The DNA sequence of argI from Escherichia coli K12.

The argI gene from E. coli K12 has been sequenced. It contains an open reading frame of 1002 bases which encodes a polypeptide of 334 amino acids. Three such polypeptides are required to form the functional catalytic trimer (c3) of ornithine transcarbamoylase (OTCase-1, EC 2.1.3.3). The molecular mass of the mature trimer deduced from the amino acid sequence is 114,465 daltons. An altered form of argI was produced when a 1.6 kilobase DdeI fragment was subcloned into the HincII site of plasmid pUC8 extending the open reading frame an additional 20 nucleotides. It has been previously reported that the amino-terminal region of the respective polypeptides of argI, argF, and pyrB of E. coli possessed significant homology. In contrast, the homologous promoter/operator regions of argI and argF did not appear to share any homologies with pyrB. However, a closer scrutiny of the nucleotide sequence immediately preceding the pyrBI attenuator revealed a remarkable similarity to the argI and argF control region.     

Isolation and characterization of lambda transducing bacteriophages for argF, argI and adjacent genes.

Two genes for ornithinetranscarbamylase exist in strain Escherichia coli K-12, argI, at 85 min, and argF, at 7 min. In an attempt to compare the deoxyribonucleic acid material of these two genes, the lambda transducing phages carrying a portion of the argI region, lambda dvalS argI, lambda pvalS, and lambda dvalS pyrB, and of the argF region, lambda dargF, have been isolated. Their structure, including that of phi 80dargF previously isolated, was studied by the method of heteroduplex mapping. In this paper, the results of this mapping are reported.     

Molecular basis for modulated regulation of gene expression in the arginine regulon of Escherichia coli K-12.

We compare the nucleotide sequences of the regulatory regions of five genes or groups of genes of the arginine regulon of Escherichia coli K-12: argF, argI, argR, the bipolar argECBH operon and the carAB operon. All these regions harbour one or two copies of a conserved 18 bp sequence which appears to constitute the basic arginine operator sequence (ARG box). We discuss the influence of ARG box copy number, degree of dyad symmetry, base composition, and position relative to the cognate promoter site on the derepression-repression ratios of the genes of the regulon. A novel hypothesis, based on structural considerations, is also put forward to account for the absence ot attenuation control.     

Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages.

Among the Enterobacteriaceae, Escherichia coli K-12 is the only strain known to have two structural genes (argF and argI) for ornithine carbamoyltransferase. The two gene products interact to form a family of four functional isoenzymes, respectively designated FFF, FFI, FII, and III. The FFF and III isoenzymes exhibit nearly identical kinetic parameters in the conditions applied. FFF is more thermolabile than III; this allows the straightforward characterization of new transducing phages carrying either argF or argI. The bearing of the available information regarding ornithine carbamoyltransferase isoenzymes on the evolution of the ancestral E. coli chromosome is reconsidered.     

Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB.

The complete nucleotide sequence of argF is presented, together with that of an operator-constitutive mutant. ArgF is compared with the other gene coding for ornithine carbamoyltransferase (OTCase) in E. coli K-12, argI, and with pyrB, encoding the catalytic monomer of aspartate carbamoyltransferase (ATCase). ArgF and argI appear very closely related having emerged from a relatively recent ancestor gene. The relationship between OTCase and ATCase appears more distant. Nevertheless, the homology observed between the two proteins (mainly in the polar domain) suggests a common origin.     

Some regulation profiles of ornithine transcarbamylase synthesis in vitro.

The regulation profiles of OTCase (argF, argI) synthesis in vitro were investigated by using the in vitro system described in the accompanying paper. Addition of 2.6 mM arginine, crude repressor and partially purified repressor to the in vitro system demonstrated that lambdadargF-DNA-directed OTCase-FFF synthesis is more sensitive to the repressor than lambdapargI-DNA-directed OTCase-III synthesis. The effects of some low-molecular substances on FFF and III syntheses were investigated; guanosine 3'-diphosphate 5'-diphosphate (ppGpp) stimulated both syntheses while cAMP and guanosine 5'-tetraphosphate (Gpppp) were not effective on III synthesis and were slightly inhibitory for FFF synthesis. The substances had no effect on the maturation of the enzyme or on the activity of the enzyme, FFF or III, synthesized. We suggest that argF- and argI-genes are regulated in a slightly different fashion and that the operator-promotor regions are not completely identical for these two genes.     

Metabolism of arginine-specific messenger ribonucleic acid in Escherichia coli K-12.

Ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization was employed for the determination of the level of messenger RNA (mRNA) transcribed from seven of the nine genes of the arginine regulon of Escherichia coli K-12. The quantity of RNA complexing with each of the separated DNA strands of the argA, argF, argE, and argCBH operons carried on specialized transducing phages was measured. The derepressed:repressed ratio of mRNA formed in vivo was found to vary between about 3 and 4 when measured by hybridization to DNA isolated from specialized transducing phages carrying the argA, argE, argCBH, argF, and argI operons.     

Binding of the arginine repressor of Escherichia coli K12 to its operator sites.

In the arginine regulon of Escherichia coli K12 each of the eight operator sites consists of two 18-base-pair-long palindromic sequences called ARG boxes. In the operator sites for the structural genes of the regulon the two ARG boxes are separated by three base-pairs, in the regulatory gene argR they are separated by two base-pairs. The hexameric arginine repressor, the product of argR, binds to the two ARG boxes in an operator in the presence of L-arginine. From the results of various kinds of in vitro footprinting experiments with the ARG boxes of argF and argR (DNase I protection, hydroxyl radical, ethylation and methylation interference, methylation protection) it can be concluded that: (1) the repressor binds simultaneously to two adjacent ARG boxes; (2) that it binds on one face of the double helix; and (3) that it forms contacts with the major and minor grooves of each ARG box, but not with the central three base-pairs. The repressor can bind also to a single ARG box, but its affinity is about 100-fold lower than for two ARG boxes. From gel retardation experiments with 3H-labeled repressor and 32P-labeled argF operator DNA, it is concluded that the retarded DNA-protein complex contains no more than one repressor molecule per operator site and that most likely one hexamer binds to two ARG boxes. The bound repressor was shown to induce bending of argF operator DNA. The bending angle calculated from the results of gel retardation experiments is about 70 degrees and the bending center was located within the region encompassing the ARG boxes. The main features that distinguish the arginine repressor from other repressors studied in E. coli are its hexameric nature and the simultaneous binding of one hexameric molecule to two palindromic ARG boxes that are close to each other.     

Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli

We have determined the complete nucleotide sequence of the arcB gene from Pseudomonas aeruginosa strain PAO and we have purified the arcB product, the catabolic ornithine carbamoyltransferase (EC 2.1.3.3), to apparent homogeneity from the same strain. The N-terminal amino acid sequence, the total amino acid composition and the subunit size of the purified enzyme were in agreement with nucleotide sequencing results, which predict a polypeptide of 336 amino acids (Mr 38,108). Crosslinking experiments suggest that the native enzyme (apparent Mr approx. 420,000) basically consists of a trimer aggregating to form nonamers or dodecamers. The arcB gene of P. aeruginosa had strong homology with the argF and argI genes which code for the anabolic ornithine carbamoyltransferase isoenzymes in Escherichia coli; 63% of the nucleotides and 57% of the amino acids were absolutely conserved in arcB and argF. This indicates a close evolutionary relationship between these genes although their products have different physiological functions in the cell. Under conditions of induction (energy depletion) the catabolic ornithine carbamoyltransferase represented greater than or equal to 10% of the total cellular protein. Like other highly expressed Pseudomonas genes, the arcB gene was found not to use seven codons which correspond to minor or weakly interacting tRNA species in E. coli.     

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Sequence diversity within the argF, fbp and recA genes of natural isolates of Neisseria meningitidis: interspecies recombination within the argF gene

Studies of natural populations of Neisseria meningitidis using multilocus enzyme electrophoresis have shown extensive genetic variation within this species, which, it has been proposed, implies a level of sequence diversity within meningococci that is greater than that normally considered as the criterion for species limits in bacteria. To obtain a direct measure of the sequence diversity among meningococci, we obtained the nucleotide sequences of most of the argF, recA and fbp genes of eight meningococci of widely differing electrophoretic type (from the reference collection of Caugant). Sequence variation between the meningococcal strains ranged from 0-0.6% for fbp, 0-1.3% for argF, and 0-3.3% for recA. These levels of diversity are no greater than those found within Escherichia coli 'housekeeping' genes and suggest that multilocus enzyme electrophoresis may overestimate the extent of nucleotide sequence diversity within meningococci. The average sequence divergence between the Neisseria meningitidis strains and N. gonorrhoeae strain FA19 was 1.0% for fbp and 1.6% for recA. The argF gene, although very uniform among the eight meningococcal isolates, had a striking mosaic structure when compared with the gonococcal argF gene: two regions of the gene differed by greater than 13% in nucleotide sequence between meningococci and gonococci, whereas the rest of the gene differed by less than 1.7%. One of the diverged regions was shown to have been introduced from the argF gene of a commensal Neisseria species that is closely related to Neisseria cinerea. The source of the other region was unclear.     

In vitro synthesis of ornithine transcarbamylase.

An in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(lambda) and DNA of a lambda transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the additon of DNA, and was sensitive to chloramphenicol and rifampicin. Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme. This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.     

Ornithine transcarbamylase from Salmonella typhimurium: purification, subunit composition, kinetic analysis, and immunological cross-reactivity.

Ornithine transcarbamylase (OTCase) was purified to hemogeneity from a derepressed strain of Salmonella typhimurium. The optimal pH for enzyme activity is 8.0. The molecular weight of the enzyme was calculated to be 116,000, based on measurements of the sedimentation coefficient by sucrose gradient ultracentrifugation and the Stokes radius by gel filtration. Polyacrylamide gel electrophoresis of cross-linked OTCase in the presence of sodium dodecyl sulfate showed that the enzyme is composed of three identical subunits. The molecular weight of the monomer was determined to be 39,000. Steady-state kinetics indicate that the reaction mechanism is sequential. The limiting Michealis constants for carbamylphosphate and ornithine were determined to be 0.06 and 0.2 mM, respectively. The dissociation constant for carbamylphosphate was 0.02 mM. Product and dead-end inhibition patterns are consistent with an ordered Bi Bi mechanism, in which carbamylphosphate is the first substrate added and phosphate is the last product released. OTCase activity was inhibited by arginine, but relatively high concentrations were required for significant inhibition. The inhibition by arginine might be physiologically significant in the regulation of carbamlphosphate utilization; a single carbamylphosphate synthetase is responsible for the synthesis of carbamylphosphate for both arginine and pyrimidines in S. typhimurium and the inhibition by argine might serve to divert carbamlphosphate to the synthesis of pyrimidines when arginine is present at high concentrations. The crossreaction of OTCases from different microorganisms with purified antibodies raised against the homogeneous OTCase from S. typhimurium was investigated. The results of immunotitration and immunodiffusion experiments revealed a high degree of identity between the enzymes form S. typhimurium and Esherichia coli B and W. In these three cases, a single gen (argl) encodes OTCase. Wild-type E. coli K-12 and strain 3000 X 111, which carry two OTCase genes (argI, argF), also revealed similar cross-reactivity, supporting the hypothesis that argF is the product of a relatively recent duplication. The activity of OTCase from Bacillus subtilis was partially inhibited by antibodies against the enzyme from S. typhimurium, indicating unusual conservation of primary structure among widely different taxonomic groups. OTCase from Saccharomyces cerevisiae, whose molecular weight and primary structure are similar to those of the enzyme from S. typhimurium, was without detectable cross-reactivity.     

Isolation and identification of Fur binding genes in Escherichia coli

Fur (ferric uptake regulation) binding fragments were isolated by in vitro binding of purified Fur protein with Sau3AI-digested genomic DNA fragments. The Fur-bound DNA fragments were filtered on nitrocellulose paper, isolated, cloned, and sequenced. The protein binding was confirmed by gel retardation assay for five DNA fragments. The sequence data were used to identify the genes by comparison with the GenBank data. The proposed Fur binding regions lie on or near the putative promoter regions of marAB (multiple antibiotic resistance), pyrC (dihydroorotase), mreB (mecillinam resistance) and an unidentified gene (ecouw93) near argI and in the middle of the treBC (trehalose permease enzyme II) coding region. The proposed Fur binding sites of the known iron regulating operators including the genes of this work are AAT(pyrimidine) and A(purine)TT. The two conserved sequences are 10 bases apart and palindromic to each other, which might suggest the classical pattern of protein binding toward one side of the DNA in contrast to the concept of the Fur protein wrapping around the DNA.     

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.     

Isolation, cloning and characterization of argF gene DNA from Escherichia coli K-12.

A 1650 base pair (BP) fragment carrying the entire argF structural gene with its associated control regions was isolated from an EcoRI/BamHI digest of phi80argFilambda cI857 DNA. This segment was cloned using the EcoRI and BamHI cleavage sites in the plasmid pBR322. A preliminary restriction map of the argF region was prepared. RNA polymerase binding studies indicated that the argF promoter is located approx. 30 base pairs from the EcoRI terminus of the cloned DNA segment.     

Purification and characterization of an arginine regulatory protein, ArgR, from Pseudomonas aeruginosa and its interactions with the control regions for the car, argF, and aru operons.

Pseudomonas aeruginosa ArgR, a regulatory protein that plays a major role in the control of certain biosynthetic and catabolic arginine genes, was purified to homogeneity. ArgR was shown to be a dimer of two equal subunits, each with a molecular mass of 37,000 Da. Determination of the amino-terminal amino acid sequence showed it to be identical to that predicted from the derived sequence for the argR gene. DNase I footprinting showed that ArgR protects a region of 45 to 47 bp that overlaps the promoters for the biosynthetic car and argF operons, indicating that ArgR exerts its negative control on the expression of these operons by steric hindrance. Studies were also carried out with the aru operon, which encodes enzymes of the catabolic arginine succinyl-transferase pathway. Quantitative S1 nuclease experiments showed that expression of the first gene in this operon, aruC, is initiated from an arginine-inducible promoter. Studies with an aruC::lacZ fusion showed that this promoter is under the control of ArgR. DNase I experiments indicated that ArgR protects two 45-bp binding sites upstream of aruC; the 3' terminus for the downstream binding site overlaps the -35 region for the identified promoter. Gel retardation experiments yielded apparent dissociation constants of 2.5 x 10(-11), 4.2 x 10(-12), and 7.2 x 10(-11) M for carA, argF, and aruC operators, respectively. Premethylation interference and depurination experiments with the car and argF operators identified a common sequence, 5'-TGTCGC-3', which may be important for ArgR binding. Alignment of ArgR binding sites reveals that the ArgR binding site consists of two half-sites, in a direct repeat arrangement, with the consensus sequence TGTCGCN8AAN5.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.