Appl Microbiol Biotechnol (1990) 33:680-682
Applied o.. Microbiology Biotechnology © Springer-Verlag 1990
Short contribution
Cloning of a gene coding for phosphotransacetylase from Escherichia coli Hideko Yamamoto-Otake, Asahi Matsuyama, and Eiichi Nakano Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba, Japan Received 13 November 1989/Accepted 23 April 1990
Summary. A phosphotransacetylase gene (pta) has been cloned from a genomic D N A library of Escherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities o f p t a + plasmid-containing strains were amplified about 150-fold under control of the lac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pta gene was found to be downstream of a c k A by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of the purFf o I C - h i s T region of the chromosome.
We previously reported the cloning and sequencing of the gene that codes for acetate kinase ( M a t s u y a m a et al. 1989). In the Escherichia coli genetic map, pta and a c k A m a p p e d close to each other at 49.5 min on the E. coli chromosome. A class of E. coli mutants ( f a t - A ) lacking both acetate kinase and phosphotransacetylase activities has been reported (Bachmann 1983; Guest 1979). In Salmonella typhimurium ack and pta are present in a single operon (Van D y k and LaRossa 1987). Here we report the cloning of the pta gene, the probability of an ackA-pta operon in E. coli, and the determination of its physical and functional maps.
Materials and methods Introduction Phosphotransacetylase (acetyl coenzyme A (CoA): ort h o p h o s p h a t e acetyltransferase, EC2.3.1.8) reversibly catalyses the following reaction: Acetyl C o A + Pi ~ C o A + Acetyl phosphate. The acetate kinase-phosphotransacetylase system has been identified in the Enterobacteriaceae (Gilvarg and Davis 1956; Rose et al. 1954) and is thought to play an amphibolic role in the excretion and activation of acetate (Sanwal 1970; Brown et al. 1977). Phosphotransacetylase, together with acetate kinase (EC2.7.2.1), is able to activate acetate to form acetyl CoA, a key metabolic intermediate, and to couple the hydrolysis of acetyl C o A with the production of ATP. C o A thioesters serve as substrates in a large variety of enzyme-catalysed reactions and acetyl C o A is central in biological acetylation reactions. We have isolated a gene coding for p h o s p h o transacetylase to obtain further understanding of the biological and physiological roles of phosphotransacetylase and to use the reaction catalysed by this enzyme for regeneration of C o A and acetyl-CoA more effectively.
Offprint requests to: A. Matsuyama
Chemicals. Restriction endonucleases and DNA modification enzymes were purchased from Takara Shuzo Co. (Kyoto, Japan) and Boehringer (Mannheim, FRG). All other reagents were analytical grade reagents. Isolation of mutants deficient in pta. E. eoli mutants lacking phosphotransacetylase have been isolated from an E. coli K-12 derivative, E. coli 1100 (thi, endA) (Durwald and Hoffmann-Berling 1968) as described by Brown et al. (1977). Briefly, cells of E. coli 1100 were plated onto minimal medium containing fluoroacetate with pyruvate as the sole carbon source. After 3-4 days at 37° C, spontaneous mutants appeared at a freqency of about 1 in 10 610 7. The fluoroacetate-resistant derivatives were picked, purified and assayed for phosphotransacetylase activity. One mutant (AM1000) was selected as a pta- mutant for further study. DNA manipulations. The procedures and conditions used for isolation of plasmid DNA, digestion with restriction endonucleases, isolation of DNA fragments, and transformation of E. eoli were carried out by the methods of Maniatis et al. (1982). Construction of E. coli 1100 9enomic DNA library. E. coli 1100 used as a source of the genomic DNA was grown in Luria-Bertani (LB) medium (Miller 1972) at 37°C for 20 h on a rotary shaker. Chromosomal DNA of E. coli 1100 was isolated by the method of Saito and Miura (1963). The E. coli genomic DNA, partially digested with the restriction endonuclease Sau3AI, was ligated with plasmid vector pBR322, which had been digested with BamHI. E. coli AM1000 was transformed with the ligated mixture. Approximately 5000 ampicillin-resistant and tetracycline-sensitive transformants were obtained.
681
Enzyme assay. To determine the phosphotransacetylase activity acetyl CoA formed from acetyl phosphate and CoA was measured as described by Brown et al. (1977). The activity of acetate kinase was measured by coupling ATP production from acetyl phosphate and ADP to nicotinamide adenine dinucleotide phosphate (NADP ÷) reduction via hexokinase and glucose-6-phosphate dehydrogenase as described by Thomas et al. (1980). In order to determine the above enzyme activities, cell cultures (10 ml) were grown on LB medium containing ampicillin (25 ~tg/ml) at 37°C up to the early stationary phase.
Table 1. Acetate kinase (ACK) and phosphotransacetylase (PTA) activities in Escherichia coli 1100 cells dosed with plasmids carrying ackA and/or pta genes Plasmid
pPT100 pPT200 pPT300
Results and discussion Several spontaneous mutants of E. coli 1100 were selected for resistance to fluoroacetate and an isolate with trace levels of phosphotransacetylase (PTA) and with a low reversion rate was chosen as a p t a - strain for further manipulation. This mutant was designated AM1000 and used as a recipient for transformation involving c o m p l e m e n t for pta mutation. The E. coli 1100 genomic D N A library constructed on plasmid vector pBR322 was consisted o f approximately 5000 transformants. F r o m this library the clone to c o m p l e m e n t the pta mutation in AM1000 was screened for an increase in PTA activity o f sonicated crude lysates. Only one clone c o m p l e m e n t e d pta and was chosen for the expression experiments described below. Plasmid D N A was isolated from this transformant and designated pPT100, which had a 3.2-kb insert based on the restriction m a p (Fig. 1A). The specific PTA activity of this strain 1100 (pPT100) was about threefold higher than that of the control strain 1100 (Table 1). We constructed several derivatives from this plasmid to reduce the size of the insert and found that the plasmid which contained a 2.9-kb EcoRI-KpnI fragment f r o m pPT100 still c o m p l e m e n t e d pta. Therefore (S)K
~r~oo
I'
so p*K122
,
pAK222
I
KvK
PB p
~'
Ir ,
Y
(~
rl 0.SKb,
Ell
Fig. 1. A Restriction maps of the insert DNA fragments of plasmid pAK122 (Matsuyama et al. 1989) and pPT200, and the construction of pAK222. Vector fragments are not included: ~ , direction of the lac promoter. B Molecular map of 34 kb of DNA containing the ackA-pta gene from Escherichia coli 1100. Boxes above the restriction map indicate the genes identified in this region. The genes usg, hisT, folC, purF, and ackA have been previously characterized (Arps et al. 1985; Bognar et al. 1987; Nonet et al. 1987; Tso et al. 1982; Matsuyama et al. 1989): ~ under the gene boxes, direction of transcription. Abbreviations for restriction sites: B, BamHI; E, EcoRI; H, HindlII; K, KpnI; M, MluI; P, PstI; S, Sau3AI; Sc, ScaI; V, EcoRV
pAK122 pAK222
Addition a
None None IPTG None IPTG None None
Enzyme activity ACK
PTA
_b _ 2.6 (1.0) 1.4 ( 0 . 6 ) ----476.2 (183.2) 102.5 (39.4)
5.3e (2.9)d 9.6 (5.3) 224.0 (124.4) 5.2 (2.0) 4.7 (2.6) 2.0 (1.1) 78.3 (43.5)
a Isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the medium at an initial concentration of 1 mM b --, not determined c Units/mg protein; all values are the average of two experiments d Relative activity
this 2.9-kb fragment was recloned into the appropriate restriction site of the high-copy n u m b e r vector pUC18 and pUC19 to obtain pPT200 (Fig. 1A) and pPT300, respectively, allowing pta expression by the lac promotor. To determine the level of expression of the cloned pta gene, E. coli 1100 carrying pPT200 and pPT300 were cultured in LB m e d i u m at 37 ° C for 16 h and sonicated extracts were assayed for PTA activity. As shown in Table 1, expression of PTA in the strain harbouring pPT200, but not pPT300, was 150fold greater than the strain harbouring pUC18, when isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the medium. Cell extracts of strain 1100 (pPT200) cultured with the addition of I P T G were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining. A unique protein with a molecular weight of ca. 81,000 was produced in the cells of strain 1100 (pPT200), whereas a similar protein was not found in the cells of the other strains (data not shown). These data suggest that the cloned pta gene, which p r o b a b l y lacks a p r o m o t e r region, may be contained entirely in these plasmids and that the pta gene in pUC18 is expressed in a forward orientation with respect to the lac p r o m o t e r (pPT200). In the E. coli genetic map, the pta gene is located near the ackA gene at 49.5 rnin on the E. coli chromosome. By cloning the ackA gene and determining its nucleotide sequence, we recently found that the ackA open reading frame was located about 15 kb upstream of the purF-foIC-hisT region of the c h r o m o s o m e (Matsuyama et al. 1989). We therefore c o m p a r e d the restriction endonuclease maps of plasmids pAK122 containing the entire ackA gene, which p r o b a b l y had the ackA p r o m o t e r region ( M a t s u y a m a et al. 1989), and pPT200. As shown in Fig. 1A, it was revealed that these plasmid D N A s overlapped each other between the KpnI and B a m H I sites on the restriction endonuclease map. This is in agreement with the close location o f these two genes in E. cob (Bachmann 1983; Guest 1979) and al-
682 lows for precise location of the corresponding genes on the chromosomal map of E. coli K-12. The B a m H I - H i n d l I I fragment from pPT200 was inserted into the B a m H I and H i n d l I I sites o f pAK122 to obtain pAK222 (Fig. 1A). The ability of these plasmids to express the products of both a c k A and pta genes was tested by enzymatic assays. While the cells harbouring pPT200 showed inducible expression of PTA activity, the cells harbouring pAK222 showed constitutive expression of PTA activity. Under non-inducing conditions, expressions of both A C K and PTA in strain 1100 (pAK222) were about 40-fold greater than in the control strain 1100 (pUC19) (Table 1). These data suggest that a c k A and pta may form an operon in E. coli. The order of the genes determined by our experiments is consistent with the results of previous studies in which genetic techniques were used (Guest 1979). In addition, the ackA-pta region was found to be located about 13 kb upstream of the purF-foIC-hisT region of the c h r o m o s o m e (Fig. 1B). Experiments are currently in progress in our laboratory to determine the complete nucleotide sequences and the promoter region of the ackA-pta region, and to use the reaction catalysed by PTA for the recycling of acetyl CoA. Furthermore, these a c k A and pta genes may be useful tools to study acetate metabolism.
Acknowledgements. We thank Prof. A. Kimura and Dr. S. Nasuno for valuable discussion. K. Saito is acknowledged for excellent technical assistance.
References Arps PJ, Marvel CC, Rubin BC, Tolan DA, Penhoet EE, Winkler ME (1985) Structural features of the hisT operon of Escherichia coli K-12. Nucleic Acids Res 12:5297-5315 Bachmann BJ (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180-230
Bognar AL, Osborne C, Shane B (1987) Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J Biol Chem 262:1233712343 Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymatic interconversions of acetate and acetyi-coenzyme A in Escherichia coli. J Gen Microbiol 102:327-336 D~irwald H, Hoffmann-Berling H (1968) Endonuclease I-deficient and ribonuclease I-deficient Escherichia coli mutants. J Mol Biol 34:331-346 Gilvarg C, Davis BD (1956) The role of the tricarboxylic acid cycle in acetate oxidation in Escherichia coli. J Biol Chem 222:307-319 Guest JR (1979) Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants. J Gen Microbiol 115 :259-271 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Matsuyama A, Yamamoto H, Nakano E (1989) Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene. J Bacteriol 171:577-580 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Nonet ML, Marvel CC, Tolan DR (1987) The hisT-purF region of the Escherichia coli K-12 chromosome. J Biol Chem 262:12209-12217 Rose IA, Grunberg-Manago M, Korey SR, Ochoa S (1954) Enzymatic phosphorylation of acetate. J Biol Chem 211:737-756 Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619-629 Sanwal BD (1970) Allosteric controls of amphibolic pathways in bacteria. Bacteriol Rev 34:20-39 Thomas TD, Turner KW, Crow VL (1980) Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation. J Bacteriol 144:672-682 Tso JY, Zalkin H, Cleepment M van, Yanofsky C, Smith JM (1982) Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase. J Biol Chem 257:3525-3531 Van Dyk TK, LaRossa RA (1987) Involvement of ack-pta operon products in alpha~ketobutyrate metabolism by Salmonella typhimurium. Mol Gen Genet 207:435-440
Applied o.. Microbiology Biotechnology © Springer-Verlag 1990
Short contribution
Cloning of a gene coding for phosphotransacetylase from Escherichia coli Hideko Yamamoto-Otake, Asahi Matsuyama, and Eiichi Nakano Research and Development Division, Kikkoman Corporation, 399 Noda, Noda-shi, Chiba, Japan Received 13 November 1989/Accepted 23 April 1990
Summary. A phosphotransacetylase gene (pta) has been cloned from a genomic D N A library of Escherichia coli 1100, a derivative of strain K-12. The phosphotransacetylase activities o f p t a + plasmid-containing strains were amplified about 150-fold under control of the lac promoter. The molecular weight of the phosphotransacetylase was estimated to be about 81,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The pta gene was found to be downstream of a c k A by a combination of restriction analysis and plasmid subcloning. It is located about 13 kb upstream of the purFf o I C - h i s T region of the chromosome.
We previously reported the cloning and sequencing of the gene that codes for acetate kinase ( M a t s u y a m a et al. 1989). In the Escherichia coli genetic map, pta and a c k A m a p p e d close to each other at 49.5 min on the E. coli chromosome. A class of E. coli mutants ( f a t - A ) lacking both acetate kinase and phosphotransacetylase activities has been reported (Bachmann 1983; Guest 1979). In Salmonella typhimurium ack and pta are present in a single operon (Van D y k and LaRossa 1987). Here we report the cloning of the pta gene, the probability of an ackA-pta operon in E. coli, and the determination of its physical and functional maps.
Materials and methods Introduction Phosphotransacetylase (acetyl coenzyme A (CoA): ort h o p h o s p h a t e acetyltransferase, EC2.3.1.8) reversibly catalyses the following reaction: Acetyl C o A + Pi ~ C o A + Acetyl phosphate. The acetate kinase-phosphotransacetylase system has been identified in the Enterobacteriaceae (Gilvarg and Davis 1956; Rose et al. 1954) and is thought to play an amphibolic role in the excretion and activation of acetate (Sanwal 1970; Brown et al. 1977). Phosphotransacetylase, together with acetate kinase (EC2.7.2.1), is able to activate acetate to form acetyl CoA, a key metabolic intermediate, and to couple the hydrolysis of acetyl C o A with the production of ATP. C o A thioesters serve as substrates in a large variety of enzyme-catalysed reactions and acetyl C o A is central in biological acetylation reactions. We have isolated a gene coding for p h o s p h o transacetylase to obtain further understanding of the biological and physiological roles of phosphotransacetylase and to use the reaction catalysed by this enzyme for regeneration of C o A and acetyl-CoA more effectively.
Offprint requests to: A. Matsuyama
Chemicals. Restriction endonucleases and DNA modification enzymes were purchased from Takara Shuzo Co. (Kyoto, Japan) and Boehringer (Mannheim, FRG). All other reagents were analytical grade reagents. Isolation of mutants deficient in pta. E. eoli mutants lacking phosphotransacetylase have been isolated from an E. coli K-12 derivative, E. coli 1100 (thi, endA) (Durwald and Hoffmann-Berling 1968) as described by Brown et al. (1977). Briefly, cells of E. coli 1100 were plated onto minimal medium containing fluoroacetate with pyruvate as the sole carbon source. After 3-4 days at 37° C, spontaneous mutants appeared at a freqency of about 1 in 10 610 7. The fluoroacetate-resistant derivatives were picked, purified and assayed for phosphotransacetylase activity. One mutant (AM1000) was selected as a pta- mutant for further study. DNA manipulations. The procedures and conditions used for isolation of plasmid DNA, digestion with restriction endonucleases, isolation of DNA fragments, and transformation of E. eoli were carried out by the methods of Maniatis et al. (1982). Construction of E. coli 1100 9enomic DNA library. E. coli 1100 used as a source of the genomic DNA was grown in Luria-Bertani (LB) medium (Miller 1972) at 37°C for 20 h on a rotary shaker. Chromosomal DNA of E. coli 1100 was isolated by the method of Saito and Miura (1963). The E. coli genomic DNA, partially digested with the restriction endonuclease Sau3AI, was ligated with plasmid vector pBR322, which had been digested with BamHI. E. coli AM1000 was transformed with the ligated mixture. Approximately 5000 ampicillin-resistant and tetracycline-sensitive transformants were obtained.
681
Enzyme assay. To determine the phosphotransacetylase activity acetyl CoA formed from acetyl phosphate and CoA was measured as described by Brown et al. (1977). The activity of acetate kinase was measured by coupling ATP production from acetyl phosphate and ADP to nicotinamide adenine dinucleotide phosphate (NADP ÷) reduction via hexokinase and glucose-6-phosphate dehydrogenase as described by Thomas et al. (1980). In order to determine the above enzyme activities, cell cultures (10 ml) were grown on LB medium containing ampicillin (25 ~tg/ml) at 37°C up to the early stationary phase.
Table 1. Acetate kinase (ACK) and phosphotransacetylase (PTA) activities in Escherichia coli 1100 cells dosed with plasmids carrying ackA and/or pta genes Plasmid
pPT100 pPT200 pPT300
Results and discussion Several spontaneous mutants of E. coli 1100 were selected for resistance to fluoroacetate and an isolate with trace levels of phosphotransacetylase (PTA) and with a low reversion rate was chosen as a p t a - strain for further manipulation. This mutant was designated AM1000 and used as a recipient for transformation involving c o m p l e m e n t for pta mutation. The E. coli 1100 genomic D N A library constructed on plasmid vector pBR322 was consisted o f approximately 5000 transformants. F r o m this library the clone to c o m p l e m e n t the pta mutation in AM1000 was screened for an increase in PTA activity o f sonicated crude lysates. Only one clone c o m p l e m e n t e d pta and was chosen for the expression experiments described below. Plasmid D N A was isolated from this transformant and designated pPT100, which had a 3.2-kb insert based on the restriction m a p (Fig. 1A). The specific PTA activity of this strain 1100 (pPT100) was about threefold higher than that of the control strain 1100 (Table 1). We constructed several derivatives from this plasmid to reduce the size of the insert and found that the plasmid which contained a 2.9-kb EcoRI-KpnI fragment f r o m pPT100 still c o m p l e m e n t e d pta. Therefore (S)K
~r~oo
I'
so p*K122
,
pAK222
I
KvK
PB p
~'
Ir ,
Y
(~
rl 0.SKb,
Ell
Fig. 1. A Restriction maps of the insert DNA fragments of plasmid pAK122 (Matsuyama et al. 1989) and pPT200, and the construction of pAK222. Vector fragments are not included: ~ , direction of the lac promoter. B Molecular map of 34 kb of DNA containing the ackA-pta gene from Escherichia coli 1100. Boxes above the restriction map indicate the genes identified in this region. The genes usg, hisT, folC, purF, and ackA have been previously characterized (Arps et al. 1985; Bognar et al. 1987; Nonet et al. 1987; Tso et al. 1982; Matsuyama et al. 1989): ~ under the gene boxes, direction of transcription. Abbreviations for restriction sites: B, BamHI; E, EcoRI; H, HindlII; K, KpnI; M, MluI; P, PstI; S, Sau3AI; Sc, ScaI; V, EcoRV
pAK122 pAK222
Addition a
None None IPTG None IPTG None None
Enzyme activity ACK
PTA
_b _ 2.6 (1.0) 1.4 ( 0 . 6 ) ----476.2 (183.2) 102.5 (39.4)
5.3e (2.9)d 9.6 (5.3) 224.0 (124.4) 5.2 (2.0) 4.7 (2.6) 2.0 (1.1) 78.3 (43.5)
a Isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the medium at an initial concentration of 1 mM b --, not determined c Units/mg protein; all values are the average of two experiments d Relative activity
this 2.9-kb fragment was recloned into the appropriate restriction site of the high-copy n u m b e r vector pUC18 and pUC19 to obtain pPT200 (Fig. 1A) and pPT300, respectively, allowing pta expression by the lac promotor. To determine the level of expression of the cloned pta gene, E. coli 1100 carrying pPT200 and pPT300 were cultured in LB m e d i u m at 37 ° C for 16 h and sonicated extracts were assayed for PTA activity. As shown in Table 1, expression of PTA in the strain harbouring pPT200, but not pPT300, was 150fold greater than the strain harbouring pUC18, when isopropyl-fl-D-thiogalactopyranoside (IPTG) was added to the medium. Cell extracts of strain 1100 (pPT200) cultured with the addition of I P T G were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue staining. A unique protein with a molecular weight of ca. 81,000 was produced in the cells of strain 1100 (pPT200), whereas a similar protein was not found in the cells of the other strains (data not shown). These data suggest that the cloned pta gene, which p r o b a b l y lacks a p r o m o t e r region, may be contained entirely in these plasmids and that the pta gene in pUC18 is expressed in a forward orientation with respect to the lac p r o m o t e r (pPT200). In the E. coli genetic map, the pta gene is located near the ackA gene at 49.5 rnin on the E. coli chromosome. By cloning the ackA gene and determining its nucleotide sequence, we recently found that the ackA open reading frame was located about 15 kb upstream of the purF-foIC-hisT region of the c h r o m o s o m e (Matsuyama et al. 1989). We therefore c o m p a r e d the restriction endonuclease maps of plasmids pAK122 containing the entire ackA gene, which p r o b a b l y had the ackA p r o m o t e r region ( M a t s u y a m a et al. 1989), and pPT200. As shown in Fig. 1A, it was revealed that these plasmid D N A s overlapped each other between the KpnI and B a m H I sites on the restriction endonuclease map. This is in agreement with the close location o f these two genes in E. cob (Bachmann 1983; Guest 1979) and al-
682 lows for precise location of the corresponding genes on the chromosomal map of E. coli K-12. The B a m H I - H i n d l I I fragment from pPT200 was inserted into the B a m H I and H i n d l I I sites o f pAK122 to obtain pAK222 (Fig. 1A). The ability of these plasmids to express the products of both a c k A and pta genes was tested by enzymatic assays. While the cells harbouring pPT200 showed inducible expression of PTA activity, the cells harbouring pAK222 showed constitutive expression of PTA activity. Under non-inducing conditions, expressions of both A C K and PTA in strain 1100 (pAK222) were about 40-fold greater than in the control strain 1100 (pUC19) (Table 1). These data suggest that a c k A and pta may form an operon in E. coli. The order of the genes determined by our experiments is consistent with the results of previous studies in which genetic techniques were used (Guest 1979). In addition, the ackA-pta region was found to be located about 13 kb upstream of the purF-foIC-hisT region of the c h r o m o s o m e (Fig. 1B). Experiments are currently in progress in our laboratory to determine the complete nucleotide sequences and the promoter region of the ackA-pta region, and to use the reaction catalysed by PTA for the recycling of acetyl CoA. Furthermore, these a c k A and pta genes may be useful tools to study acetate metabolism.
Acknowledgements. We thank Prof. A. Kimura and Dr. S. Nasuno for valuable discussion. K. Saito is acknowledged for excellent technical assistance.
References Arps PJ, Marvel CC, Rubin BC, Tolan DA, Penhoet EE, Winkler ME (1985) Structural features of the hisT operon of Escherichia coli K-12. Nucleic Acids Res 12:5297-5315 Bachmann BJ (1983) Linkage map of Escherichia coli K-12, edition 7. Microbiol Rev 47:180-230
Bognar AL, Osborne C, Shane B (1987) Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation of expression by an upstream gene. J Biol Chem 262:1233712343 Brown TDK, Jones-Mortimer MC, Kornberg HL (1977) The enzymatic interconversions of acetate and acetyi-coenzyme A in Escherichia coli. J Gen Microbiol 102:327-336 D~irwald H, Hoffmann-Berling H (1968) Endonuclease I-deficient and ribonuclease I-deficient Escherichia coli mutants. J Mol Biol 34:331-346 Gilvarg C, Davis BD (1956) The role of the tricarboxylic acid cycle in acetate oxidation in Escherichia coli. J Biol Chem 222:307-319 Guest JR (1979) Anaerobic growth of Escherichia coli K12 with fumarate as terminal electron acceptor. Genetic studies with menaquinone and fluoroacetate-resistant mutants. J Gen Microbiol 115 :259-271 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Matsuyama A, Yamamoto H, Nakano E (1989) Cloning, expression, and nucleotide sequence of the Escherichia coli K-12 ackA gene. J Bacteriol 171:577-580 Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Nonet ML, Marvel CC, Tolan DR (1987) The hisT-purF region of the Escherichia coli K-12 chromosome. J Biol Chem 262:12209-12217 Rose IA, Grunberg-Manago M, Korey SR, Ochoa S (1954) Enzymatic phosphorylation of acetate. J Biol Chem 211:737-756 Saito H, Miura K (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim Biophys Acta 72:619-629 Sanwal BD (1970) Allosteric controls of amphibolic pathways in bacteria. Bacteriol Rev 34:20-39 Thomas TD, Turner KW, Crow VL (1980) Galactose fermentation by Streptococcus lactis and Streptococcus cremoris: pathways, products, and regulation. J Bacteriol 144:672-682 Tso JY, Zalkin H, Cleepment M van, Yanofsky C, Smith JM (1982) Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase. J Biol Chem 257:3525-3531 Van Dyk TK, LaRossa RA (1987) Involvement of ack-pta operon products in alpha~ketobutyrate metabolism by Salmonella typhimurium. Mol Gen Genet 207:435-440
