose-mediated transcriptional activation.

Key words: Bacillus subtilis, CcpA, phosphotransacetylase.
 

INTRODUCTION

The Gram-positive spore-forming bacterium, Bacillus subtilis, can grow on various carbon sources. In a rich medium containing an excess amount of a carbohydrate such as glucose, B. subtilis typically secretes acids such as pyruvate and acetate, which results in a decrease in the culture pH (1). After all the glucose has been exhausted, the acids secreted into the medium are oxidized again, the culture pH increasing again (1). Escherichia coli also excretes acetate during aerobic growth on glucose, which is inhibitory for exponential growth (2). In both B. subtilis and E. coli, acetate is formed from acetyl CoA mainly via the phosphotransacetylase (Pta) [EC 2.3.1.8] and acetate kinase (Ack) [EC 2.7.2.1] pathway (Pta-Ack pathway), with the production of ATP (Fig. 1).

E. coli cells can utilize acetate as the sole carbon source. It is believed that two independent pathways are involved in the activation of acetate. One pathway is the Pta-Ack pathway, which is also used in acetate excretion, and the second one is catalyzed by acetyl CoA synthetase (Acs) [EC 6.2.1.1] (Fig. 1). It was proposed that the Pta-Ack pathway is used with high concentrations of acetate, and Acs is used with low concentrations of acetate (3). In B. subtilis, on the other hand, it appears that only AcsA is involved in the utilization of acetate (4), suggesting that different modes of regulation of acetate metabolism may be involved in B. subtilis. In addition to the role of the Pta-Ack pathway in acetate metabolism, acetyl phosphate has been implicated in the regulation of two-component signal transduction systems as a phosphoryl donor for response regulators, including CheY, PhoB, NtrC, and OmpR (5-7).

Despite the importance of the Pta-Ack pathway in metabolism and the signal transduction pathway, only limited information is available on the regulation of the Pta-Ack pathway. In B. subtilis, it has been found that the ackA and acsA genes are affected by a CcpA protein which was shown to be involved in the catabolite repression of many genes and operons (8-10). Pta has also been purified and characterized in B. subtilis (11), but no genetic approach has been taken as to regulation of the pta gene. Recently, the complete genome sequence of B. subtilis was reported (12), and now a sequence for pta is available in the genome database. But this pta gene was only defined by sequence homology and there is no evidence that this pta-like gene really encodes a Pta.

In this study, we report the cloning of a pta gene and the purification of its protein product from over-expressing E. coli cells. On analysis of the pta mutant, we concluded that the pta gene encodes a Pta. From the results of pta-lacZ fusion studies, we found that the transcription of the gene is activated by glucose, and that this activation is dependent on an intact ccpA gene and a cre sequence located in the pta upstream region.
 

MATERIALS AND METHODS

Bacterial Strains and Plasmids

The bacterial strains used in this study are described in Table I. E. coli DH5a was used for plasmid construction and for overexpression of the pta gene for purification of Pta. All B. subtilis strains were derivatives of JH642.

Media and Growth

LB medium (13) was routinely used for cultivating E. coli and B. subtilis. Difco sporulation medium (DSM) (14) was used for growth and maintenance of B. subtilis. Antibiotics were used at the following concentrations: ampicillin at 100 mg/ml, spectinomycin at 100 mg/ml, and erythromycin and lincomycin at 1.0 and 25 mg/ml, respectively, for selection of the erm gene. Competent cells were obtained in Spizizen's medium as described by Albano et al. (15). To make DSMG, sterile 50% glucose was added, to a final concentration of 1.0%, to sterile DSM. TSS minimal medium (16) was made with 0.2% NH₄Cl as the nitrogen source. Casamino acids (Difco) were added to the TSS medium to 1.0% as a 10% sterile stock solution as indicated.

The growth rates of strains were measured using Bioscreen C (Labsystems, Finland). An inoculum was prepared by growing the cells on TSS (containing 1% casamino acids) agar plates at 30°C overnight with appropriate antibiotics. Then the cells were harvested by washing the plate surface with 1.0 ml of the same broth, and then liquid medium (without antibiotics) was inoculated to an initial absorbance level at 600 nm of <0.001. The increase in turbidity was monitored automatically every 10 min at 600 nm at 37°C. Data for determination of the growth rate were taken between A₆₀₀ of 0.01 and 0.1. Analysis of data was performed using the SigmaPlot 4.0 program.

Chemicals

Restriction enzymes, T4 polynucleotide kinase, Expand^TM reverse transcriptase, DNase-free RNase, malate dehydrogenase, and citrate synthase were purchased from Boehringer Mannheim. The substrates used in the Pta assay and molecular weight markers for gel filtration were purchased from Sigma. [g-³²P]ATP was purchased from Amersham.

DNA Manipulations

The oligonucleotide primers used for PCR and site-directed mutagenesis were purchased from Genotech (Taejon, Korea) and are shown in Table II. Recombinant techniques, site-directed mutagenesis and DNA sequencing were performed by standard methods (17). B. subtilis chromosomal DNA was isolated as described by Cutting and Horn (18). Preparation and transformation of competent B. subtilis cells were performed as described by Albano et al. (15).

Purification of Pta

The open reading frame of a pta gene fragment was amplified by PCR using primers PT5 and PT6, and a 1.07-kb BamHI-PstI fragment obtained from the PCR product was cloned into plasmid pQE31 (Qiagen) so that six histidine codons were fused into the 5' end of the pta gene, resulting in pQEpta. E. coli DH5a transformed with pQEpta was grown in 1 liter of LB medium containing 50 mg/ml of ampicillin. Then expression of pta was induced by the addition of 1 mM IPTG at the mid-exponential phase, and growth was continued for 14 h. Cells were harvested by centrifugation and resuspended in lysis buffer [50 mM sodium phosphate (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 1 mM PMSF]. After incubation with lysozyme (final, 1 mg/ml) on ice for 1 h, the cells were disrupted by sonication, followed by clarification by centrifugation at 20,000 x g for 30 min. The supernatant was mixed with 5 ml of a 50% suspension of Ni-NTA resin (Qiagen) previously equilibrated with the same buffer, followed by gentle stirring for 1 h at 4°C. The suspension was poured into a Spectra/Chrom^TM disposable minicolumn (Spectrum) and washed with 10 ml of lysis buffer containing 20 mM imidazole. The protein was eluted with 76 mM imidazole in the lysis buffer. All steps were performed at room temperature unless otherwise indicated. Fractions were analyzed by SDS-PAGE. The purified protein was aliquoted and stored at –65°C. When crude lysates were required, cells were harvested by centrifugation, washed with 0.1 M potassium phosphate (pH 7.6), and lysed by sonication. Cell debris was removed by centrifugation at 12,000 x g for 10 min.

Determination of the Native Molecular Weight

The native molecular weight of the purified protein was determined by gel filtration on a Sephacryl S-300-HR column (Pharmacia; 1.5 x 60 cm). The molecular mass markers used were as follows: b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine albumin (66 kDa), carbonic anhydrase (29 kDa), and cytochrome c (12.4 kDa).

Enzyme Assays

The activity of Pta in the forward reaction (acetyl phosphate–>acetyl CoA) was estimated by using the coupled reaction (19). The assay mixture contained 225 mM Tris-Cl (pH 7.8), 15 mM malic acid, 4.5 mM MgCl₂, 3.75 mM CoA, 22.5 mM NAD⁺, 12 units of malate dehydrogenase, 1.1 units of citrate synthase, and 10 mM acetyl phosphate. The reaction was started by the addition of the enzyme solution, and the initial velocity was determined at 340 nm. The specific activity of Pta was expressed in U/mg. Protein concentrations in cell extracts were determined by the Bradford method (20), using a kit from BioRad, with bovine serum albumin as the standard.

Primer Extension Analysis

Total RNA isolated from JH642 cells, which had been grown in DSMG (DSM +1% glucose) until T₁, was subjected to primer extension. RNA was prepared using a High Pure RNA Isolation Kit (Boehringer Mannheim). A 21-mer primer (PT11), which is complementary to nucleotides +71 to +91, was used for cDNA synthesis. The primer was labeled with [g-³²P]ATP with T4 polynucleotide kinase. 50 pmol of the labeled primer was mixed with 48 mg of RNA in 13 ml of distilled water. The mixture was heated at 65°C for 10 min and then immediately cooled on ice. Then the annealing mixture was mixed with 5 ml of 5 x reverse transcriptase buffer, 1 ml of a four deoxynucleotide mixture (20 nmol/ml of each deoxynucleotide), 2 ml of actinomycin D (0.5 mg/ml) (Sigma), 2 ml of DTT (100 mM), and 25 units of Expand^TM reverse transcriptase. The reaction mixture was incubated at 42°C for 100 min. After the RNA had been digested with RNase, 8 ml of formamide stop buffer was added to the reaction mixture. Following that, the extended cDNA was analyzed by electrophoresis on a 6% sequencing gel. The DNA sequencing reaction was performed using the same oligonucleotide as the primer as the size standard.

Construction of a pta-lacZ Fusion

The pta upstream region, nucleotides –372 to +148 relative to the pta transcriptional start site, with flanking EcoRI and BamHI restriction sites, was amplified with PCR using primers PT7 and PT8, and then cloned into the spoVG-lacZ fusion vector, pDG1728 (21), to generate pBS9853. The DNA sequence was determined to verify that no changes had occurred during the PCR amplification. The fusion construct was linearized with ScaI and then integrated into the amyE locus in a single copy by means of a double crossover, selecting for spectinomycin resistance. Integration into the amyE locus was confirmed by examining the amylase activity on an LB agar plate containing 1% soluble starch. The amylase activity was visualized by staining the plate with a staining solution comprising 0.2% potassium iodide and 0.1% iodine. For construction of isotopic integration of a pta-lacZ fusion, a 6.14-kb NsiI-SmaI fragment bearing the pta-lacZ fusion and the spc gene conferring spectinomycin resistance was eluted from pBS9853 and then cloned into pGEM-7Zf(+) (Promega), generating pBS9854. The resulting plasmid was recombined into the chromosome by means of a single crossover, selecting for spectinomycin resistance, giving strain BS9854. Integration into the pta locus was confirmed by PCR using primers YW1 (complementary to downstream of the ywfl gene, which is located upstream of the pta gene, Fig. 3) and LZ1 (complementary to the 5' end of the spoVG-lacZ fusion).

b-Galactosidase Assay

The inoculum for the b-galactosidase assay was prepared by growing cells on a DSM agar plate at 30°C overnight with appropriate antibiotics. The cells were then harvested by washing the plate surface with 1.0 ml of prewarmed DSM, and liquid medium was inoculated to an initial absorbance level at 600 nm of about 0.01. Cultures were grown at 37°C and 300 rpm, and growth was monitored by measuring A₆₀₀. When the absorbance (A₆₀₀) of the culture reached about 0.1 (approximately 1.5 h), samples were taken at appropriate intervals (30 min to 1 h) for the b-galactosidase assay. T₀ was defined as the end of exponential growth. The assays were performed with toluenized cells, as described by Nicholson and Setlow (22). The specific activity was expressed as Miller units (13).

Construction of a pta Null Mutant

To construct a pta mutant, a 1.44-kb DNA fragment containing the pta gene was amplified by PCR using primers PT1 and PT2, and then cloned into pGEM-T (Promega), giving pBSpta. Then, a deletion-insertion mutation of the pta gene was constructed by replacing the 0.18-kb BglII-EcoRI fragment of pBSpta with a 1.2-kb BamHI-EcoRI fragment of the erm gene, cloned from pDG1728 (21) by PCR with primers ER1 and ER2. The resulting plasmid, pBS9844 (Fig. 3), was integrated into the chromosome by means of a double crossover, creating strain BS9844. The orientation of the erm gene in the resulting construct is opposite to that of the pta gene. Mutation of the pta gene was confirmed by PCR using PT3 and PT4, which were designed to detect the integration of the erm gene into pta. Chromosomal DNA isolated from the pta mutant (BS9844) was used as a template DNA for PCR.

Construction of a ccpA Null Mutant

To construct a ccpA mutant, a 2.08-kb DNA fragment containing the ccpA gene was amplified by PCR using primers CC1 and CC2, with flanking XbaI and BamHI restriction sites, and then cloned into pGEM-7Zf(+), giving pBSccpA. Then, a deletion-insertion mutation of the ccpA gene was constructed by replacing the 0.21-kb ClaI-PstI fragment of pBSccpA with a 1.1-kb ClaI-PstI fragment of the erm gene, cloned from pDG1728 by PCR with primers ER2 and ER3. The resulting plasmid, pBS9901 (Fig. 3), was integrated into the chromosome by means of a double crossover, creating strain BS9901. Mutation of the ccpA gene was confirmed by PCR using CC3 (complementary to just upstream of the ClaI site located in the ccpA gene) and ER3 (complementary to the 5' end of the erm gene), which were designed to detect the integration of the erm gene into ccpA. Chromosomal DNA isolated from a ccpA mutant (BS9901) was used as a template DNA for PCR.

Sporulation Assay

Cells were cultured in DSM at 37°C until approximately 20 h after the end of exponential growth. Serial dilutions were made in 10 mM potassium phosphate buffer (pH 7.4) containing 50 mM KCl and 1 mM MgSO₄, and then plated on LB agar (for JH642) or DSM (for pta mutant BS9844) before and after heat treatment (80°C for 10 min). We used DSM agar plates for counting colonies in the case of the pta mutant (BS9844) because the growth rate of this strain in LB medium was extremely low. The sporulation frequency is the ratio of spores per milliliter to viable cells per milliliter.

Site-Directed Mutagenesis

For in vitro site-directed mutagenesis of the cre region, an Altered Sites® II in vitro Mutagenesis System (Promega) was used. A 0.52-kb EcoRI-BamHI fragment from pBS9853, which was used for construction of the lacZ fusion, was inserted into the pALTER-1 vector and then mutagenesis was performed following the instructions of the manufacturer. All mutations were confirmed by DNA sequencing. The oligonucleotides directing mutations are shown in Table II.
 

FIGURES

Fig. 1. Pathways of acetate metabolism in B. subtilis
Fig. 2. Purification of Pta and gel permeation chromatography
Fig. 3. Genetic map of the pta (top) and ccpA (bottom) region, and plasmids carrying different parts of this region
Fig. 4. Determination of the transcriptional start site for the pta gene by primer extension analysis
Fig. 5. DNA sequence of the pta upstream region
Fig. 6. Expression of b-galactosidase from the pta-lacZ fusion in cells grown in DSM
Table 1. Bacterial strains used in this study
Table 2. Oligonucleotide primers used in this study
Table 3. Growth of B. subtilis strains in TSS minimal medium

RESULTS AND DISCUSSION

Purification of Pta

The putative pta gene in B. subtilis encodes a protein of 323 amino acid residues with a calculated molecular mass of 34,758 Da. To determine whether or not the putative pta open reading frame actually encodes a protein that has Pta activity, we cloned the pta gene by PCR and purified the protein from over-expressing E. coli cells as an N-terminal His₆ tag. The purified protein gave a single protein band when analyzed by SDS-PAGE, corresponding to a molecular mass of 36 kDa, which is close to the calculated molecular mass of 34,758 Da (Fig. 2A). The activity of the protein was determined in the forward reaction, i.e. towards acetyl CoA formation, using a coupled reaction, as described under "MATERIALS AND METHODS." The protein showed specific activity of 1,150 units/mg, indicating that this open reading frame actually encodes a Pta. The Pta of B. subtilis was previously isolated and characterized by Rado and Hoch (11), and the molecular mass of the purified protein was found to be 90 kDa. Since the molecular mass of the subunit was found to be 36 kDa, it was not easy to determine the oligomeric state of the intact enzyme. Thus we performed gel filtration chromatography again with the purified protein, the estimated molecular mass being 76,000 Da (Fig. 2B), indicating that the Pta of B. subtilis probably exists as a dimer under our experimental conditions.

Characterization of the pta Mutant

In an effort to determine the in vivo function of the pta in B. subtilis, we constructed a mutant in which a 0.18-kb internal fragment of the gene was replaced by the erm gene (Fig. 3), resulting in strain BS9844 (Table I). To test for Pta activity, we grew the wild-type (JH642) and pta mutant (BS9844) at 37°C in DSMG until 1 h after the end of the exponential phase (T₁). Crude lysates were prepared by sonication of cells and centrifugation to remove cellular debris, and then assayed for the ability to convert acetyl phosphate to acetyl CoA through coupled reactions. While the wild-type strain (JH642) showed 16 units of enzymatic activity per mg of protein, the pta mutant (BS9844) exhibited no detectable activity, indicating that strain BS9844 is deficient in the Pta. This result also suggests that only one copy of the gene showing Pta activity is present on the chromosome of B. subtilis.

The ability of the pta mutant cells to produce spores was also examined. Both the wild-type (JH642) and mutant (BS9844) were grown in DSM until approximately T₂₀, and then the sporulation frequency was determined from the percentage of colonies that survived heat treatment. Strain BS9844 sporulated at a frequency of 92% while the wild-type cells sporulated at a frequency of 76%, indicating that insertional inactivation of the pta did not have a negative effect on sporulation in DSM. In E. coli, it was shown that loss of the pta gene function results in defective survival on glucose starvation, and it was speculated that this impaired ability might be due to the absence of acetyl phosphate, because the ackA mutant is normal as to the ability to survive glucose starvation (23). If we consider sporulation as a kind of survival process caused by nutrient deprivation, it is interesting that the pta mutant of B. subtilis exhibits a normal (or even higher) sporulation frequency.

Generally, the pta mutant (BS9844) exhibited good growth in DSM and TSS (containing 1% casamino acids) minimal medium, although the growth rates were somewhat lower than those of JH642. A previous study by Grundy et al. (8) showed that the addition of 1% glucose to TSS minimal medium (with 1% casamino acids) greatly inhibited growth of the ackA mutant. In the case of the pta mutant, however, the addition of 1% glucose (or fructose) to the TSS minimal medium increased the growth rate of strain BS9844, although the growth stimulation was not as remarkable as for the wild type (Table III). Interestingly, LB medium could not support the vegetative growth of strain BS9844.

Transcript Mapping

Primer extension analysis was performed using primer PT11, which is complementary to nucleotides +71 to +91 (Fig. 5), to determine the putative transcription start point of the pta. The major 5' end of the mRNA was identified at 37 nucleotides upstream of the pta start codon (Fig. 4). Sequences similar to those of the –35 and –10 regions of a sigma-A-dependent promoter could be identified just upstream of the pta transcription start point (Fig. 5). The TG motif of the –16 region, which is found in a large number of Gram-positive bacterial promoters (24), was absent in the pta promoter.

Regulation of pta Transcription

In many bacterial species, the genes for pta and ack are close to each other on the chromosome, and it seems that these two genes are co-transcribed. The close arrangement of the pta and ack genes has been detected in Clostridium acetobutylicum (25), E. coli (26), Methanosarcina thermophila (27), and Sinorhizobium meliloti (28). However, in B. subtilis, the pta and ackA genes are separated by about 848-kb on the chromosome, suggesting that a different mode of regulation may be involved for each gene. To study pta gene expression, the upstream region of the pta gene bearing the 5' end of the pta gene and the promoter region was cloned in front of the promoterless spoVG-lacZ fusion in pDG1728. The resulting plasmid, pBS9853, was linearized and integrated at the amyE locus by double-crossover recombination, giving strain BS9853. Expression of the pta-lacZ fusion in strain BS9853 was monitored during growth in DSM. As shown in Fig. 6A, pta-lacZ expression decreased continuously during the entire growth stage, decreasing to about 5 Miller units between T₃ and T₄. However, the addition of glucose to a final concentration of 1% as to DSM significantly induced the pta-lacZ expression as the cells entered the stationary phase (T₀), a maximum level (about 60 Miller units) being reached around T₁. A similar induction pattern was observed when 1% fructose or 2% glycerol was added to DSM, although the addition of glycerol resulted in weaker induction than that of fructose or glucose (Fig. 6A). The same pta-lacZ fusion was also integrated isotopically into the original pta locus (see "MATERIALS AND METHODS"), and a similar induction pattern of transcription was observed. Mutations in spo0A or spo0H also had little or no effect on the induction pattern of pta-lacZ expression indicating that Spo0A or Spo0H does not appear to play a role in the regulation of pta expression (data not shown).

In order to examine the possibility that CcpA, which has been shown to act as either a repressor or an activator of gene expression (29), also might be responsible for the activation of pta-lacZ expression, a fusion construct was introduced into a ccpA mutant strain (BS9901), giving strain BS9902. Expression of the pta-lacZ fusion in strain BS9902 was not activated by the addition of glucose unlike in the case of the wild type (Fig. 6B), suggesting that the CcpA protein is critically involved in the induction of the pta-lacZ fusion by glucose.

Identification and Mutagenesis of the cre-Like Sequence

Since it appeared that glucose-induced pta-lacZ expression required the CcpA protein, we searched for cis-acting sequences called catabolite-responsive element (cre), which has been functionally identified in a number of genes or operons affected by a CcpA (30). A putative cre sequence, located upstream of the tentative promoter site, at positions –49 to –62, was identified (Fig. 5). This cre-like sequence is very similar to the consensus sequence originally proposed by Weickert and Chambliss (31) based on the results of mutational analyses of the catabolite-repression-mediating sequence in amyE. To examine the functional role of the cre-like sequence located in the pta upstream region, site-directed mutagenesis was performed. We made three different single-base changes in the cre-like sequence: PC1 (–55G to T), PC2 (–53T to A), and PC3 (–50A to C) (Fig. 5). To analyze the effects of the mutations on pta expression, pta-lacZ fusions containing a mutagenized cre-like sequence were constructed and integrated into the chromosome as a single copy at the amyE locus. As shown in Fig. 6C, the pta-lacZ expression of strain BS9905 bearing the PC1 mutation was not activated by the addition of glucose to DSM. Glucose induction of the pta-lacZ fusion in strain BS9905 was comparable to that in the ccpA mutant (BS9902) (Fig. 6B). The PC2 mutation also abolished the pta-lacZ activation by glucose, though the basal level of b-galactosidase activity was higher than that of strain BS9905 bearing the PC1 mutation (Fig. 6C). On the other hand, the pta-lacZ expression was still activated by the addition of glucose in strain BS9906 bearing the PC3 mutation. This result was expected because the PC3 mutation was designed to increase the homology to the consensus sequence of the cre sequence (31) (Fig. 5). All of these results suggest that the cre sequence in the pta upstream region functions as an active cis-element for glucose-induced activation of the pta gene, which is mediated by CcpA.

Although the pta and ackA genes are located separately on B. subtilis chromosomal DNA, it seems that they share a common mechanism for transcriptional regulation. The following observations support this conclusion: (i) The transcription of both the pta and ackA genes is activated in the presence of excess glucose (8). (ii) The cre sequences are present in the upstream region of the promoter, and this element was found to be indispensible for transcriptional activation mediated by glucose (9). (iii) The transcriptional activation of both genes was abolished in the ccpA mutant (8). All of these results indicate that these two genes are positively regulated by the CcpA protein with excess glucose. Since the catabolite repression of the acsA gene is also mediated by CcpA (10), it is very interesting that all three genes, i.e. pta, ackA, and acsA, for interconversion between acetyl CoA and acetate are regulated by the CcpA protein. It has been shown that a number of genes encoding carbon catabolic enzymes and some of the genes encoding enzymes of the central metabolic pathway contain the cre sequence (32), suggesting that global regulation of carbon flux is mediated by the CcpA protein in B. subtilis. Like the CcpA protein, E. coli Cra is also a member of the LacI-GalR family of transcription factors, and represses the carbon catabolic pathway and activates the carbon anabolic pathway (33). Thus the protein structure and physiological role of the Cra protein may be homologous to those of CcpA, although the mechanism of sensory transduction for activation or repression is different in each case (33).

We wish to thank P. Stragier for the plasmids and strains, and D.E. Chang for the technical assistance. We also thank J.G. Pan and J.K. Lee for reading the manuscript.

. ___

¹ This work was supported by a grant, KG1341, from the Ministry of Science and Technology of Korea.
² To whom correspondence should be addressed. Phone: +82-42-860-4412, Fax: +82-42-860-4594, E-mail: shpark@kribb4680.kribb.re.kr
Abbreviations: IPTG, isopropyl-b-D-thiogalactopyranoside; PMSF, phenylmethylsulfonyl fluoride.
 

REFERENCES

1. Speck, E.L. and Freese, E. (1973) Control of metabolite secretion in Bacillus subtilis. J. Gen. Microbiol. 78, 261-275
2. Bauer, K.A., Ben-Bassat, A., Dawson, M., de la Puente, V.T., and Neway, J.O. (1990) Improved expression of human interleukin-2 in high-cell-density fermentor cultures of Escherichia coli K-12 by a phosphotransacetylase mutant. Appl. Environ. Microbiol. 56, 1296-1302
3. Kumari, S., Tishel, R., Eisenbach, M., and Wolfe, A.J. (1995) Cloning, characterization, and functional expression of acs, the gene which encodes acetyl coenzyme A synthetase in Escherichia coli. J. Bacteriol. 177, 2878-2886
4. Grundy, F.J., Waters, D.A., Takova, T.Y., and Henkin, T.M. (1993) Identification of genes involved in utilization of acetate and acetoin in Bacillus subtilis. Mol. Microbiol. 10, 259-271
5. Lukat, G.S., McCleary, W.R., Stock, A.M., and Stock, J.B. (1992) Phosphorylation of bacterial response regulator proteins by low molecular weight phospho-donors. Proc. Natl. Acad. Sci. USA 89, 718-722
6. Feng, J., Atkinson, M.R., McCleary, W., Stock, J.B., Wanner, B.L., and Ninfa, A.J. (1992) Role of phosphorylated metabolic intermediates in the regulation of glutamine synthetase synthesis in Escherichia coli. J. Bacteriol. 174, 6061-6070
7. Wanner, B.L. and Wilmes-Riesenberg, M.R. (1992) Involvement of phosphotransacetylase, acetate kinase, and acetyl phosphate synthesis in control of the phosphate regulon in Escherichia coli. J. Bacteriol. 174, 2124-2130
8. Grundy, F.J., Waters, D.A., Allen, S.H., and Henkin, T.M. (1993) Regulation of the Bacillus subtilis acetate kinase gene by CcpA. J. Bacteriol. 175, 7348-7355
9. Turinsky, A.J., Grundy, F.J., Kim, J.H., Chambliss, G.H., and Henkin, T.M. (1998) Transcriptional activation of the Bacillus subtilis ackA gene requires sequences upstream of the promoter. J. Bacteriol. 180, 5961-5967
10. Grundy, F.J., Turinsky, A.J., and Henkin, T.M. (1994) Catabolite regulation of Bacillus subtilis acetate and acetoin utilization genes by CcpA. J. Bacteriol. 176, 4527-4533
11. Rado, T.A. and Hoch, J.A. (1973) Phosphotransacetylase from Bacillus subtilis: purification and physiological studies. Biochim. Biophys. Acta 321, 114-125
12. Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P., Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S.C., Bron, S., Brouillet, S., Bruschi, C.V., Caldwell, B., Capuano, V., Carter, N.M., Choi, S.K., Codani, J.J., Connerton, I.F., Danchin, A., et al. (1997) The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature 390, 249-256
13. Miller, J. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
14. Schaeffer, P., Millet, J., and Aubert, J.-P. (1965) Catabolic repression of bacterial sporulation. Proc. Natl. Acad. Sci. USA 54, 704-711
15. Albano, M., Hahn, J., and Dubnau, D. (1987) Expression of competence genes in Bacillus subtilis. J. Bacteriol. 169, 3110-3117
16. Fisher, S.H. and Sonenshein, A.L. (1977) Glutamine-requiring mutants of Bacillus subtilis. Biochem. Biophys. Res. Commun. 79, 987-995
17. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor, Cold Spring Harbor, NY
18. Cutting, S.M. and Vander Horn, P.B. (1990) Genetic analysis in Molecular Biological Methods for Bacillus (Harwood, C.R. and Cutting, S.M., eds.) pp. 27-74, John Wiley & Sons, New York
19. Prü244F, B.M. and Wolfe, A.J. (1994) Regulation of acetyl phosphate synthesis and degradation, and the control of flagellar expression in Escherichia coli. Mol. Microbiol. 12, 973-984
20. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254
21. Guérout-Fleury, A., Frandsen, N., and Stragier, P. (1996) Plasmids for ectopic integration in Bacillus subtilis. Gene 180, 57-61
22. Nicholson, W.L. and Setlow, P. (1990) Sporulation, germination and outgrowth in Molecular Biological Methods for Bacillus (Harwood, C.R. and Cutting, S.M., eds.) pp. 391-450, John Wiley & Sons, New York
23. Nyström, T. (1994) The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival. Mol. Microbiol. 12, 833-843
24. Voskuil, M.I., Voepel, K., and Chambliss, G.H. (1995) The –16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol. Microbiol. 17, 271-279
25. Boynton, Z.L., Bennett, G.N., and Rudolph, F.B. (1996) Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824. Appl. Environ. Microbiol. 62, 2758-2766
26. Kakuda, H., Hosono, K., Shiroishi, K., and Ichihara, S. (1994) Identification and characterization of the ackA (acetate kinase A)-pta (phosphotransacetylase) operon and complementation analysis of acetate utilization by an ackA-pta deletion mutant of Escherichia coli. J. Biochem. 116, 916-922
27. Singh-Wissmann, K. and Ferry, J.G. (1995) Transcriptional regulation of the phosphotransacetylase-encoding and acetate kinase-encoding genes (pta and ack) from Methanosarcina thermophila. J. Bacteriol. 177, 1699-1702
28. Summers, M.L., Denton, M.C., and McDermott, T.R. (1999) Genes coding for phosphotransacetylase and acetate kinase in Sinorhizobium meliloti are in an operon that is inducible by phosphate stress and controlled by PhoB. J. Bacteriol. 181, 2217-2224
29. Henkin, T.M. (1996) The role of CcpA transcriptional regulator in carbon metabolism in Bacillus subtilis. FEMS Microbiol. Lett. 135, 9-15
30. Hueck, C.J. and Hillen, W. (1995) Catabolite repression in Bacillus subtilis: a global regulatory mechanism for the gram-positive bacteria? Mol. Microbiol. 15, 395-401
31. Weickert, M.J. and Chambliss, G.H. (1990) Site-directed mutagenesis of a catabolite repression operator sequence in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 87, 6238-6242
32. Hueck, C.J., Hillen, W., and Saier, M.H., Jr. (1994) Analysis of a cis-active sequence mediating catabolite repression in gram-positive bacteria. Res. Microbiol. 145, 503-518
33. Saier, M.H., Jr. (1996) Cyclic AMP-independent catabolite repression in bacteria. FEMS Microbiol. Lett. 138, 97-103

(Full Text online)

Back to Automation in Microbiology main page

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005