Molecular cloning and sequencing of the gene for CDP-diglyceride synthetase of Escherichia coli

The cds gene of Escherichia coli codes for the enzyme CDP-diglyceride synthetase. We now report the construction of plasmids which carry cds. Using these plasmids, we have sequenced 1274 base pairs of DNA, including a 750-base pair open reading frame which is the coding region of the cds gene. This DNA sequence allows the deduction of the primary peptide sequence for CDP-diglyceride synthetase. The protein is very hydrophobic, and, assuming no processing or modification, has a molecular weight of 27,570. Furthermore, there is a second open reading frame immediately after cds, implying that cds may be part of an operon. We have also constructed a runaway replication cds-plasmid that directs approximately 50-fold overproduction of CDP-diglyceride synthetase. This overproduction has been utilized in the purification of the enzyme to homogeneity, as described in the accompanying paper (Sparrow, C.P., and Raetz, C.R.H., J. Biol. Chem. 260, 12084-12091). Finally, the molecular cloning work reported herein allows the exact placement of the cds gene on the E. coli genetic map.     

The Escherichia coli orthologue of the Salmonella ushB gene (ushB(c)) produces neither UDP-sugar hydrolase activity nor detectable protein, but has an identical sequence to that of Escherichia coli cdh

Salmonella ushB, which encodes a membrane-bound UDP-sugar hydrolase, has an Escherichia coli orthologue (ushB(c)) which does not detectably produce this activity. In this report, we show that ushB(c) does not produce any detectable protein either, despite being transcribed normally. Remarkably, ushB(c) is shown to have 100% sequence identity with E. coli cdh, previously characterised as encoding an active CDP-diglyceride hydrolase, an apparent contradiction with implications regarding enzyme evolution. We suggest that a useful gene designation is cdh (ushB(c)) rather than either ushB(c) or cdh, alone.     

Nucleotide sequence and high-level expression of the major Escherichia coli phosphofructokinase

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfkA, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli. The region which has been sequenced comprises the complete pfkA--tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate-binding protein, sbp, and for a membrane-bound enzyme, CDP-diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA-sbp-cdh-tpi. The pfkA gene has been subcloned into a high-copy-number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 X 10(3)-nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40-50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using 'reverse genetics'.     

Genes from Pseudomonas sp. strain BS involved in the conversion of L-2-amino-Delta(2)-thiazolin-4-carbonic acid to L-cysteine

DL-2-amino-Delta(2)-thiazolin-4-carbonic acid (DL-ATC) is a substrate for cysteine synthesis in some bacteria, and this bioconversion has been utilized for cysteine production in industry. We cloned a DNA fragment containing the genes involved in the conversion of L-ATC to L-cysteine from Pseudomonas sp. strain BS. The introduction of this DNA fragment into Escherichia coli cells enabled them to convert L-ATC to cysteine via N-carbamyl-L-cysteine (L-NCC) as an intermediate. The smallest recombinant plasmid, designated pTK10, contained a 2.6-kb insert DNA fragment that has L-cysteine synthetic activity. The nucleotide sequence of the insert DNA revealed that two open reading frames (ORFs) encoding proteins with molecular masses of 19.5 and 44.7 kDa were involved in the L-cysteine synthesis from DL-ATC. These ORFs were designated atcB and atcC, respectively, and their gene products were identified by overproduction of proteins encoded in each ORF and by the maxicell method. The functions of these gene products were examined using extracts of E. coli cells carrying deletion derivatives of pTK10. The results indicate that atcB and atcC are involved in the conversion of L-ATC to L-NCC and the conversion of L-NCC to cysteine, respectively. atcB was first identified as a gene encoding an enzyme that catalyzes thiazolin ring opening. AtcC is highly homologous with L-N-carbamoylases. Since both enzymes can only catalyze the L-specific conversion from L-ATC to L-NCC or L-NCC to L-cysteine, it is thought that atcB and atcC encode L-ATC hydrolase and N-carbamyl-L-cysteine amidohydrolase, respectively.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat

The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.     

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.     

Molecular cloning and primary structure of cDNA encoding the catalytic domain of rat liver aspartyl-tRNA synthetase

A cDNA clone encoding rat liver aspartyl-tRNA synthetase was isolated by probing a lambda gt11 recombinant cDNA expression library with antibodies directed against the corresponding polypeptide from sheep liver. The 1930-base pairs-long cDNA insert allowed the expression in Escherichia coli of an active enzyme of mammalian origin. The nucleotide sequence of that cDNA, corresponding to the DRS1 gene, was determined. The open reading frame of DRS1 corresponds to a protein of Mr = 57,061, in good agreement with the previously determined molecular weight of the purified enzyme. The deduced amino acid sequence shows extensive homologies with that of yeast cytoplasmic aspartyl-tRNA synthetase, more than 50% of the residues being identical. In rat liver, aspartyl-tRNA synthetase occurs in two distinct forms: a dimeric enzyme and a component of a multienzyme complex comprising the nine aminoacyl-tRNA synthetases specific for arginine, aspartic acid, glutamic acid, glutamine, isoleucine, leucine, lysine, methionine, and proline. The primary structure of the DRS1 gene product is discussed in relation to the occurrence of two distinct forms of that enzyme.     

Chloroform-soluble nucleotides in Escherichia coli. Role of CDP-diglyceride in the enzymatic cytidylylation of phosphomonoester acceptors

CDP-diglyceride, the precursor of all the phospholipids in Escherichia coli, is cleaved in vitro to phosphatidic acid and CMP by a membrane-bound hydrolase. Since the physiological function of CDP-diglyceride hydrolase is unknown, we have explored the possibility that this enzyme acts in vivo as either a phosphatidyl- or cytidylyltransferase. To distinguish between these two alternatives, partially purified hydrolase was incubated with CDP-diglyceride in the presence of 50% H218O. Analysis of the reaction products by 31P NMR showed that 18O is incorporated exclusively into CMP, suggesting that the enzyme is a cytidylyltransferase. This conclusion is further supported by the following experimental results: (i) the hydrolase catalyzes the transfer of CMP from CDP-diglyceride to Pi; (ii) numerous phosphomonoesters, such as glycerol 3-phosphate, phosphoserine, and glucose 1-phosphate also function as CMP acceptors, but the corresponding compounds lacking the phosphate residues are not substrates for the enzyme; and (iii) CDP-diglyceride hydrolase exchanges [32P]phosphatidic acid for the phosphatidyl moiety of CDP-diglyceride and 32Pi for the beta-phosphate residue of CDP, indicating the involvement of a novel CMP-enzyme complex. These data suggest a biosynthetic role for CDP-diglyceride hydrolase, and extend the possible functions of CDP-diglyceride in the E. coli envelope.     

Cloning of the Pseudomonas aeruginosa gene encoding CDP-diglyceride synthetase

The CDP-diglyceride synthetase (CDS)-encoding gene (cds) from Pseudomonas aeruginosa PAO1 was cloned and sequenced. The gene possessed an open reading frame of 813 bp capable of encoding a putative polypeptide of 271 amino acids (aa) (28 699 Da). The deduced aa sequence of CDS revealed a 67% similarity (45% identity) to Escherichia coli CDS.     

Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus

The de novo purine biosynthetic enzymes 5-amino-4-imidazolecarboxamide-ribonucleotide (AICAR) transformylase (EC 2.1.2.3), IMP cyclohydrolase (EC 3.5.4.10) and glycineamide-ribonucleotide (GAR) synthetase (EC 2.1.2.2) are encoded by the purHD locus of Escherichia coli. The DNA sequence of this locus revealed two open reading frames encoding polypeptides of Mr 57,335 and 45,945 (GAR synthetase), respectively, that formed an operon. The DNA sequence, maxicell and complementation analyses all supported the concept that the Mr 57,335 polypeptide is the product of the purH gene and encodes a bifunctional protein containing both AICAR transformylase and IMP cyclohydrolase activities. The 5' end of the purHD mRNA was determined by primer extension mapping and contains two regions of dyad symmetry capable of forming 'hairpin' loops where the formation of the one would prevent the formation of the other but not vice versa. Regulation by the purR gene product was explained by the discovery of a purR binding site in the purHD control region.     

Purification and properties of the membrane-bound CDP-diglyceride synthetase from Escherichia coli

The enzyme CDP-diglyceride synthetase (CTP: phosphatidate cytidylyltransferase; EC 2.7.7.41) has been purified to 90% homogeneity from Escherichia coli cells that overproduce the enzyme 50-fold through the use of recombinant DNA technology. The purification required the use of different detergents at each step, illustrating the refractory hydrophobic nature of this protein. Apparent physical effects of EDTA on the enzyme were also utilized in the purification. The enzyme has an apparent minimum subunit mass of 27,000 daltons, as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The amino acid composition of the protein was determined, and it correlates well with the theoretical protein product of the cds gene, the sequence of which is reported in the accompanying paper (Icho, T., Sparrow, C. P., and Raetz, C. R. H. (1985) J. Biol. Chem. 260, 12078-12083). The pure enzyme displays surface dilution kinetics when assayed in the presence of Triton X-100. As previously suggested on the basis of studies using partially purified preparations, the enzyme mechanism is sequential, and computer-calculated kinetic constants are reported herein. The substrate specificity of the enzyme is also investigated. This is the first time this enzyme has been purified to homogeneity from any source, despite the fact that it is essential for phospholipid biosynthesis in all organisms.     

Molecular cloning and structure of the gene for 7 beta-(4-carboxybutanamido)cephalosporanic acid acylase from a Pseudomonas strain.

A Pseudomonas strain produced an enzyme capable of deacylating 7 beta-(4-carboxybutanamido)cephalosporanic acid to 7-aminocephalosporanic acid in response to glutaric acid. The gene for the enzyme was cloned within the PstI site of pBR325 as a 7.35-kilobase-pair DNA segment from a mutant of this strain whose enzyme is produced constitutively. The gene expression in the primary clone appeared to be low in Escherichia coli but was significantly enhanced by reducing the size of the initial segment coupled with E. coli promoters. Subsequent subcloning resulted in localization of the gene to a 2.45-kilobase-pair fragment. Three clone-specific polypeptides with molecular weights of ca. 16,000, 54,000, and 70,000 were shown by maxicell analysis. The former two corresponded to the small and large subunits of the purified enzyme from the Pseudomonas strain, and the third polypeptide was suggested to be their precursor. This was supported by DNA sequence study together with amino acid sequencing of the amino terminus of both subunits: the sequences for the small and large subunits were localized contiguously in this order on the structural gene without termination codons between them. The nucleotide sequence also disclosed the presence of a signallike sequence preceding that for the small subunit, consistent with the previous observation that the enzyme might be periplasmic in the Pseudomonas strain. Those results suggest a process for the formation of an active enzyme complex from a precursor through two steps of processing.     

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.     

Revised sequence of the tetracycline-resistance gene of pBR322

A revised sequence of the tetracycline-resistance gene of pBR322 is reported. The change, the presence of an additional CG base pair at position 526, adjusts the published sequence to allow an open reading frame from nucleotides 86-1273 (new number) and increases the size of the plasmid to 4363 bp. The predicted polypeptide encoded by this region would contain 396 amino acid residues and have a calculated Mr of 41518. A polypeptide of the predicted size has been reported previously when pBR322 is used as template in the maxicell system.     

Molecular cloning and sequence analysis of complementary DNA encoding rat mammary gland medium-chain S-acyl fatty acid synthetase thio ester hydrolase

Poly(A)+ RNA from pregnant rat mammary glands was size-fractionated by sucrose gradient centrifugation, and fractions enriched in medium-chain S-acyl fatty acid synthetase thio ester hydrolase (MCH) were identified by in vitro translation and immunoprecipitation. A cDNA library was constructed, in pBR322, from enriched poly(A)+ RNA and screened with two oligonucleotide probes deduced from rat MCH amino acid sequence data. Cross-hybridizing clones were isolated and found to contain cDNA inserts ranging from approximately 1100 to 1550 base pairs (bp). A 1550-bp cDNA insert, from clone 43H09, was confirmed to encode MCH by hybrid-select translation/immunoprecipitation studies and by comparison of the amino acid sequence deduced from the DNA sequence of the clone to the amino acid sequence of the MCH peptides. Northern blot analysis revealed the size of the MCH mRNA to be 1500 nucleotides, and it is therefore concluded that the 1550-bp insert (including G X C tails) of clone 43H09 represents a full- or near-full-length copy of the MCH gene. The rat MCH sequence is the first reported sequence of a thioesterase from a mammalian source, but comparison of the deduced amino acid sequences of MCH and the recently published mallard duck medium-chain S-acyl fatty acid synthetase thioesterase reveals significant homology. In particular, a seven amino acid sequence containing the proposed active serine of the duck thioesterase is found to be perfectly conserved in rat MCH.     

Methionyl-tRNA synthetase from Bacillus stearothermophilus: structural and functional identities with the Escherichia coli enzyme.

The metS gene encoding homodimeric methionyl-tRNA synthetase from Bacillus stearothermophilus has been cloned and a 2880 base pair sequence solved. Comparison of the deduced enzyme protomer sequence (Mr 74,355) with that of the E. coli methionyl-tRNA synthetase protomer (Mr 76,124) revealed a relatively low level (32%) of identities, although both enzymes have very similar biochemical properties (Kalogerakos, T., Dessen, P., Fayat, G. and Blanquet, S. (1980) Biochemistry 19, 3712-3723). However, all the sequence patterns whose functional significance have been probed in the case of the E. coli enzyme are found in the thermostable enzyme sequence. In particular, a stretch of 16 amino acids corresponding to the CAU anticodon binding site in the E. coli synthetase structure is highly conserved in the metS sequence. The metS product could be expressed in E. coli and purified. It showed structure-function relationships identical to those of the enzyme extracted from B. stearothermophilus cells. In particular, the patterns of mild proteolysis were the same. Subtilisin converted the native dimer into a fully active monomeric species (62 kDa), while trypsin digestion yielded an inactive form because of an additional cleavage of the 62 kDa polypeptide into two subfragments capable however of remaining firmly associated. The subtilisin cleavage site was mapped on the enzyme polypeptide, and a gene encoding the active monomer was constructed and expressed in E. coli. Finally, trypsin attack was demonstrated to cleave a peptidic bond within the KMSKS sequence common to E. coli and B. stearothermophilus methionyl-tRNA synthetases. This sequence has been shown, in the case of the E. coli enzyme, to have an essential role for the catalysis of methionyl-adenylate formation.