Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.     

Mutagenesis of the folC gene encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli

The folC gene of Escherichia coli, cloned in a pUC19 vector, was mutagenized by progressive deletions from both the 5' and the 3' ends and by TAB linker insertion. A number of 5'-deleted genes, which had the initiator ATG codon removed, produced a truncated gene product, in reduced amounts, from a secondary initiation site. The most likely position of this site at a GTG codon located 35 codons downstream of the normal start site. This product could complement the folC mutation in E. coli strain SF4 as well as a strain deleted in the folC gene. The specific activity of extracts of the mutant enzyme are 4-16% that of the wild type enzyme for the folylpolyglutamate synthetase activity and 6-19% for the dihydrofolate synthetase activity. The relative amount of protein expressed by the mutant, compared to the wild type, in maxicells was comparable to the relative specific activity, suggesting that the kcat of the mutant enzyme is similar to that of the wild type. Mutants with up to 14 amino acids deleted from the carboxy terminal could still complement the folC deletion mutant. Seven out of ten linker insertions dispersed through the coding region of the gene showed complementation of the folC mutation in strain SF4 but none of these insertion mutants were able to complement the strain containing a deleted folC gene. None of the carboxy terminal or linker insertion mutants had a specific activity greater than 0.5% that of the wild type enzyme. The dihydrofolate synthetase and folylpolyglutamate synthetase activities behaved similarly in all mutants, both retaining a large fraction of the wild type activity in the amino terminal deletions and both being very low in the carboxy terminal deletions and linker insertion mutants. These studies are consistent with a single catalytic site for the two activities catalyzed by this enzyme.     

Folylpoly-gamma-glutamate synthetase-dihydrofolate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product

The Escherichia coli gene for folylpolyglutamate synthetase-dihydrofolate synthetase was localized to plasmids pLC22-45, 24-31, and 28-44 of the Clarke-Carbon E. coli colony bank (Clarke, L., and Carbon, J. (1976) Cell 9, 91-99) by screening the bank by replica mating with an E. coli folC mutant. The folC gene was subcloned from pLC22-45 and inserted into a high copy number plasmid containing the lambda replication control region under the control of the temperature-sensitive cI857 repressor and into a high expression plasmid containing the lambda PL promoter and the cI857 repressor. The folC structural gene was located on a 1.52-kilobase PvuI fragment, sufficient to code for a protein of maximum Mr 55,000. E. coli transformants containing the recombinant plasmids, when induced by culturing at 42 degrees C, had folylpolyglutamate synthetase and dihydrofolate synthetase levels that were 100- to 400-fold higher than in wild type strains and which represented up to 4% of the soluble cell protein. The E. coli folylpolyglutamate synthetase-dihydrofolate synthetase has been purified to homogeneity from the transformants. Both activities are catalyzed by a single protein of Mr 47,000. Some kinetic properties of the enzymes and a new spectrophotometric method for assaying dihydrofolate synthetase activity are described.     

Molecular characterization of Lactobacillus plantarum genes for beta-ketoacyl-acyl carrier protein synthase III (fabH) and acetyl coenzyme A carboxylase (accBCDA), which are essential for fatty acid biosynthesis

Genes for subunits of acetyl coenzyme A carboxylase (ACC), which is the enzyme that catalyzes the first step in the synthesis of fatty acids in Lactobacillus plantarum L137, were cloned and characterized. We identified six potential open reading frames, namely, manB, fabH, accB, accC, accD, and accA, in that order. Nucleotide sequence analysis suggested that fabH encoded beta-ketoacyl-acyl carrier protein synthase III, that the accB, accC, accD, and accA genes encoded biotin carboxyl carrier protein, biotin carboxylase, and the beta and alpha subunits of carboxyltransferase, respectively, and that these genes were clustered. The organization of acc genes was different from that reported for Escherichia coli, for Bacillus subtilis, and for Pseudomonas aeruginosa. E. coli accB and accD mutations were complemented by the L. plantarum accB and accD genes, respectively. The predicted products of all five genes were confirmed by using the T7 expression system in E. coli. The gene product of accB was biotinylated in E. coli. Northern and primer extension analyses demonstrated that the five genes in L. plantarum were regulated polycistronically in an acc operon.     

Organization and nucleotide sequences of the genes encoding the biotin carboxyl carrier protein and biotin carboxylase protein of Pseudomonas aeruginosa acetyl coenzyme A carboxylase.

The genetic organization of the Pseudomonas aeruginosa acetyl coenzyme A carboxylase (ACC) was investigated by cloning and characterizing a P. aeruginosa DNA fragment that complements an Escherichia coli strain with a conditional lethal mutation affecting the biotin carboxyl carrier protein (BCCP) subunit of ACC. DNA sequencing and RNA blot hybridization studies indicated that the P. aeruginosa accB (fabE) homolog, which encodes BCCP, is part of a 2-gene operon that includes accC (fabG), the structural gene for the biotin carboxylase subunit of ACC. P. aeruginosa homologs of the E. coli accA and accD, encoding the alpha and beta subunits of the ACC carboxyltransferase, were identified by hybridization of P. aeruginosa genomic DNA with the E. coli accA and accD. Data are presented which suggest that P. aeruginosa accA and accD homologs are not located either immediately upstream or downstream of the P. aeruginosa accBC operon. In contrast to E. coli, where BCCP is the only biotinylated protein, P. aeruginosa was found to contain at least three biotinylated proteins.     

Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae

Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.     

Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy

In some bacteria, such as Escherichia coli, the addition of L-glutamate to dihydropteroate (dihydrofolate synthetase activity) and the subsequent additions of L-glutamate to tetrahydrofolate (folylpolyglutamate synthetase (FPGS) activity) are catalyzed by the same enzyme, FolC. The crystal structure of E. coli FolC is described in this paper. It showed strong similarities to that of the FPGS enzyme of Lactobacillus casei within the ATP binding site and the catalytic site, as do all other members of the Mur synthethase superfamily. FolC structure revealed an unexpected dihydropteroate binding site very different from the folate site identified previously in the FPGS structure. The relevance of this site is exemplified by the presence of phosphorylated dihydropteroate, a reaction intermediate in the DHFS reaction. L. casei FPGS is considered a relevant model for human FPGS. As such, the presence of a folate binding site in E. coli FolC, which is different from the one seen in FPGS enzymes, provides avenues for the design of specific inhibitors of this enzyme in antimicrobial therapy.     

Cloning, heterologous expression, and sequencing of the Proteus vulgaris glnAntrBC operon and implications of nitrogen control on heterologous urease expression

Abstract The glnAntrBC operon of Proteus vulgaris was cloned and heterologously expressed in Escherichia coli . The nucleotide sequence was determined. An open reading frame of 1407 bp was identified as the glnA gene and the deduced amino acid sequence showed 82% identity with the E. coli glutamine synthetase protein. Heterologous expression of the glnA gene in E. coli restored glutamine synthetase (GS) activity in a GS-negative mutant and a 52 kDa protein was detected and addressed as the GS subunit of P. vulgaris . Adjacent to the glnA gene the regulatory genes ntrB and ntrC were identified. Their coding regions comprised 1053 and 1452 bp, respectively, and the deduced gene products NR_II (NtrB) and NR_I (NtrC) shared 72% identity with the corresponding E. coli proteins. Heterologous expression in E. coli revealed only a 54 kDa protein which was shown to be NR_I. NR_II was not detectable using the methods employed.     

Characterization of a mutation affecting the function of Escherichia coli folylpolyglutamate synthetase-dihydrofolate synthetase and further mutations produced in vitro at the same locus

The folC gene from mutant strain SF4 was cloned into a pUC19 plasmid. Expression of the mutant gene from the lac promoter of the plasmid complemented the auxotrophy for methionine of the SF4 strain. The only difference in sequence between the mutant and wild-type genes was a G925A base change resulting in an A309T amino acid change. The mutant enzyme had a 30-fold higher Km for 10-formyltetrahydrofolate as well as a 60-fold higher Km for glutamate and a 200-fold higher Km for dihydropteroate of the dihydrofolate synthetase activity. Site-specific mutagenesis was used to substitute other amino acids at codon 309. Mutants with glycine, isoleucine, and valine substitutions at this position, when expressed from multicopy plasmids, complemented the SF4 strain. The glycine mutant had properties similar to the wild-type enzyme, whereas the isoleucine and valine mutants had properties similar to the threonine mutant, SF4. Mutant genes with arginine, glutamate, and leucine substitutions, which did not complement the SF4 strain, could complement a folC deletion strain, but produced smaller colonies on complex plates and did not grow on minimal medium. In the deletion strain, an increasing requirement for folate product supplements was observed as the folylpolyglutamate synthetase-dihydrofolate synthetase activities of the complementing mutants decreased.     

Regulation of nitrogenase activity in Anabaena variabilis by modification of the Fe protein

The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329. A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment. The nucleotide sequence of a 1.8 kb segment encoding the GS was determined. This segment showed an open reading frame which would encode a polypeptide of 444 amino acids. The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E. coli GS.     

Genetic evidence for a repressor of synthesis of cytosine deaminase and purine biosynthesis enzymes in Escherichia coli.

Addition of purines to the growth medium of Escherichia coli represses synthesis of cytosine deaminase (codA) and enzymes of purine de novo synthesis. After Tn10 mutagenesis, mutants displaying derepressed levels of cytosine deaminase in the presence of hypoxanthine were isolated. One of these had simultaneously acquired resistance to the hypoxanthine analog 6-mercaptopurine. The mutation purR6::Tn10 was shown to affect de novo synthesis of the purine enzymes glutamine phosphoribosylpyrophosphate amidotransferase (purF) and phosphoribosyl glycinamide synthetase (purD). The mutation was mapped by P1 transduction at 36 min on the E. coli linkage map. A plasmid containing the purR region was obtained by complementation of the purR6::Tn10 mutation. By comparing the restriction maps of the cloned fragment and the E. coli chromosome, the purR gene was found to be located very close to the lpp gene (36.3 min).     

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.     

Sequence and functional analysis of the cloned Neisseria meningitidis CMP-NeuNAc synthetase

The CMP-N-acetylneuraminic acid (CMP-NeuNAc) synthetase gene of Neisseria meningitidis group B is located on a 2.3-kb EcoRI fragment within the cps gene cluster. Nucleotide sequence determination of the gene encoding the CMP-NeuNAc synthetase revealed a 515-bp open reading frame that can encode a 18.9-kDA protein. A computer data base scan revealed a 59.4% identity to the CMP-NeuNAc synthetase gene of E. coli K1. Enzymatic activity was confirmed in vitro and in vivo. Transformation of the CMP-NeuNAc defective E. coli K1 strain EV5 with the meningococcal CMP-NeuNAc synthetase could complement the defect in E. coli.     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

Detection of elastase production in Escherichia coli with the elastase structural gene from several non-elastase-producing strains of Pseudomonas aeruginosa.

The elastase structural gene from Pseudomonas aeruginosa IFO 3455 has been cloned and sequenced. Using this gene as a probe, we cloned the DNA fragments (pEL3080R, pEL10, and pEL103R) of the elastase gene from non-elastase-producing strains (P. aeruginosa IFO 3080, N-10, and PA103 respectively). These three Pseudomonas strains showed no detectable levels of elastase antigenicity by Western blotting (immunoblotting) or by elastase activity. When elastase structural genes about 8 kb in length were cloned into pUC18, an Escherichia coli expression vector, we were able to detect both elastase antigenicity and elastolytic activity in two bacterial clones (E. coli pEL10 and E. coli pEL103R). However, neither elastolytic activity nor elastase antigenicity was detected in the E. coli pEL3080R clone, although elastase mRNA was observed. The partial restriction map determined with several restriction enzymes of these three structural genes corresponded to that of P. aeruginosa IFO 3455. We sequenced the three DNA segments of the elastase gene from non-elastase-producing strains and compared the sequences with those from the elastase-producing P. aeruginosa strains IFO 3455 and PAO1. In P. aeruginosa N-10 and PA103, the sequences were almost identical to those from elastase-producing strains, except for several nucleotide differences. These minor differences may reflect a microheterogeneity of the elastase gene. These results suggest that two of the non-elastase-producing strains have the normal elastase structural gene and that elastase production is repressed by regulation of this gene expression in P. aeruginosa. Possible reasons for the lack of expression in these two strains are offered in this paper. In P. aeruginosa IFO 3080, the sequence had a 1-base deletion in the coding region, which should have caused a frameshift variation in the amino acid sequence. At present, we have no explanation for the abnormal posttransciptional behavior of this strain.