Biosynthesis of Arginine and Polyamines
Daniel Charlier 1 and Nicolas Glansdorff 2*
[SECTION EDITOR, GEORGES COHEN]
Posted September 9, 2004
1Erfelijkheidsleer en Microbiologie (MICR), Vrije Universiteit Brussel, Pleinlaan, 2, B-1050 Brussels, Belgium
1Phone: +32-2-6291342, Fax: +32-2-6291345, E-mail:
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2Corresponding author: J. M. Wiame Institute for Microbiological Research, 1, ave E. Gryzon, B-1070 Brussels, Belgium
2Phone: +32-2-5267275, Fax: +32-2-5267273, E-mail:
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In their historical article published in one of the first issues of the Journal of Molecular Biology, F. Jacob and J. Monod ( 210) brought forth two concepts which were to remain cornerstones of molecular biology: the messenger RNA (mRNA) and the operon. This breakthrough was largely based on studies of lactose catabolism and phage development, but the paper already emphasized two systems which were going to become paradigms in their own right: the biosyntheses of the amino acids tryptophan and arginine. While tryptophan biosynthesis would soon epitomize the concept of operon in biosynthetic metabolism and later lead to the discovery of attenuation control, the biosynthesis of arginine was considered at the outset as a model system to study control of gene expression at a higher level of complexity. Indeed, the arginine genes are not clustered on the chromosome in a single functional unit ( 18, 33, 319; Fig. 1). Furthermore, the occurrence of a precursor common to the biosyntheses of arginine and the pyrimidines—carbamoylphosphate—raised very early a question of basic physiological interest: which metabolic controls were coordinating the production of this substance? Another important metabolic interconnection was disclosed with the discovery that arginine and ornithine are precursors in the biosynthesis of polyamines.
Early investigations on arginine biosynthesis brought to light basic features of metabolic regulation. Interpreting a series of experiments suggesting that the synthesis of N-acetylornithinase was antagonized by arginine, Vogel ( 529) proposed the use of the term "repression" for a "relative decrease resulting from the exposure of cells to a given substance, in the rate of synthesis of a particular apoenzyme," a phenomenon for which a few examples had already been reported at the time ( 529). Experiments on the kinetics of ornithine carbamoyltransferase (OTCase) 1 formation in batch cultures and in chemostats ( 160), in bradytrophic mutants, and in cells grown in arginine-free rich medium ( 368) established that not only added arginine but also endogenously produced amino acid sets the pace of enzyme synthesis in the pathway. The concept of "repressor" as a regulatory molecule mediating the phenomenon of repression, however, was to emerge from studies on lactose metabolism and prophage control ( 210) before being extended to biosynthetic metabolism.
The first indications concerning the mechanism involved in repression of enzyme synthesis in the arginine pathway came with the isolation of derepressed ( argR) regulatory mutants in Escherichia coli ( 157, 300). The dominance of the argR + allele in transient merozygotes and stable diploids ( 304, 306) suggested that the argR product was an aporepressor, in keeping with the conclusions already drawn for the lac and trp genes from similar conjugation experiments ( 94, 379). As the arginine genes were scattered and their expression not strictly coordinated (see Table 2), Maas ( 300) and Gorini et al. ( 157) proposed that argR coded for a unique aporepressor interacting with a family of slightly dissimilar operators. For such physiological entities consisting of scattered genes controlled by the same regulatory molecule, Maas and Clark ( 304) coined the word "regulon."
Table 2Repression response in the arginine regulon of E. coli K-12. |
The research of the next 40 years has borne out this model, which can now be drawn in considerable detail. When methods bypassing the problems posed by the scattering of the genes in several functional units and the absence of suitable attachment sites for the construction of transducing phages were elaborated, the isolation of specific operator and promoter mutants became possible, and the way was paved toward the isolation of individual genes. These developments provided the tools for the demonstration in vivo and in vitro that regulation operates essentially at the level of gene transcription (see Level of Control and Nature of the Corepressor, below). The advent of cloning and sequencing methods led to the characterization of the arginine operators and of the repressor itself. By a curious twist the results bearing out this "classical" type of mechanism came as a surprise to many, since there was no evidence for regulation by attenuation, in contrast with the conclusions reached in the meantime for many other amino acid pathways ( 562). It has been speculated that stretches of adjacent arginine codons might constitute an obstacle for efficient ribosome translocation because of the intrinsic instability of codon-anticodon interactions involving inosine in the anticodon or for other reasons linked to the usage of arginine codons ( 103). Moreover, despite the existence of RNA aptamers able to recognize individual molecules of arginine or arginine-rich peptides ( 142, 317), no arginine-specific "riboswitch" control mechanism has been reported, in contrast with the involvement of the chemically related amino acid lysine in such regulatory mechanisms ( 427, 528).
Several findings of general interest were disclosed by studying the arginine regulon: (i) the discovery of informational suppression by streptomycin ( 159); (ii) one of the two first cases of divergent operons expressed from two promoters facing each other over an internal control region ( 127, 213); (iii) the occurrence of gene duplication mechanisms reactivating silent genes by the formation of tandem or inverted repeats ( 24, 75, 84) and a particularly striking instance of gene amplification ( 93, 221, 222); (iv) the first unambiguous demonstration that a transposon (IS 3) may carry an outward promoter ( 81); (v) the organization of the control region of the carbamoylphosphate synthase operon ( carAB) in tandem promoters that are respectively controlled by arginine and the pyrimidines, a striking example of differential control of the same genes by different metabolites ( 40, 403); (vi) the discovery of a cryptic, inducible acetylornithine transaminase which later turned out to operate in a degradative pathway also operating in other bacteria ( 452) and provided the first indication ever that a repressor could also be involved in activation of gene expression ( 19); (vii) the occurrence in E. coli K-12 of a duplicate ornithine carbamoyltransferase (OTCase) gene, argF, one of the very first cases of laterally acquired genes ever described ( 149, 154, 520); and (viii) the homology relating argF and argI to the structural gene ( pyrB) for the catalytic subunit of aspartate carbamoyltransferase (ATCase), one of the most elegant cases in support of the theory of enzyme recruitment ( 201, 522).
The most significant advances of the last 10 to 15 years concern the arginine repressor, its structure and mode of action in both E. coli and Salmonella typhimurium (official name, Salmonella enterica serovar Typhimurium), the sequence analysis of all arg structural genes in E. coli and Salmonella, the resulting evolutionary inferences (see the sections describing specific enzymes), and the dual regulation of the carAB operon. A most exciting discovery was the joint involvement of regulatory molecules such as the argR (or xerA) and the carP (or xerB, pepA) gene products in a totally unrelated cellular process: the site-specific recombination that resolves ColE1 dimers into monomers (see Arginine Repressor and Operator-Repressor Interactions, and The carAB Operon, a Dual-Control System, below). Significant advances have also been made concerning arginine transport and its regulation, which is mostly distinct from ArgR-mediated repression. Reviews on arginine metabolism in prokaryotes in general ( 101, 106, 176) and in Bacillus subtilis in particular ( 22) have been published.
This section provides an overall picture of the pathways, their interconnections, the regulatory circuits involved, and the resulting interferences between them. At the end of this section we draw attention to observations which disclose metabolic interconnections that either were not obvious at first sight or have remained as yet unexplained.
Arginine biosynthesis proceeds in eight steps, according to the scheme depicted in Fig. la, starting from glutamate. The first four intermediates are acetylated ( 532). Except for N-acetylornithine, they penetrate the membrane only weakly (mutants more permeable to N-acetylglutamate than the wild type have been repeatedly described). This N-acetylation prevents the cyclization which, in the proline pathway, leads from glutamate- γ-semialdehyde to Δ1-pyrroline carboxylate. In Enterobacteriaceae, in a few other bacteria, and in Sulfolobus solfataricus ( 440, 442, 444, 515, 556), N-acetylornithine is deacylated by an acetylornithinase, whereas in other bacteria and in fungi, the acetyl group is recycled on glutamate by an ornithine-glutamate acetyltransferase, an enzyme discovered in Micrococcus glutamicus by Udaka and Kinoshita ( 511). In some organisms, this acetyltransferase (encoded by gene argJ) displays both acetylglutamate synthetase and ornithine acetyltransferase activity ( 440, 442) despite the apparent lack of homology between argA and argJ; in others it remains monofunctional (see reference 444 and N-Acetylglutamate Synthetase, below). From ornithine onward, the intermediates of the pathway appear to be common to all organisms investigated (however, see OTCase, below, for the intriguing situation occurring in Bacteroides and Xylella). Ornithine and citrulline are taken up easily, but argininosuccinate rather poorly.
Two strategies of feedback inhibition, by which arginine (or ornithine [ 440]) regulates the flow of metabolic intermediates through the pathway, correspond to the alternative fates of the acetyl group. In organisms with an acetyltransferase, N-acetyl-glutamokinase or the acetyltransferase itself is inhibited ( 115, 440, 509, 515); in some of these organisms, N-acetylglutamate synthetase is inhibited as well ( 175). In contrast, in members of the Enterobacteriaceae, N-acetylglutamate synthetase alone appears to be the target enzyme ( 538).
The synthesis of the eight enzymes is repressed by arginine to various extents in E. coli ( 157, 300, 538) and in S. enterica serovar Typhimurium ( 4, 141, 239). A single regulatory gene ( argR) accounts for this repression in both organisms ( 4, 141, 157, 239, 300). We will compare the enteric argR with its Bacillus homologue in detail below.
Carbamoylphosphate is a precursor common to arginine and the pyrimidines. In both E. coli and S. enterica serovar Typhimurium, it is produced by a single synthetase, carbamoylphosphate synthetase (CPSase), with glutamine as the physiological amino group donor ( 2, 393, 396). This situation contrasts with the existence of separate enzymes specific for arginine and pyrimidine biosynthesis in Bacillus subtilis and fungi ( 22, 101, 106, 112, 113). The enzyme is inhibited by UMP; this inhibition is antagonized by ornithine and IMP ( 2, 14, 390). Since arginine controls the formation of ornithine through feedback inhibition of N-acetylglutamate synthetase, the antagonistic effects of UMP and ornithine ensure a balanced distribution of carbamoylphosphate between the tributory pathways. In both organisms, CPSase is cumulatively repressed by arginine and the pyrimidines ( 2, 393, 396). The arginine repressor is involved in this control in both E. coli and S. enterica serovar Typhimurium ( 5, 239, 394).
Pyrimidines interfere with the regulation of the arginine pathway at the level of carbamoylphosphate utilization. The growth of E. coli Pyr mutants blocked after the ATCase step in a chemostat limited by uracil results in a marked derepression of the arginine pathway ( 150); this effect is not seen in pyrB mutants (lacking ATCase). A similar effect has been observed in both E. coli and S. enterica serovar Typhimurium: pyrimidine bradytrophic mutants are derepressed for both the pyrimidine and the arginine pathways ( 239; A. Piérard and N. Glansdorff, unpublished data). Conversely, adding uracil to wild-type cells growing in minimal medium results in partial derepression of arginine biosynthetic enzymes, presumably because a slightly excessive inhibition of CPSase by UMP diminishes the supply of carbamoylphosphate for arginine biosynthesis ( 158). Arginine represses the synthesis of ATCase about twofold, even in an argR nonsense mutant. This is probably due to diversion of excess carbamoylphosphate toward the pyrimidine pathway when the pool of ornithine is low ( 392). Purines also exert a certain repression on CPSase synthesis (see The carAB Operon, a Dual-Control System, below).
An interesting type of indirect suppression may result from an interaction between the arginine and proline pathways ( 209, 263): proAB auxotrophs of E. coli and S. enterica serovar Typhimurium blocked in the conversion of glutamate into glutamate-γ γ-semialdehyde can revert to the Pro + phenotype by the presence of mutations inactivating the argD gene, encoding N-acetylornithine- δ-transaminase. argD mutants display a leaky arginineless phenotype, probably because another transaminase takes over the missing function. When such cells are grown without extraneous arginine, the feedback inhibition of N-acetylglutamate synthetase is lifted and the whole pathway is derepressed. Under those circumstances, enough N-acetylglutamate- γ-semialdehyde is produced and deacylated by the relatively nonspecific N-acetylornithinase to feed the proline pathway. In the presence of arginine, this indirect suppression is abolished and the cells return to the Pro – phenotype. Several authors (see N-Acetylglutamate Synthetase, below) have taken advantage of this effect of arginine on argD proAB mutants to isolate derepressed mutants or strains in which N-acetylglutamate synthetase has become resistant to arginine.
An interesting discovery brought to light that, in E. coli, N-acetylornithine transaminase—the enzyme coded for by argD—appears identical with N-succinyl- l,l-diaminopimelate:alpha-ketoglutarate aminotransferase ( dapC), an enzyme of lysine biosynthesis ( 274). The full metabolic and evolutionary implications of this finding remain to be investigated. For homology relationships between enzymes of arginine and lysine biosynthesis in other organisms, see the sections below on N-Acetylglutamylphosphate Reductase, N-Acetylornithine-δ-Transaminase, and N-Acetylornithinase.
Polyamine biosynthesis has been particularly well studied in E. coli, and the cognate genes have been identified in the Salmonella genome as well, including those involved in transport functions ( Fig. 1b). Detailed information is also available for Pseudomonas aeruginosa and Bacillus subtilis ( 355, 356, 455, 456, 457). In E. coli, the diamine putrescine can be made either directly by decarboxylation of ornithine or indirectly by decarboxylation of arginine into agmatine, followed by hydrolysis of agmatine into putrescine and urea by an agmatine ureohydrolase ( 349, 350). Urea is not degraded by E. coli and is actually an indication of the flux through the agmatine pathway ( 347). Putrescine and an aminopropyl group from enzymatically decarboxylated S-adenosylmethionine give rise to the triamine spermidine. The tetramine spermine is normally not found in E. coli. Cadaverine is produced by decarboxylation of lysine. Cadaverine and decarboxylated S-adenosylmethionine give rise to N-3-aminopropyl-1,5-diaminopentane.
Each of the amino acids arginine and ornithine is a substrate for two kinds of decarboxylases ( 483) (see below). In each case, the so-called biosynthetic and "constitutive" decarboxylase is the only one to be found in cells grown at physiological pH in minimal medium. At low pH, in anaerobiosis, at high substrate concentration, and in rich media, distinct biodegradative decarboxylases are induced. These enzymes were shown to constitute a defense mechanism against acidity; since the original observation that a mutant lacking arginine decarboxylase is unable to grow at low pH values (E. F. Becker, Jr., Fed. Proc. 26:812, 1967), much more information has become available on this topic (see Polyamine Biosynthesis and Transport, below). Lysine decarboxylase had originally been considered to be exclusively an inducible enzyme, but evidence for another lysine decarboxylase activity, produced under normal growth conditions, has been published ( 246, 289, 542, 558). Many strains of E. coli appear to lack the biodegradative ornithine decarboxylase ( 15).
In the presence of a concentration of arginine high enough to inhibit ornithine formation, putrescine is made via agmatine exclusively, whereas in unsupplemented medium, the route from ornithine is preferred ( 348). In minimal medium, the amount of putrescine and spermidine is equivalent to 25 to 30% of the arginine in the cell protein ( 487).
Controlling arginine biosynthesis in Enterobacteriaceae entails some interesting effects which were by no means easy to predict. A remarkable chain of events indeed takes place when arginine is added to a growing culture ( 128, 241). Arginine curtails the synthetic flow of ornithine, which is itself a precursor of hydroxamate siderophores; a temporary iron deprivation ensues which causes incomplete methylthiolation of the isopentenyladenosine residue in tRNA; this raises the level of expression in aromatic amino acid biosynthesis by relieving attenuation control of gene expression; this leads to an enhanced and compensatory synthesis of the high-affinity iron-chelator enterochelin. Therefore, addition of arginine to a growing culture—and not only deprivation of this amino acid—may be regarded as a metabolic stress as well!
Another interesting aspect is the growth inhibition which results from the accumulation of ornithine and possibly acetylornithine under certain circumstances ( 98). The basis of this effect has not yet been elucidated, but it has been used for the forward selection of arginine auxotrophs blocked at early steps of the pathway ( 98). Polyamines have been reported to counteract accumulation of ornithine by a mechanism that is as yet unexplained ( 62). Arginine was found to stimulate the growth of hemA mutants; derivatives in which this stimulatory effect is abolished appear identical to previously described alu mutants defective for uptake of exogenous 5-aminolevulinic acid, a precursor in tetrapyrrole synthesis ( 524). This phenomenon also remains unexplained. However, in P. aeruginosa hemA transcription is regulated among others by the anaerobic regulator ANR ( 258), for which ArgR can act as a helper ( 298). As E. coli ArgR can also act as a coactivator, it is tempting to suggest that the stimulatory effect of arginine on growth of an E. coli hemA mutant could possibly be due to arginine/ArgR-mediated activation of transcription of a leaky mutant hemA allele (P. Cornelis, personal communication).
This section summarizes what we know about the enzymes involved in the arginine pathway of E. coli and S. enterica serovar Typhimurium; homologous genes were identified in both organisms, except argF (encoding a supplementary OTCase), which is lacking in Salmonella. In several instances, our understanding of enzymology and related observations on metabolite flow have been exploited to select mutants affected in the regulation of enzyme activity or enzyme synthesis. The rationale for these selection procedures is also presented in this section. Though this module should not be considered as an exhaustive comparative review of arginine biosynthesis in the microbial world, some salient (or curious) observations will be reported, which were as yet unknown when the last extensive survey of this kind was published ( 106).
Table 1 gives the common and systematic names of each arginine biosynthetic enzyme and the reaction it catalyzes. The symbols and map positions of the corresponding gene(s) are reported in Fig. la. All arginine genes had been sequenced before the complete sequences of the E. coli and Salmonella genomes were reported; the original references can be found below.
Table 1Enzymes of arginine and polyamine biosynthesis. |
Using resting cells, Vyas and Maas ( 538) showed that N-acetylglutamate synthetase was inhibited by arginine. After Haas et al. ( 175) found that glycerol stabilized the enzyme in extracts of P. aeruginosa, Leisinger and Haas ( 281) and Marvil and Leisinger ( 312) were able to examine the properties of the E. coli synthetase. The enzyme consists of a single type of subunit of molecular weight (MW) of 50,000, but the actual MW depends on the protein concentration; arginine and N-acetylglutamate stabilize or induce the formation of a hexameric form. Fifty percent inhibition is achieved with 0.02 mM l-arginine. N-Acetylglutamate synthetase from S.enterica serovar Typhimurium is also very sensitive to arginine ( 4). The analog O-( l-norvalyl-5)-isourea is as effective an inhibitor as arginine. Indospicin inhibits the enzyme to some extent.
Ennis and Gorini ( 129) showed that feedback inhibition efficiently controlled the flux of arginine precursors only when the level of arginine biosynthetic enzymes was kept low, a status that is normally achieved by repression in E. coli K-12 or W and is an intrinsic property of E. coli B (see The E. coli B Paradox, below). Derepressed ( argR) mutants excrete arginine.
Desensitized argA mutants could be selected owing to the growth-inhibitory effect exerted by arginine on argD pro double mutants incubated in minimal medium without proline supplement (see Arginine and Polyamine Biosynthesis: Outline, Metabolic Interferences, and Interconnections ). This inhibition is due to repression of the first five enzymes of the pathway and to feedback inhibition of N-acetylglutamate synthetase. When applied in two steps, the selection first gives slow-growing argR mutants ( 209, 239), from which feedback-insensitive mutants can be obtained by further selection and screening for colonies able to excrete proline even in the presence of arginine ( 126). Alternatively, desensitized mutants can be detected directly in a population of mutagenized argD pro argR mutants as proline excreters ( 126).
Malamy and coworkers ( 415) have implemented a genetic strategy combining a plasmid bearing feedback-resistance-conferring argA mutations and several arginine operators (thus titrating the repressor) with plasmids overexpressing the argI and carAB genes to construct strains capable of vigorous arginine production.
Curiously, the argA sequence ( 53) shows no significant similarity to that of the argJ gene from Neisseria gonorrhoeae ( 311) and Bacillus stearothermophilus ( 440), which however encodes a protein with both acetylglutamate synthetase and ornithine-glutamate acetyltransferase activity. It would therefore appear that the ArgA and ArgJ proteins belong to different evolutionary families ( 440, 444). Another surprising observation is the weak similarity between E. coli ArgA and the cognate fungal enzyme ( 1, 444). Interesting similarities were noted between the N-terminal parts of bacterial ArgA and ArgB (suggesting an evolutionary relationship between these consecutive enzymes, in keeping with the "retrograde" evolution hypothesis [ 200]), while examination of the C-terminal part of ArgA suggested an ancient evolutionary link with a family of small acetyl coenzyme A (CoA)-dependent acetyltransferases ( 444).
N-Acetylglutamate and ATP are the substrates of N-acetylglutamokinase ( 153, 534). From gel electrophoresis of the proteins synthesized by UV-irradiated cells infected with λd argECBH transducing phages, the molecular mass of the argB product was estimated to be around 29 kDa ( 289), which is in keeping with the value predicted from the argB nucleotide sequence ( 383). The argB gene of B. stearothermophilus is homologous, and the active enzyme was shown to be a dimer ( 440, 443). Similar and thus presumably homologous sequences are being found in a growing number of genomes, including fungal ones ( 37, 383). The labile intermediate, N-acetylglutamylphosphate, has not been isolated in the free state, and it is not known whether the kinase and the subsequent N-acetylglutamylphosphate reductase, which are encoded by adjacent genes in both E. coli and S.enterica serovar Typhimurium, form some kind of complex in vivo. In this respect, it is interesting that the two equivalent proteins in Saccharomyces cerevisiae and Neurospora crassa are produced from a single unit of genetic expression ( 37, 211, 334, 540, and other references in Physiological and Evolutionary Considerations, below). The structures of the enzyme and of its catalytic mechanism have been investigated in considerable detail by the group of V. Rubio ( 147, 309, 416). These studies provide critical information on amino acid kinases in general and also bring to light interesting similarities between acetylglutamate kinase and carbamate kinase.
The enzyme N-acetylglutamylphosphate reductase catalyzes the reduction of N-acetylglutamylphosphate to the corresponding semialdehyde. It has been partially purified ( 535). The molecular mass predicted from the sequence of the cloned gene is 35,911 Da ( 383). Cognate sequences appear throughout the microbial world including fungi ( 20, 37, 191, 383, 440, 443). Curiously, Thermus thermophilus harbours two argC exemplars, rather distantly related ( 20, 255). One of the argC genes is clustered with an OTCase and an ornithine-glutamate acetyltransferase gene with unidentified reading frames, one of them co-regulated with arg genes ( 447). It remains to be determined whether this situation is peculiar to Thermus or whether unsuspected genes involved in the arg regulon also exist in other organisms, perhaps E. coli itself. The second " argC" gene would appear to be involved in the novel pathway for lysine biosynthesis discovered in Thermus (see next section).
N-Acetylornithine- δ-transaminase, the product of the argD gene, catalyzes the formation of N-acetylornithine and α-ketoglutarate from N-acetylglutamate semialdehyde and glutamate ( 513). The same enzyme also exhibits an ornithine- δ-transaminase activity, which probably plays no physiological role ( 30). Surprisingly, this enzyme turned out to be identical with N-succinyl- l,l-diaminopimelate: α-ketoglutarate aminotransferase ( dapC), an enzyme of lysine biosynthesis in E. coli ( 274). As the argD gene is part of the arginine regulon, this dual biosynthetic role raises interesting questions from the metabolic and evolutionary points of view. In T. thermophilus, an argD homologue named lysJ coclusters with an argE homologue named lysK; both N 2-acetylornithine and N 2-acetyllysine are substrates for LysJ. The cognate enzymes have been proposed to function in a novel pathway for lysine biosynthesis that would convert α-aminoadipate into lysine by a series of steps resembling those involved in ornithine biosynthesis ( 338, 339). It seems that the second argC and a homolog of argB identified in Thermus ( 255) and further studied by Nishida et al. ( 366) code for proteins in this novel pathway. It is possible that the same pathway is present in Archaea ( 52).
The transaminase was purified and crystallized ( 31), with the objective of comparing it with the purified product of the argM gene. E. coli W ( 19) and E. coli K-12 (T. Eckhardt, Ph.D. thesis, Eidgenossische Technische Hoschschule, Zurich, Switzerland, 1975) indeed contain a cryptic function, originally ascribed to an argM gene, unlinked to argD, which can be expressed by selecting for suppressors of argD mutants. The active argM enzyme is inducible by arginine, and the comparison of argR – with argR + derivatives of argD argM + strains suggested that induction was mediated by the argR product itself ( 19). The respective MWs of the argM and argD transaminases of E. coli W were originally reported as 61,000 and 119,000, respectively dimers and tetramers of a 31-kDa subunit ( 31), but the identification of the cognate genes (see reference 452) showed these values to be underestimates due to proteolysis in the preparations. The argM protein displayed ornithine- δ-transaminase activity as well ( 30). The data already suggested a common origin for the two genes, at a time when only the sequence of argD was known, and provided the first very early indication that a repressor could also be involved in gene activation ( 186). The inducibility of argM suggested that this gene was a cryptic element of an arginine degradative pathway that was silent in the E. coli strains analyzed at the time. In keeping with this idea, the argM enzyme proved able to transaminate succinylornithine, an intermediate in a newly discovered catabolic pathway responsible for the breakdown of arginine in a number of bacteria including Klebsiella pneumoniae (formerly K. aerogenes MK53), another member of the Enterobacteriaceae ( 517). Our understanding of this curious situation improved considerably with the discovery of a complete arginine catabolic pathway proceeding via succinylated intermediates in E. coli, where argM is now named astC ( 452). The same gene was independently described under the cstC denomination ( 133). The E. coli argD and astC gene products show 58.6% amino acid sequence identity (73.5% similarity). This arginine succinyltransferase pathway is indeed controlled by the argR product, which plays here the role of a transcriptional coactivator. In E. coli, the ast operon is under complex expression control, ArgR being necessary to achieve maximal expression but not essential ( 252). In Salmonella spp., however, ArgR is required for ast expression ( 293).
Genes similar to E. coli argD are widespread in the microbial world. In particular, E. coli argD and the S. cerevisiaecognate biosynthetic gene are homologous ( 182). Interestingly, they are also homologous to the genes encoding ornithine aminotransferase in yeasts and animals ( 186). The origin of this homology can be understood in terms of enzyme recruitment ( 220, 563).
N-Acetylornithinase is characteristic of the so-called linear pathway for arginine biosynthesis (see Arginine and Polyamine Biosynthesis: Outline, Metabolic Interferences, and Interconnections ). A superficially similar reaction has been found in organisms recycling the N-acetyl group, such as Saccharomyces cerevisiae and Thermus sp. strain ZO5; it could be ascribed to a carboxypeptidase (see reference 116 and below). N-Acetylornithinase hydrolyzes N-acetylornithine into ornithine and acetate ( 536). It is dependent on Co 2+ and a thiol compound (preferably glutathione) for maximal activity. The enzyme has been purified and found to be a dimer of 43-kDa subunits; the MW calculated from the gene nucleotide sequence is 42,320 ( 43, 324). A detailed mechanistic analysis of the reaction has been published ( 216).
ArgE appears homologous to a lysine biosynthetic enzyme, succinyldiaminopimelate desuccinylase (the dapE product), which again suggests enzyme recruitment ( 43). lt also seems homologous to carboxypeptidase G2 from Pseudomonas spp. and to an aminoacylase from B. stearothermophilus (see reference 441). It is possible that some of the sequences annotated in bacterial genomes as encoding an "acetylornithinase" may not play this role; a case in point is P. aeruginosa, whose genome contains both a typical argJ gene (coding for an ornithine-glutamate acetyltransferase) and an argE-like sequence ( 472) annotated as argE. A similar situation occurs in the genome of the archaeon Archaeoglobus fulgidus. In Thermus thermophilus, which makes an ornithine-glutamate acetyltransferase (ArgJ), there is also an ArgE homologue, active on acetylornithine and acetyllysine but encoded by a gene of a putative lysine operon (see previous section of this module and references 338 and 339).
E. coli N-acetylornithinase readily deacylates substrates other than N-acetylornithine, including N-acetylglutamate semialdehyde, N-acetylarginine, N-acetyl- and N-formylmethionine, and N-acetylhistidine ( 21, 536). The action of the enzyme on N-acetylglutamate semialdehyde explains the suppression of proAB mutants by argD mutations (see Arginine and Polyamine Biosynthesis: Outline, Metabolic Interferences, and Interconnections ). Several interesting applications derive from the somewhat relaxed specificity of this enzyme. Using mutants resistant to N-acetylnorvaline, Kelker and Maas ( 236) obtained argR + revertants from derepressed mutants and argE auxotrophs from the wild type. Baumberg ( 21) obtained regulatory mutants with mutations in the arginine pathway by selecting for derivatives of his auxotrophs able to utilize N-acetylhistidine in the presence of ornithine and arginine. Sakanyan et al. ( 441) cloned the gene for an aminoacylase of B. stearothermophilus by complementing E. coli argE mutants. This strategy could be used to characterize other enzymes displaying aminoacylase activity.
With the collateral CPSase, OTCase is the best known of the regulon. The reaction is usually observed in the forward direction, by far the more favored one, but arsenolytic cleavage of citrulline can be used to measure the reaction in the reverse direction ( 277).
A peculiarity of E. coli K-12 is that it contains two OTCase genes ( 154), argF and argI, whose products interact to form a family of four trimeric isoenzymes ( 276). The argI gene or its equivalent is the only one to be found in other E. coli strains ( 212) or other members of the Enterobacteriaceae ( 278), S.enterica serovar Typhimurium in particular ( 477). The kinetic parameters of the E. coli argF and argI isoenzymes are very similar, but the argF protein is much more thermolabile ( 278).
The symbol argI has been used later by some authors to designate the gene for arginase, a catabolic enzyme ( 262). To our knowledge this possible source of confusion has not been corrected.
The kinetic properties of the ArgI protein in E. coli W ( 277) and S. enterica serovar Typhimurium ( 7) have been investigated extensively. The reaction displays a preferred sequence of substrate binding: carbamoylphosphate is the first to bind and P i is the last to be released, as in the reaction catalyzed by ATCase ( 406). In contrast to the E. coli isoenzymes, S. enterica serovar Typhimurium OTCase is moderately inhibited (58%) by arginine at relatively high concentrations (5 mM). This inhibition is unlikely to be of physiological importance, in contrast to the situation prevailing in Agrobacterium tumefaciens ( 527). Knight and Jones ( 253) have shown that the OTCase of E. coli W displays a kinetically complex activation by orotate which may be of physiological value in coordinating the flow of metabolites through the arginine and pyrimidine pathways.
An extremely strong and specific inhibition of the reaction is exerted by the bisubstrate analog phosphonoacetylornithine (PALO) ( 387). E. coli is impervious to PALO but not to the oligopeptide Gly-Gly-PALO ( 386, 388), which penetrates via the oligopeptide permease, the product of the opp gene. PALO is then liberated by an intracellular peptidase and inhibits OTCase. opp and, to a certain extent, argR mutants are resistant to Gly-Gly-PALO. These investigations may have therapeutic implications, since they provide an example of "illicit" uptake, by which a substance unable to penetrate the cell membrane does so when it is covalently associated with a pervasive carrier ( 386).
The relatively inefficient reverse reaction (i.e., phosphorolysis of citrulline) can be demonstrated in vivo; strains defective in CPSase grow very slowly on citrulline used as a source of carbamoylphosphate for the pyrimidine pathway ( 462). This property was used to select for mutants with high OTCase specific activity, such as argG bradytrophs (in the presence of citrulline, they accumulate this amino acid and at the same time derepress OTCase) and constitutive argF or argI mutants specifically derepressed by operator mutations or chromosomal rearrangements, some of which turned out to be gene amplifications ( 93, 221, 222, 279). The same strategy was applied to Saccharomyces cerevisiae to obtain cis-dominant constitutive OTCase mutants, the first example of an operator mutant ever isolated in a eukaryote ( 328).
The occurrence of hybrid argF-argI isoenzymes suggested that the constituent polypeptides were the homologous products of a duplication ( 276). Heteroduplex analysis of λ argF and λ argI transducing phages ( 245) and comparison based on part of the cognate amino acid ( 146) and nucleotide ( 340, 402) sequences supported this idea. The full extent of the similarity could be appreciated when the nucleotide sequences of argF ( 522) and argI ( 25) were determined. The two genes display 86% amino acids and 71.8% nucleotides in common, not unlike the trpA genes of E. coli and S. enterica serovar Typhimurium (85.1 and 72.5%, respectively). It seems that argF would have been inherited from a related species by lateral gene transfer—one of the first convincing instances of this kind of event to have been described—as indicated by the presence of two IS 1 elements flanking that gene in E. coli K-12 ( 203, 564) and by the relatively high GC content of argF ( 520). In keeping with this hypothesis, the other genes found between the two IS sequences also proved to have a high GC content (see reference 149).
The comparison between OTCase and the polypeptide constituting the catalytic subunit of ATCase is of considerable interest from the evolutionary point of view. The overall similarity is 35 to 40% in terms of amino acids and shows up in similarly structured domains, mainly in the polar moiety (the carbamoylphosphate binding domain) and helical regions joining the polar to the equatorial (or carboxy-terminal) domain (aspartate binding) of ATCase ( 201, 522). The two proteins therefore appear to have a common origin. An ancestral, possibly ambiguous carbamoyltransferase gene ( 201, 522, 563) could have been duplicated in near-tandem copies, which would have diverged in the course of evolution but may have remained associated on the chromosome in certain organisms as suggested by the strong linkage between argI and pyrB in E. coli and S. enterica serovar Typhimurium. Sequence comparisons between OTCases and ATCases of a large number of organisms ( 267, 268, 546) reinforce the notion of a common, ancient origin for both carbamoyltransferases, as already suggested above. This analysis however shows that the phylogeny of carbamoyltransferases is not congruent with the organismal tree of life; the data suggest that the last common ancestor population already contained at least two copies of OTCase and ATCase genes and that different copies were subsequently lost in different lineages ( 267, 268).
Very elegantly, Houghton et al. ( 201) have shown that fusing the OTCase polar domain to the carboxy-terminal domain of ATCase produces a hybrid protein able to carbamoylate aspartate but not ornithine.
The sequences of a large number of OTCase genes and the three-dimensional structure of several OTCases have been determined, including that of E. coli ( 174, 223, 315, 525, 526). All appear homologous, whether involved in anabolism or catabolism. In general, anabolic OTCases are trimeric, with the exception of the enzyme found in Pyrococcus furiosus, which is a dodecamer and presents a striking illustration of the key role played by oligomerization in the stabilization of enzymes ( 91, 280, 315, 526). The OTCase of the piezo-psychrophilic bacterium Moritella abyssi may also exist in vivo mostly under a dodecameric form, which is more stable than the trimeric form ( 554).
The putative OTCase genes found in Bacteroides and Xylella fastidiosa raise an intriguing question. Though the enzyme—the structure of which has been determined for Bacteroides ( 111)—was shown to be essential for arginine synthesis in vivo, the carbamoylation of ornithine could not be demonstrated in vitro; moreover, in spite of a reasonable overall sequence conservation, certain residues usually found at the active site are not conserved. Either the reaction requires an as yet uncharacterized factor (cofactor or another protein) or its chemical pathway is actually different ( 111).
The comparison between the cold-adapted OTCase from M. abyssi and its homologues from E. coli and thermophilic microorganisms is also of general interest because of the comparatively low catalytic efficiency of the cold-adapted enzyme ( k cat/ K m), a property shared by the dihydrofolate reductase of the related species Moritella profunda. This feature appears to result from a marked trade-off between affinity and catalytic activity and may actually be typical of many psychrophilic enzymes, indicating that there are natural limits to enzyme optimization at low temperature ( 554, 555).
Argininosuccinate synthetase, encoded by the argG gene, catalyzes the conversion of citrulline, aspartate, and ATP into argininosuccinate, AMP, and PP i. The enzyme has been purified from mammals and from Saccharomyces cerevisiae ( 190, 417). The yeast enzyme is a tetramer of identical MW 49,000 subunits, and the E. coli enzyme appears to consist of a basic polypeptide with a similar MW (48 kDa) as estimated from denaturing gels loaded with extracts of minicells producing a plasmid-encoded argG protein ( 357). This finding is in keeping with the E. coli argG nucleotide sequence ( 521), which predicts a polypeptide of 49,925 Da. The sequence appears homologous to those of many microorganisms—including yeasts—and humans. The three-dimensional structure of ArgG has been determined for E. coli ( 274) and Thermus thermophilus ( 161, 162). Catalysis by the mesophilic enzyme proceeds with a large conformational change which does not appear to occur in the thermophilic homologue.
argG transcription is not only repressed by arginine but is also activated by the cyclic AMP (cAMP)-CAP complex; this activation occurs at the same DNA site as for the divergently transcribed metY gene, coding for one of the two methionine tRNA initiators. Likewise, metY is repressed by ArgR; this unexpected situation suggests a link between carbon metabolism and translation initiation ( 259).
Argininosuccinase, encoded by the argH gene, hydrolyzes argininosuccinate into arginine and fumarate. The cognate sequence has been found in many prokaryotes and eukaryotes. The purified mammalian argininosuccinase is a tetramer of 50-kDa subunits ( 417), as it also appears to be in a cyanobacterium ( 502). The E. coli enzyme appears similar, since extracts of UV-irradiated cells infected with a λ transducing phage carrying the argH gene display a 55-kDa polypeptide when examined by electrophoresis under denaturing conditions on polyacrylamide gels ( 289). The sequence ( 32) bears high similarity to that of the yeast cognate gene and predicts a molecular mass of about 50 kDa (1,374 nucleotides).
Curiously, the piezo-psychrophilic Vibrionaceae Moritella abyssi (strain 2693) and M. profunda (strain 2674) harbor a gene very similar to E. coli argH in its proximal part but extended by a sequence able to complement an argA mutation in E. coli ( 556). It is not known whether these organisms have a genuine, separate acetylglutamate synthetase.
In both E. coli and S. enterica serovar Typhimurium, a single CPSase which utilizes glutamine as the natural amino group donor provides carbamoylphosphate for arginine and pyrimidine biosynthesis ( 2, 393, 396). For various reasons, this remarkable catalytic machine is presently the most interesting enzyme of the system; it therefore deserves extensive discussion. Moreover, the three-dimensional structures of various liganded forms of this large, highly regulated enzyme from E. coli have now been solved ( 332, 492, 494, 495, 496). Several reviews on structural and biochemical aspects of CPSase have appeared recently ( 195, 196, 325, 420, 421).
The synthesis of carbamoylphosphate proceeds in four steps: (i) formation of enzyme-bound carboxyphosphate; (ii) reaction between this complex and glutamine; (iii) transfer of the amido group of glutamine to activated CO 2 and formation of enzyme-bound carbamate; and (iv) phosphorylation of carbamate in the presence of a second molecule of ATP and liberation of the carbamoylphosphate formed ( 13; for a more recent review see reference 196). As all the intermediates in this reaction are highly reactive, the reaction mechanism must be perfectly timed and synchronized, and the intermediates must supposedly be shielded from the bulk of the solvent.
Powers-Lee and coworkers have proposed the nucleotide switch model as an alternative mechanism for the production of carbamoyl phosphate ( 256). In this model, ammonia is supposed to attack the carboxy phosphate intermediate directly, without involvement of carbamate. The nucleotide switch mechanism has, however, been shown to be inconsistent with the results of isotopic labeling and pulse-chase experiments ( 419, 438).
Both in E. coli and in S. enterica serovar Typhimurium, CPSase consists of two subunits, a heavy one (MW 118,000) able to catalyze the synthesis of carbamoylphosphate from HCO 3, ATP, and NH 3 (but not from glutamine) and a small one (MW 41,000) which carries the glutamine-binding site and displays a glutaminase activity in vitro ( 3, 503). The ammonium ion is a low-affinity nitrogen donor for the enzyme ( 226); the reaction with the ammonium ion may become growth supporting in mutants deficient in glutaminase activity when the ammonium ion concentration in the medium is high ( 145, 327) or when the large subunit is produced in vast excess ( 276, 436). The small and large subunits are encoded by the adjacent carA and carB genes ( 3, 327). In both organisms, carA and carB (collectively named pyrA in older articles on Salmonella species) form an operon oriented from carA to carB; some nonsense carB mutants display curious antipolar effects as yet unexplained ( 99, 145). The complete sequences of the two genes in E. coli ( 369, 403) and that of carA in S. enterica serovar Typhimurium ( 247) had been determined; the operon sequences are now confirmed and completed by genome sequencing ( 319).
Sequence comparisons and structural analyses indicate that E. coli CPSase consists of domains and subdomains that can be observed in other amidotransferases as well, but the finely tuned action of CPSase results from the manner in which these domains are connected and cooperate. The three active sites are widely separated on the enzyme, but appear to be connected by a molecular tunnel, nearly 100 Å long, through which the reaction intermediates are supposed to be translocated, as further supported by isotopic competition and protein modification ( 204, 205, 248). Though the glutaminase and catalytic domains of the enzyme can function autonomously, they cross-talk, and the partial reactions are coordinated by a reciprocal link between glutamine hydrolysis and carbamoylphosphate synthesis ( 329, 331, 436).
From glutamine bound to the small subunit, the amido group is transferred, likely as nascent NH 3, to the site of the large subunit, which also accepts the ammonium ion as a nitrogen donor. CPSase is thus basically similar to other amidotransferases involved in the synthesis of amino acids, purine, pyrimidines, or cofactors ( 183, 391, , 420, 565). Possibly, this family of enzymes results from the combination of a primordial glutaminase with various synthetases originally utilizing NH 3 as the nitrogen donor. The glutaminase reaction of CPSase proceeds through two tetrahedral intermediates and a covalent glutamyl thioester intermediate, which could be directly observed in the structure of a mutant enzyme ( 493). The residue Cys269 serves as the active site nucleophile that is activated by His353 ( 229, 335, 352, 435). The structure also suggests that ammonia might be translocated through a molecular tunnel, over a distance of about 45 Å from its site of production to the site of utilization. The observation that ammonia produced by the enzyme from glutamine does not mix with ammonia added to the solvent supports this proposal.
There is a high degree of similarity (39% identical residues) between the amino- and carboxy-terminal moieties of the large, catalytic subunit, and it has been suggested that carB results from the duplication of a smaller ancestral gene ( 369). As the enzyme is allosteric (see below), Nyunoya and Lusty ( 369) have suggested that the two halves of the carB molecule fold as separate domains capable of conformational interactions, each domain carrying one of the two ATP-binding sites recognized on CPSase ( 36, 409). Biochemical studies, site-directed mutagenesis, and subcloning of separate domains confirmed this view ( 168, 169, 170, 171, 172; also see references 330 and 418 and references therein); moreover, they indicate (i) that the amino- and carboxy-terminal halves of the carB subunit are involved in the formation of carboxyphosphate and in the phosphorylation of carbamate, respectively ( 407), and (ii) that the two catalytic ATP sites interact with each other ( 168). A single mutation in the ATP-binding site of the carbamate phosphorylation domain may uncouple functional interactions between the two catalytic domains of the large subunit and also between the carbamate phosphorylation domain and the glutaminase ( 168, 299). Residues critical for catalytic action were identified by site-directed mutagenesis in both the carA- and carB-encoded subunits ( 168, 217, 299, 330, 335, 407, 466). These findings and proposals are now underscored by the structural investigations which indicate that the large subunit is folded in two topologically equivalent parts each consisting of four major components (called A to D). They are reminiscent of biotin carboxylase and other members of the ATP-grasp superfamily ( 139, 539). The N-terminal part is the carboxyphosphate-forming part (residues 1–553), whereas the C-terminal half (residues 554–1073) is responsible for CP formation. The domains A to C of the two halves of the large subunit superimpose very well and form a pseudo-homodimeric structure. The two ATP binding pockets are structurally similar with comparable chemical interactions between the enzyme and the substrate at both sites, situated between the B and C subdomains. In contrast, the two D domains diverge widely in structure. In the N terminus it constitutes the oligomerization domain, whereas in the C-terminal half it forms a Rossman fold and is responsible for the binding of the allosteric effectors.
CPSase is a highly regulated enzyme. It is subject to feedback inhibition by UMP ( 2, 390). This inhibition is antagonized by IMP ( 14), an effect with probable significance for the coordination of purine and pyrimidine pathways (also operative at the level of enzyme production [ 121, 296]), and, most importantly, it is antagonized by ornithine ( 390). Since arginine curtails ornithine formation by feedback inhibition of N-acetylglutamate synthase, the UMP-ornithine antagonism amounts to a double feedback by arginine and UMP. In the presence of ornithine, UMP exerts a partial inhibition; in the absence of ornithine, the inhibition is almost total. All of these allosteric ligands act primarily on the binding of Mg-ATP, and various mutant studies and photoaffinity labeling indicate that they all interact with the 18-kDa carboxy-terminal domain of the large subunit ( 47, 48, 56, 73, 74, 110, 117, 342, 397, 398, 426, 437, 503). The crystal structures of E. coli CPSase complexed with ADP, Mn 2+, K +, ornithine, and P i on the one hand, and IMP on the other hand, indicate that ornithine binds at the interface of the carbamoylphosphate (C) and allosteric (D) domains, whereas the overlapping IMP/UMP binding site is entirely contained within the D domain ( 492, 494, 495, 496). Thus, binding of ornithine establishes a direct connection between the allosteric effector-binding region and the active site of the carbamoylphosphate domain. It has been shown previously that ornithine has a stronger effect on the carbamoyl phosphate-dependent ATP synthesis (reverse reaction) than on the other partial reactions ( 47). A series of hydrogen bonds connect the IMP-binding pocket to the active active site for carbamate phosphorylation. Mutant studies indicate that residue Thr1042 of the D domain, a residue directly involved in binding of ornithine, and Ser948 of the nucleotide effector binding site play crucial roles in the coupling of activation and inhibition pathways ( 117, 426).
CPSase is thus controlled by several allosteric effectors belonging to different pathways. Anderson and Marvin ( 10, 12) have shown that the effectors affect the distribution of the enzyme among at least three conformational states, one of them stabilized by the positive effectors and favorable to substrate binding, and another stabilized by the inhibitor UMP. Mutational alterations of CPSase are thus likely to result in a variety of phenotypes. Some E. coli carB mutants are sensitive to uracil, whereas others exhibit either a pseudoarginineless phenotype or an arginine-sensitive one; still others are sensitive to both arginine and uracil ( 145, 327). Several of these mutant carB genes have been sequenced, the corresponding enzymes have ben purified and characterized, and a relationship between the mutant phenotypes and enzymatic and regulatory properties has been presented ( 117).
supJ-hisT double mutants of S. enterica serovar Typhimurium are also uracil sensitive ( 38). It is not yet known whether the primary effect of the mutation is structural or regulatory; an explanation in terms of attenuation control ( 38) now appears unlikely (see The carAB Operon, a Dual-Control System, below).
From investigations with S. enterica serovar Typhimurium, Abdelal et al. ( 5) concluded that the arginine sensitivity of some car mutants was due to repression by arginine of OTCase synthesis; indeed, in such mutants (but not in the wild type), an interaction between the synthetase and the transferase would appear to be critical for proper assembly and function of the carA and carB gene products. These observations would of course be less surprising if the formation of multienzyme complexes between the two proteins were a normal feature of carbamoylphosphate metabolism (see Physiological and Evolutionary Considerations, below). According to the same authors ( 6), arginineless car mutants display this phenotype because arginine curtails the formation of N-acetylornithine, which would appear to antagonize the maturation of the mutant synthetase. Later on ( 181), the same group reported two remarkable observations that may be related to these findings: (i) a cold-sensitive car mutant of S. enterica serovar Typhimurium affects an amino acid residue that appears to be critical for proper folding of the enzyme (indeed, the growth temperature affects the properties of the mutant synthetase); and (ii) to a lesser extent, the characteristics of the wild-type enzyme also appear to be influenced by the growth temperature. In this context of mutienzyme complexes it is worth mentioning that though the oligomeric state of E. coli CPSase has no effect on the catalytic turnover of the enzyme, changes in the oligomeric state might be an interesting means to affect the distribution of CPSase to the arginine and pyrimidine pathways through differential interactions with either OTCase or ATCase (see also Physiological and Evolutionary Considerations, below). Indeed, the oligomeric state of CPSase depends on the enzyme concentration and the presence of effector molecules; UMP favors the formation of a dimer, whereas ornithine and IMP promote the formation of the tetramer ( 10, 11, 410). Recently, it has been shown that dimers are formed by interactions across the regulatory domains and tetramers are formed by interactions of two dimers across the oligomerization domains ( 249, 341).
Gigot ( 144) observed that from a strain carrying a deletion in carB, it is possible to select derivatives growing in the presence of cyanate. Such mutants display an ammonia-dependent CPSase activity; surprisingly, the synthesis of the corresponding enzyme is repressible by uracil. The responsible gene has not yet been characterized.
In addition to its general toxic effects, cyanate, a well-known carbamoylating agent, appears to inhibit the synthase specifically ( 167).
Most of the structural genes of the arginine pathway have been identified and localized in E. coli and S. enterica serovar Typhimurium by conventional selection and mapping techniques. These results have been confirmed and completed by genome sequencing and in silico annotation ( 33, 319). Forward selection methods have also been devised for mutants blocked in steps A, B, C ( 98), and E ( 236). As in fungi ( 17, 351), car mutants could be recovered from a dihydroorotaseless ( pyrC) strain by selecting for derivatives unable to accumulate the toxic intermediate carbamoylaspartate (D. Charlier, Masters thesis, Vrije Universiteit Brussel, Brussels, Belgium, 1974). The toxicity of carbamoylaspartate is not understood; it was later been observed in S. enterica serovar Typhimurium as well ( 507).
With the exception of argF, which is a peculiarity of E. coli K-12, both E. coli and S. enterica serovar Typhimurium display very similar arrangements of arg loci ( Fig. 1a). The only genes to be clustered are argECBH and carAB. argECBH in E. coli was shown by physical methods to be a divergent operon consisting of two arms, argE and argCBH ( 375, 408), with an internal operator region flanked by two convergent promoters ( 399, 400), bearing out earlier genetic analyses ( 127, 213). The genome sequence ( 319) predicts a similar organization in Salmonella.
E. coli mutants affected in the regulatory gene argR were originally obtained by selecting for canavanine-resistant derivatives ( 157, 300); the same strategy was later used to select trans-dominant mutant repressors ( 500). Remarkably, canavanine does not inhibit growth of S. enterica serovar Typhimurium. argR mutants of E. coli B have also been obtained by selecting for resistance to homoarginine ( 389), which does not affect the K-12 strain. argR mutants of both S. enterica serovar Typhimurium and E. coli could also be isolated by indirect suppression of the proline auxotrophy induced by arginine in argD mutants (see N-Acetylglutamate Synthase, above). argR mutants of E. coli were also recovered as strains resistant to a mixture of 2-thiouracil and arginine ( 394), whose toxic effect ( 26) can now be explained by the repression of the tandem pair of car promoters by arginine and pyrimidines (see The carAB Operon, a Dual-Control System, below). This combination is particularly useful to select for argR derivatives of arg auxotrophs; the use of auxotrophs also circumvents selection for permease mutants. Another useful method is the selection from histidine or methionine auxotrophs of derivatives able to use the cognate N-acetyl amino acid in the presence of ornithine or arginine (see N-Acetylornithinase, above). A last method of general interest for the selection of derepressed mutants involves selecting for derivatives of leaky mutants (a carA mutant was used [ 394]) that exhibit no further growth lag when transferred from minimal medium supplemented with arginine (and, in this case, uracil as well) to minimal medium. The method has been applied with success to isolate cis-dominant constitutive mutants of arg genes in Saccharomyces cerevisiae ( 211). Finally, the dual activity of the E. coli ArgR protein (alias XerA), an obligate accessory partner in site-specific DNA recombination at ColE1 cer (see also Arginine Repressor and Repressor-Operator Interactions, below), allowed the isolation of argR mutants deficient for the resolution reaction and transcriptional control, by Tn 5 transposon mutagenesis ( 470, 471). Noteworthy, a very handy argR:: fol allele specifying trimethoprim resistance, that can easily be transferred by generalized transduction, was constructed by gene replacement ( 132).
Because several arg genes are unlinked to each other, isolation of cis-acting regulatory mutants with mutations in the arginine regulon required ad hoc strategies.
Selecting for suppressors of polar argC or argB mutations gave rise to tandem duplications connecting argH or argB plus argH to other promoters or creating new ones at their novel joints ( 24, 75). The same approach provided a deletion ( Δ sup102), which disclosed the existence of an internal operator region between argE and argC and of two flanking promoters facing each other, argEp and argCBHp ( 127).
Point mutations of the O c (operator-constitutive) type were obtained by various means, including relief from repression of streptomycin-induced suppression of argI or argC nonsense mutations ( 213, 215). In the second case, the mutants were partially constitutive for both argE and argCBH, even though the mutations were mapped between argE and argC. The existence of a divergent operon was inferred from this finding, in keeping with the properties of the sup102 deletion (see Structure of Control Regions, below). Other argECBH operator-constitutive mutants were obtained by localized mutagenesis ( 199), selecting for derivatives of his auxotrophs able to achieve high enough levels of N-acetylornithinase to deacetylate N-acetylhistidine even when the arginine regulon was repressed ( 44, 51, 97). This method proved particularly rewarding since it also provided mutations increasing the translational rate of argE ( 45). The same method gave apparently argE-specific operator-constitutive mutants; these were later shown to harbor secondary promoters relatively insensitive to repression ( 103, 399) (see Structure of Control Regions, below).
cis-dominant mutants enhancing argE expression were also obtained by selecting for derivatives of the sup102 deletion mutant able to utilize N-acetylornithine or N-acetylarginine as the source of arginine. Some of these strains were of the operator-constitutive type and also affected argH; other were promoter-up mutations and involved the insertion of a mobile promoter such as IS 3 ( 81, 151, 399).
argF and argI operator mutants or unstable rearrangements causing argF constitutivity were isolated from a car deletion mutant as derivatives able to use citrulline as a source of carbamoylphosphate for pyrimidine biosynthesis ( 221, 222, 279, 522) (see OTCase, above). Some of these rearrangements are gene amplifications whose occurrence is linked to the status of the F factor ( 93, 221, 222)
Last but not least, the construction of operon and protein fusion strains in which the lacZ gene was expressed from a foreign promoter ( 60) provided a general approach to isolate regulatory mutants ( cis- and trans-acting types) that has been applied to the argECBH ( 27) and carAB operons ( 80, 243, 432).
This variety of structural and regulatory mutants provided the biological material that allowed subsequent cloning and sequence analysis of all the genes of the regulon (at least in E. coli) by a variety of approaches, which involved extensive applied lambdology ( 152, 244, 278, 318, 411), before the systematic use of plasmid cloning vectors and the synthesis of amplicons could be generalized. The use of well-defined λd arg transducing phages was critical in establishing the reality of transcriptional repression, both in vivo and in vitro (see below).
Various models for regulation of arginine biosynthesis, including translational control, differential mRNA stabilities, and attenuation control, have been proposed in the past. Most of these observations could not be confirmed, however, or turned out to be artifacts. The bulk of data available today clearly establishes that arginine is the corepressor of the regulon, that the arginine repressor (ArgR) is the master regulator, and that regulation of enzyme production is mainly at the level of transcription initiation. No attenuation type of control is involved, which may reflect inadequacy of arginine codons to serve as basis for such a mechanism ( 103).
Control of mRNA translation rather than modulation of mRNA synthesis had been advocated very early as a mechanism for the regulation of arg genes ( 321, 531, 537). When transducing phages carrying the argECBH genes became available, RNA-DNA hybridization allowed a direct test of these hypotheses. Rogers et al. ( 430) and Cunin and Glansdorff ( 105) showed that the amount of pulse-labeled argECBH hybridizable RNA varied considerably over the range of conditions investigated. Other studies confirmed the notion of transcriptional control in vivo (see reference 358 for argA, argE, and argI and reference 395 for carAB) and in vitro with argA, argECBH, argF, argI, and carAB DNA ( 107, 125, 285, 395, 458, 459). The kinetics of enzyme repression originally interpreted by Lavallé and De Hauwer ( 272, 273; see also reference 42) in terms of regulated translational arrest appear, rather, to reflect the progressive decay of enzyme accumulated before the onset of repression ( 395).
More precise investigations with low-background DNA probes for the two arms of the argECBH divergent operon established that the amplitude of variation for hybridizable RNA levels, even if considerable, remains three- to fourfold lower than for enzyme specific activities ( 102). Zidwick et al. ( 567) were able to reproduce the phenomenon in vitro. Such a discrepancy, however, was not observed for the carAB operon; there, a close correspondence was obtained between enzyme and RNA levels ( 395).
One interpretation of the argECBH mRNA-enzyme discrepancy would have been attenuation control: in repression, the cells would contain a relatively higher proportion of leader mRNA not contributing to enzyme synthesis. However, Bény et al. ( 28), using several probes, showed that transcription of argCBH in a purified in vitro system was not restricted to a proximal leader sequence and that, in vivo, no preferential transcription of operator-proximal sequences occurred under conditions of repression. Moreover, sequence data and inspection of control regions ( 83) provide no evidence for attenuation control.
Another explanation, advocated by Vogel and coworkers ( 322, 537), assumed the discrepancy to be due to a greater stability of the mRNA in derepression than in repression. Indeed, restricting the arginine supply of an arginine auxotroph increased the chemical half-life of argECBH hybridizable RNA by a factor of 3 to 4. However, further estimates of the chemical half-life of argECBH mRNA ( 261) and analysis of the functional decay of argF argI mRNA do not support the notion of a specific effect of arginine on the stability of the cognate mRNA ( 178).
At present, there is no completely satisfactory explanation for the argECBH mRNA-enzyme discrepancy, but the data remain compatible with the notion that in repressed cells, transcripts of a distal portion of the cluster, well beyond the control region, would be less abundant than those of more proximal segments, perhaps owing to rho-promoted premature transcription termination ( 568). In our opinion, however, the mRNA-enzyme discrepancy may result at least in part from another cause. In argC, there is a weak secondary argE promoter ( argEp2) active under conditions of repression (see Structure of Control Regions, next section); formation of translationally inactive RNA-RNA duplexes by mRNA molecules respectively initiated at argCp and at argEp2 could account for the relative excess of unproductive RNA present in repressed cells.
It is now clear that arginine and not arginyl-tRNA is the corepressor of the arginine regulon. Hirschfield et al. ( 193) showed very early that mutants of arginyl-tRNA synthetase impaired in their charging ability and therefore accumulating arginine displayed reduced levels of OTCase but, most importantly, remained repressible; no correlation was found between repression and the level of charging. Moreover, Leisinger and Vogel ( 282) and Celis and Maas ( 71) showed that the charging profiles of the five isoacceptor tRNA Args appeared to be the same in repressed and derepressed cells. Subsequent reports on derepressed synthetase mutants or on derepression resulting from inhibition of the synthetase by arginine precursors ( 49) could not be confirmed or were shown to involve artifacts (see Arginyl-tRNA Synthetase, below).
Direct confirmation that arginine is able to play the role of corepressor came from in vitro experiments. In a purified system for transcription of the argECBH genes ( 376), addition of arginine and partially purified repressor free of arginyl-tRNA synthetase provoked repression of hybridizable RNA synthesis ( 107). In similar experiments, Lissens et al. ( 290) found that free arginine and repressor repressed transcription of carAB in vitro to the same extent as in vivo. G. Bény (Ph.D. thesis, Vrije Universiteit Brussel, Brussels, Belgium, 1984) was unable to observe any effect of tRNA Arg or of the synthetase itself on repression of argECBH transcription in vitro. More recent work established that purified repressor (ArgR) specifically binds to arg and car operators in the presence of arginine ( 83, 85, 285, 294, 498; see also below); that, in some genes at least, binding of liganded repressor and RNA polymerase are mutually exclusive ( 85); and that ArgR inhibits transcription initiation at the argF ( 285) and carp2 (Charlier, unpublished data) promoters in an arginine-dependent manner in a pure in vitro transcription assay, containing no other proteins than ArgR and RNA polymerase. Moreover, the three-dimensional structures of liganded C-terminal core of E. coli ArgR indicate the binding of six arginine molecules at the trimer-trimer interface ( 518) (see also Arginine Repressor and Repressor-Operator Interactions, below).
Early experiments ( 156) suggesting that ornithine was able to counteract the repressive effect of arginine in a chemostat operated under conditions of partial derepression could not be reproduced. In chemostats in which the dilution rate was controlled with great accuracy, no effect of ornithine or citrulline could be observed (reference 98 and unpublished results from this laboratory). Furthermore, argG bradytrophs accumulating citrulline remain derepressed ( 279). Zidwick et al. ( 567) showed that l-ornithine, l-citrulline, and d-arginine had no effect on in vitro synthesis of argECBH mRNA and of two of the cognate enzymes. More recently, it has been shown in vitro that l-arginine and l-canavanine prevent the exchange of trimers among hexameric ArgR molecules, whereas lysine, citrulline, and homoarginine do not, suggesting that the latter molecules do not bind the repressor ( 518), unless the binding specificity of the repressor is modified by mutation of the cofactor binding pocket ( 364; see also Arginine Repressor and Repressor-Operator Interactions, below).
Recently transcription of argG was shown to be activated by the cAMP-CAP complex ( 259) whereas ppGpp, the chemical messenger of the stringent response, partially inhibits transcription initiation at the carp1 promoter ( 40; see also The carAB Operon, a Dual-Control System, below). Starvation for an amino acid, therefore, would not only turn off the synthesis of RNA but also affect the synthesis of RNA precursors. The fact that transcription of the pyrBI operon, encoding ATCase, is also subject to stringent control ( 506) underscores this link. In contrast, the synthesis of acetylornithinase ( argE) and argininosuccinase ( argH) is enhanced by ppGpp ( 568). These investigations corrected an earlier report by Yang et al. ( 560) which concluded that synthesis of acetylornithinase was inhibited by ppGpp, and they are in keeping with the positive effect of ppGpp observed on the synthesis of enzymes encoded by argA and argI ( 138, 235, 506) and on amino acid biosynthetic enzymes in general. By performing in vitro experiments, Zidwick et al. ( 568) showed that this effect did not involve argR and found no evidence that it was exerted at the level of transcription. Further experiments from the same laboratory (M. G. Williams, Ph.D. thesis, University of Minnesota, Minneapolis, 1985) indicated that ppGpp acts at least in part by reducing the frequency of translational errors, in keeping with earlier observations ( 138).
A feature common to all genes of the E. coli regulon, including the catabolic ast operon and transport genes ( artP and artJ; see Arginine Transport, below), is the presence of one or more "ARG box" sequences, a partially conserved 18-bp sequence that displays hyphenated symmetry. All the biosynthetic operators, including the negatively autoregulated argR itself and the carAB operon, contain a tandem pair of ARG boxes that overlap the promoters to various extents ( Fig. 2 and Fig. 3). These ARG boxes ( 399, 400) were first shown to constitute operator sites by examination of the nucleotide sequence changes in operator mutants of argECBH ( 103, 399), argF ( 522), and carAB ( 432). A consensus sequence was derived from the compilation of all biosynthetic operators ( 541; Fig. 2) and its interaction with purified ArgR was studied in vitro ( 90, 479, 541). Homologous pairs of ARG boxes were also identified in front of the carAB and argR genes of S. enterica serovar Typhimurium ( 294); additional ones can be recognized in front of arg genes in the genome sequence ( 319). They exhibit 91.7 to 100% sequence identity with the cognate E. coli pairs of ARG boxes. The compilation indicates 4 highly conserved bp in each half (T3, G4, A6, T7) and a 4-bp AT-rich center ( Fig. 2). Generally, a naturally occurring ARG box is composed of one half-site, which shows a strong match to the consensus, combined with a weaker half. Analyses of sequence conservation and mutant studies indicate that the inner halves (near the center of the operator) are more important for repressor binding and repressibility than the outermost halves (summarized in reference 557, which compares the situation encountered in E. coli and in an extreme psychrophilic Moritella species). In E. coli and S. enterica serovar Typhimurium, all tandem ARG boxes are separated by 3 bp exclusively composed of weak base pairs (AT and TA), except for argR, in which there is only 2 bp in both organisms and in E. coli B as well ( 285, 294, 499). Strikingly, this 2-bp spacer contains one strong (GC) base pair, a feature also observed in the biosynthetic operators of various gram-positive Bacillus and Streptomyces species (summarized by Song et al. [ 464]). The importance of the spacer is indicated by the occurrence of O c type mutations and by in vitro binding studies ( 83, 399, 464) (see also Arginine Repressor and Repressor-Operator Interactions, below).
In E. coli argG, a third ARG box is present 101 bp upstream from the pair of boxes found in the promoter region ( 83). Though this box clearly binds ArgR in vitro ( 83), it appears not to be involved in repression of the argG gene; in contrast, it was shown to participate in regulation of the upstream, divergently transcribed metY gene ( 259). A potential single ARG box can also be identified in the S. enterica serovar Typhimurium argG control region (66.7% identity with the E. coli argG box), 98 bp upstream of the tandem pair. Several ARG box-like sequences are also present in the control region of the arginine- and ArgR-inducible ast operon encoding enzymes of the arginine succinyl transferase pathway for arginine catabolism in E. coli and in S. enterica serovar Typhimurium ( 252, 293). A single, functional ARG box sequence is present in the ColE1 cer site for site-specific resolution ( 471). Finally, in silico analyses of the E. coli genome sequence have further indicated the occurrence of a candidate ARG box sequence in front of the artPIQM and artJ genes encoding arginine-specific ABC transporters ( 308), but their functionality remains to be experimentally demonstrated.
Except for E. coli argR and for the carAB operon, each expression unit appears to be transcribed mainly from one promoter region, sometimes from a close cluster of starting points. The argA promoter has not yet been identified experimentally, however. The control region of carAB contains a pair of tandem promoters: P2 is regulated by arginine and overlaps a pair of ARG boxes; P1, located 67 bp upstream, is regulated by pyrimidines and purines (see The carAB Operon, a Dual-Control System, below). The argR gene is autoregulated; in E. coli, it is transcribed from two promoters: a constitutive one (P2), which accounts for about a third of the total repressor that can be produced, and P1, downstream, which overlaps a pair of ARG boxes ( 285). It is conceivable that the constitutive production of repressor is necessary to reach the maximal degree of repression observed in E. coli K-12 ( 499). The control regions of the argR genes of E. coli K-12 and E. coli B are identical ( 499), but in Salmonella spp., only P1 is present ( 294).
The control region of the divergent argECBH operon deserves a special discussion. The first example of divergently arranged genes transcribed from closely spaced promoters was the cI-cro cluster of bacteriophage λ ( 491), but the bioABFCD and argECBH operons provided the first instances of control regions consisting of an internal operator flanked by two facing promoters. The properties of the deletion Δ sup102 clearly indicated this arrangement: the deletion destroyed part of argB and the whole of argC, abolished argE expression without affecting the structural gene itself, and made the expression of argH partly constitutive ( 127). This formal genetic evidence, joined to the discovery of point mutations derepressing the whole cluster and located between argE and argC ( 213), was fully confirmed at the sequence level (see below). This somewhat unexpected arrangement of controlling elements had been considered at an early stage as an unlikely possibility for the bio gene cluster ( 166); it was nevertheless proved to apply to that case as well ( 372). Other examples of divergent operons have been described since then; most of them, however, display back-to-back or overlapping promoters ( 23).
The biological significance of this face-to-face arrangement is not clear, although some speculations have been considered ( 23). Obviously, as the internal operator overlaps the two facing promoters to similar extents, we would expect coordinate expression of the two wings of the cluster. This is indeed the case for argE and argCBH, but the situation became clear only after a fine dissection of the regions adjacent to the control region was performed. The respective repression coefficients of argE and argCBH are at first sight markedly different: 9 and 23 in terms of mRNA, and 17 and 60 in terms of specific activities ( 102). This paradox was explained by the discovery of a weak secondary argE promoter ( argEp2) located in argC, 180 nucleotides upstream from the major transcription initiation site for argE. argE transcription initiated from argEp2 can be detected by S1 nuclease mapping in repressed cells ( 103; J. Piette, Ph.D. thesis, Vrije Universiteit Brussel, Brussels, Belgium, 1983) and accounts for about half the specific activity of enzyme E under these conditions (A. Boyen, unpublished data from this laboratory). When this background is considered, no difference remains between the individual repression responses of argE and argCBH (see Table 2), although they kept being recorded as uncoordinate on the basis of the early evidence ( 23). Expression from argEp2 appears relatively insensitive to repression, as confirmed by the partially constitutive acetylornithinase synthesis observed in " argEp2-up" mutants ( 103;Piette, Ph.D. thesis). These observations are in keeping with previous data on trp-lac fusions ( 422; see also The carAB Operon, a Dual-Control System, below) which suggest that steric hindrance between RNA polymerase and repressor is necessary to achieve efficient repression. Furthermore, results of experiments by Sens et al. ( 459) on the order of addition of S30 extracts from argR + and argR – cells to in vitro-coupled translation-transcription systems suggested that steric hindrance is also sufficient to explain repression. This has now been confirmed by DNase I footprinting and single-round in vitro transcription assays (see The carAB Operon, a Dual-Control System, and Arginine Repressor and Repressor-Operator Interactions, both below). Do converging promoters interfere with each other? Initiation at either of the primary promoters overlapping the operator region probably occurs in an alternate fashion. Interferences between convergent promoters appear when the distance between them increases; derepression of argCp decreases transcription from argEp2 even when the latter is strengthened by up mutations ( 103; Piette, Ph.D. thesis). Likewise, derepression of argCp antagonizes expression from the outward promoter of IS 3 inserted 190 nucleotides in front of the argCBH transcription start point ( 81). It is not clear whether these interferences result from collisions between polymerases, from interactions between complementary mRNA molecules initiated at argCp and argEp2, from the unavailability of argEp2 when being traversed by frequent transcriptional waves initiated at argCp, or from local modifications of DNA topology generated by transcriptional activity, as demonstrated for the divergent pair of ilvY and ilvC promoters ( 423).
The sequence analysis of several mutations allowed a fine-structure dissection of the genetic determinants intertwined in the argECBH control region ( 103, 399) ( Fig. 2). One of the most interesting results of this study concerns mutations that simultaneously depress the activity of the argCBH promoter and enhance the efficiency of argE translation ( 44, 45). The mutations are confined to the limits of argCp but also modify the sequence of the ribosome-binding sites for argE translation, between the Shine-Dalgarno box and the 5' end of argE mRNA. The sequence data strongly suggest that maximal translation efficiency depends directly on the composition of proximal RNA sequences not necessarily included in secondary structures. They support the notion of a proximal mRNA "consensus" sequence containing elements interacting with ribosomal components ( 451).
argCp presents a paradox: the –35 region contains the consensus sequence TTGACA only 15 nucleotides away from the –10 region, yet it is not a weak promoter. It is possible that the sequence TTGTTG, 18 nucleotides from the Pribnow box, plays a role in polymerase-promoter interactions.
In E. coli and S. enterica serovar Typhimurium the arginine repressor is the master regulator of the biosynthetic pathway, but in both organisms ArgR is also involved in activation of the catabolic ast operon ( 252, 293). Similarly, the ArgR homologues from various gram-positive bacteria are capable of repressing transcription of the biosynthetic genes and taking part in the activation of various catabolic genes. It is not yet clear whether arginine repressors can directly stimulate transcription or rather function as coactivator, but clearly arginine-dependent activation of the arginase operons in B. subtilis requires the rocR gene product, a transcriptional regulator of the NtrC/NifA family ( 140); moreover, in E. coli and Salmonella, not only NtrC but also IHF and CAP bind to the ast control region ( 252, 293). Surprisingly, E. coli ArgR was found to fulfill still another, unrelated function: in what appears to be a striking case of protein recruitment, and under the denomination of the XerA protein, it is involved, in conjunction with PepA (XerB), in the resolution of ColE1 dimers into monomers by its ability to juxtapose two distant cer sites (each bearing an ARG box), with the cutting and rejoining reactions being carried out in the vicinity by the XerC and XerD proteins ( 471). This joining of distant sites is reminiscent of the pattern observed when ArgR binds to the argG control region ( 83) (see above).
After some early attempts that led to only partial purification ( 107, 237, 510, 513) but were nevertheless useful in the first in vitro analysis of arginine-mediated repression, the argR product was purified to homogeneity from E. coli K-12, E. coli B, and S. enterica serovar Typhimurium from appropriate hyperproducing strains ( 285, 294, 499). A particularly gratifying feature of this protein is that it can be precipitated by adding l-arginine at an early stage of the purification. In all three organisms, the arginine repressor (ArgR) was found to be a hexamer of identical subunits with molecular masses of about 17,000 Da. The functional significance of the single amino acid difference between E. coli K-12 and E. coli B repressors is discussed in the next section. There is 95% identity between the E. coli and S. enterica serovar Typhimurium argR products ( 294).
At first, scrutinizing the sequence of the argR genes for potential secondary structures did not disclose any of the motifs recognized in various DNA-binding proteins, but mutational studies of the E. coli repressor ( 57, 90, 499) and the analysis of proteolytic fragments ( 164) suggested the existence of a N-terminal DNA-binding domain and of a C-terminal domain for binding of arginine and oligomerization. These proposals have been confirmed by structural determinations of the separate domains. Crystals of the full-length E. coli repressor (156 amino acids) did not diffract sufficiently well, but X-ray crystallographic analysis of the C-terminal domain (residues 80-156), to 2.8 Å resolution for the unliganded hexamer and to 2.2 Å for the arginine-bound form, revealed a compact dimer of trimers which stack back to back ( 518). Each C-terminal domain exhibits an α/ β fold composed of a four-stranded antiparallel β-sheet and two antiparallel α-helices. Six arginine molecules bind at the trimer-trimer interface; each cofactor molecule makes contacts with two subunits within one trimer and a third subunit in the opposing trimer. Bound arginine thus works as a molecular glue that crosslinks the trimers, but this tightening of the interactions appears to play no role in the arginine-mediated transition to the high-affinity operator-binding form of the E. coli repressor ( 479; see also below). Seven residues of the arginine binding pocket—Gln106, Asp113, Thr124, Gly127, Asp128, Asp129, and Thr130—play a major role in cofactor binding; they are well conserved in heterologous ArgR proteins. The importance of the residues Asp128 and Asp129 in arginine binding was confirmed by site-directed mutagenesis ( 57), and a D128N substitution mutant protein was shown to bind citrulline rather than arginine ( 364). Operator DNA binding of the repressor from Thermotoga neapolitana is rather arginine independent ( 122, 464), and this is, at least in part, due to substitution of Gln106 (position 107 in the Thermotoga repressor) by serine ( 344).
The structure of the monomeric N-terminal domain of E. coli ArgR (residues 1–73) was solved by nuclear magnetic resonance spectroscopy ( 476). The DNA-binding motif consists of a three- α-helix bundle and a β-finger and shows structural homology to the winged helix-turn-helix family (wHTH) ( 50, 270). Gln38, Ser42, and Arg43 are crucial residues in complex formation: they are exposed to the solvent and part of the recognition helix ( 476, 363). Their participation in DNA binding is further indicated by mutant studies of the repressor from E. coli ( 500) and B. stearothermophilus ( 333) and the high degree of conservation among bacterial ArgR proteins.
Despite a low overall sequence conservation, a similar fold was also observed in the crystal structures of the full-length repressor proteins from B.stearothermophilus ( 363) and B. subtilis ( 120), and the wHTH motif was predicted in the experimentally investigated arginine repressors of other gram-negative and gram-positive bacteria ( 122, 307, 428, 557) and in argR homologs detected in silico ( 308). Thus, the arginine repressor molecule and the general mode of arginine regulation are surprisingly well conserved, even in distantly related bacteria. Interestingly, an E. coli argR – mutant can be complemented, also for the dimer resolution reaction, by the homolog from B. subtilis, though the converse does not work ( 109). The homodimeric repressor from Pseudomonas aeruginosa, however, is of a different type, and belongs to the AcaC/XylS family ( 380, 381). No homologue of argR could yet be recognized in archaeal genomes.
In E. coli and S. enterica serovar Typhimurium the hexameric form is the only one to have been observed, both with and without arginine. Equilibrium sedimentation experiments indicate that the stability of E. coli ArgR hexamers is at least as strong as 2.5 nM and that both apo- and holo-ArgR form stable hexamers in the range of 1 to 10 μM total protein monomer concentration ( 479). Similarly, the repressor from the closely related psychro-piezophilic bacterium Moritella profunda was isolated as a hexameric protein ( 557), but the repressors of B. stearothermophilus ( 123), B. subtilis ( 197), and Thermotoga neapolitana ( 122) were isolated as trimers, though they form hexamers at high protein concentration and in the presence of arginine and/or DNA. Interestingly, excess operator DNA exerted an anticompetitive effect (enhanced binding instead of the more frequently observed inhibition) on complex formation in vitro with the B. subtilis repressor, an observation that may suggest the assembly of an activated form of the repressor on the operator DNA ( 197). The possibility of trimer binding, also for E. coli ArgR (in the absence of arginine and for mutant repressors), is still a matter of debate and is discussed at the end of this section.
The number of molecules of repressor (expressed as monomers) per cell would appear to be around 500 to 600 in E. coli K-12 or E. coli B; autoregulation brings this number to 300 to 400 in the presence of excess arginine ( 499). The relative inefficiency of this autoregulation appears to be due to the constitutive argR promoter P2 (see the previous section). This rather large concentration (about 1.0 μM) probably explains why "escape" synthesis of arg and car genes has been observed only when they were present in rather high copy number ( 152).
The basic features of the repressor-operator interactions have now been established. In E. coli K-12, data are available concerning all the control regions of the regulon (except argI), including several mutant derivatives, but the most detailed analyses are with the carAB operator ( 541). In S. enterica serovar Typhimurium, interactions have exclusively been studied with the carAB operator ( 294).
In vitro binding experiments analyzed by DNase I and hydroxyl radical footprinting on the operator sites of argA, argECBH, argD, argF, argG, argR, and carAB in E. coli indicate that in all cases the repressor binds symmetrically to four consecutive helical turns (corresponding to pairs of boxes separated by 3 nucleotides, or by 2 nucleotides in argR) to one face of the DNA only ( 83, 85, 285, 294, 498). This pattern was further confirmed by phosphate ethylation interference experiments on the E. coli argF operator ( 498) and more recently on the artificial fully symmetrical consensus operator sequence ( Fig. 2) derived from the compilation of all biosynthetic E. coli ARG boxes and their inverse sequences ( 541).
The close proximity to particular bases in the major and minor grooves of the E. coli and S. enterica serovar Typhimurium ARG boxes was detected by purine methylation protection and premethylation interference experiments ( 83, 294, 498). A more detailed analysis of the carAB and consensus operators by premodification binding interference (purine methylation with dimethyl sulfate, oxidation of thymine with KMnO 4, and modification of cytosine with NH 2OH), missing contact probing applied to purines and pyrimidines, base substitution (uracyl, inosine), and the use of the small, groove-specific ligands distamycin A and methylgreen, provided a high-resolution contact map of the operator that was further verified by saturation mutagenesis of half an ARG box ( 541). The emerging picture indicates that a single hexameric ArgR molecule covers a pair of adjacent ARG boxes and makes contacts with two major groove segments and the intervening minor groove of each ARG box, all aligned on one face of the helix ( Fig. 3 and Fig. 4). In contrast, the center of the operator remains largely uncontacted. This symmetrical pattern and the stoichiometry of the interaction, combined with the configuration of the repressor molecule, suggest that each ARG box is contacted by two subunits, belonging to opposite trimers. In principle there are two ways of grouping two subunits belonging to opposite trimers into a functional DNA-binding dimer: with the α3 recognition helices facing each other and the wings pointing away, or, conversely, with the α3 recognition helices pointing away and the wings facing each other. The observation that the minor groove segment at the center of the operator is uncontacted, whereas the outermost minor groove segments facing the repressor play an important role in complex formation, indicates that funtional DNA-binding dimers most likely result from the pairing of two DNA binding domains with the wings pointing towards each other, facing and contacting the center of the boxes.
Methylation protection and interference experiments on different operator sites highlight several guanines in the major groove (for numbers, see Fig. 2 and Fig. 3)—bp 4-15', 15-4', and 14-5'—whose functional importance is further emphasized by the single-substitution mutations with an O c phenotype that have been obtained in vivo in argECBH, argF, and carAB ( 399, 401, 402, 432, 522). These positions also belong to the most highly conserved base pairs in the ARG box sequences. Uracil substitution interference experiments emphasize the importance of the hydrophobic 5-methyl groups (exposed in the major groove) of thymine residues at positions 3 and 13 of the ARG boxes ( 541). Several symmetrically located negative effects of premethylation or removal of A residues in the minor groove have also been noted ( 83, 294, 541), and in a saturation mutagenesis experiment of half an ARG box in the carAB operator, the most pronounced effects upon base pair substitution were observed at positions 10-9' and 12-7', in a segment of the operator where the minor groove is facing the repressor ( Fig. 4; see also reference 541). The importance of minor groove contacts is also reflected in the strong negative binding interference effect of the minor groove-specific ligand distamycin A, and can also be inferred from the sequence conservation in these AT-rich central segments. Strong minor groove base conservation indeed strongly suggests repressor-induced DNA distortion with direct or indirect contacts ( 453). The appearance of sites hyperreactive for DNase I in each ARG box ( 83, 498) is indeed indicative of ArgR-induced DNA deformation. The calculation of the bending angle from shifts determined with the circular permutation method ( 250, 497) indicated an overall deformation by 70 to 85° for the argF and artificial symmetric consensus operator ( 479, 498). Bending occurs within each ARG box of the operator, rather than being centered in the middle of the operator ( 479), between the two boxes.
The stoichiometry of repressor-operator interactions has been estimated in E. coli by quantitative DNase I footprinting ( 83) and by gel retardation with labeled repressor ( 498). The results of both methods indicate that one hexamer binds to a pair of boxes; each hyphenated ARG box would therefore contact two repressor subunits. Gel retardation experiments suggest that the stoichiometry is the same in Salmonella species ( 294). This mode of interaction would leave one site free on the repressor molecule; as a matter of fact, DNase I footprinting experiments performed with the argG control region, which contains a single ARG box 101 bp upstream from the standard tandem pair, suggest that this third site may become occupied if a loop is formed ( 83). Moreover, stoichiometric titration experiments with a single palindromic sequence indicate a 3:1 DNA:hexamer complex, implying that all six subunits are active for DNA binding and can be occupied simultaneously ( 479).
The study of interactions of the E. coli repressor with a variety of ad hoc constructions showed the importance of correct spacing between the two boxes ( 83). Binding of ArgR to a pair of correctly spaced ARG boxes shows a chelate effect. Reducing the 3-bp spacer to 2 bp already creates a partially constitutive phenotype ( 399). Increasing the spacer length showed that equivalent positions in tandem pairs of boxes must be properly aligned on the same face of the helix for efficient recognition to occur ( 83). The repressor binds to a single box but with a much higher K d than to a pair (between 5- and 160-fold higher), the lower K d being obtained with the box showing the best fit to the consensus ( 83, 90, 294, 479, 498).
The affinity of the repressor for arginine is around 10 -4 M ( 482), whereas the K d of the liganded protein for the various operators is around 10 -9 to 10 -10 M ( 83). Therefore, and since the intracellular concentration of ArgR is rather high (10 -6 M), the efficiency of the repression will in the first place depend on the concentration of active repressor hexamers and thus on the internal concentration of the corepressor. Even full repression will be achieved by a relatively small fraction of the total population of repressor molecules ( 500). The weak repressibility of argR expression would reflect the need to maintain a sufficient number of repressor molecules to ensure efficient repression (see reference 238). On the other hand, as the arginine enzymes are still 80 to 90% repressed in cells growing in minimal medium without arginine, a moderate increase in repressor concentration may be necessary to maintain a sufficient level of active repressor.
As would be expected from a system controlled solely by transcriptional repression, physiological derepression of argR + cells, achieved by limiting the arginine supply in various ways ( 98, 300, 368), leads to maximal enzyme levels very similar to those found in stringent argR mutants. This correlation contrasts with the situation encountered in tryptophan biosynthesis, in which starvation for tryptophan results, via the attenuation control mechanism, in much higher levels than in trpR mutants.
Not surprisingly, large differences in repression coefficient measured in vivo ( Table 2) are not paralleled by comparable differences in apparent equilibrium dissociation constants measured in vitro ( 83). The extent of promoter-operator overlap could strongly influence the actual extent of repressibility ( Fig. 2; Table 2). There is a rough correlation between the repression coefficient and the extent of overlap ( argE, –35 region in the overlap, R of 200; argCBH, argE, and carAB, –35 region out of the overlap, R from 50 to 60; argD, –35 region adjacent to the region covered by the repressor, R of 16). An actual overlap appears necessary for efficient repression; not only do the binding of RNA polymerase and the binding of repressor appear to be mutually exclusive in the E. coli carAB control region ( 85), but it has also been repeatedly observed that bound arginine repressor does not greatly hinder transcription initiated upstream of it, whether from the pyrimidine-specific promoter of carAB ( 85, 403) (see below), the secondary argEp2 promoter ( 103), or the outward promoter of an IS 3 element ( 81, 83). The differences observed between in vitro binding constants and repression coefficients might also be accounted for, at least in part, by the relative positions of RNA polymerase and the repressor around the double helix.
How l-arginine allosterically activates the E. coli repressor is presently unknown, but structural studies on the homologue from B. stearothermophilus indicate that ligand binding results in a tightening of the interface between the core trimers and induces a rotation by 15° of one trimer with respect to the other ( 363). Modeling studies strongly suggest that this arginine-dependent rotation is required to allow the perfect docking of four recognition helices of the wHTH motives into four consecutive major groove segments of the bent operator, aligned on one face of the DNA ( 363) (see also below). In the unliganded form the recognition helices would collide with the phosphate backbone and/or the minor grooves. Though it is very likely that a similar model might prevail for the closely related repressor from B. subtilis (72% sequence identity), it is at the moment not evident that the trimer rotation model is a general feature of the bacterial ArgR family and would be valid for the E. coli and S. enterica serovar Typhimurium repressors, especially since no net reorientation of subunits or trimers is observed in the the hexameric E. coli ArgR core with or without l-arginine ( 518).
The configuration of the repressor-DNA complex is still a matter of conjecture. Until now, no other form of the E. coli and S. enterica serovar Typhimurium repressor has been observed than the hexameric one, but it is not known whether a hexameric form may not actually assemble on the DNA in vivo. In this context, the anticompetitive effect of excess operator DNA observed with the B. subtilis repressor is certainly noteworthy (see above). This could also explain the somewhat puzzling fact that an almost perfect linear correlation with a slope around 0.5 appears between binding constants ( K d all between 10 -10 and 10 -9 M) and the in vivo repression coefficients when plotting the logarithm of K d versus the logarithm of R ( 83). This cooperative pattern, already predicted by Berg ( 29) on the basis of a statistical sequence analysis, could arise if two repressor molecules were to bind cooperatively to adjacent boxes (which the stoichiometry experiments certainly do not suggest) or if parts of the repressor were to assemble on operator DNA (which at least in vitro appears to be unlikely for E. coli ArgR as well [ 479]). More likely, the correlation could, at least in part, be ascribed to the fact that operator DNA itself is an allosteric effector of ArgR binding ( 479; also see below). On the other hand, the correlation observed could be a coincidence or, alternatively, could reflect the assembly of pairs of hexamers respectively bound at the ARG boxes present in the promoter region and at other sites as yet undisclosed.
Interestingly, l-arginine was found to be an effector of DNA specificity (enhanced ratio of specific/nonspecific sequence binding) and not only of affinity, an observation that implies that operator DNA is itself an allosteric effector for E. coli ArgR ( 479). This is likely due to bending of the palindromic ARG box sequence, which might enforce the cooperation of two repressor subunits. The possibility of ArgR trimers (from E. coli and other organisms) binding to operator DNA is probably the most controversial matter in arginine regulation. Binding studies with mutant operators altered in the arginine binding pocket or altered in the oligomeric state led D. Sherratt and coworkers ( 57, 90) to hypothesize that the residual DNA binding of E. coli ArgR in the absence of arginine is due to binding of the trimeric form of the repressor. Two kinds of complexes ascribed to binding of trimers and hexamers of E. coli ArgR were also observed by Holtham et al. ( 197), who exploited this observation to explain the DNA concentration dependence, also observed with the B. subtilis repressor (see anticompetitive effect). However, Szwajkajzer et al. ( 479) provided an alternative explanation that does not invoke trimer binding. According to their proposal, the faster-moving species observed in gel-shift assays could correspond to a single hexamer bound in a 1/1 complex to a tandem pair of ARG boxes, while the slower-migrating species might contain two bound hexamers, each interacting with a single ARG box. This model implies that normal operator binding with wild-type repressor relies on communication among four subunits of the hexameric repressor and that this communication can be altered or lost by mutation or by solution conditions. Consistently, the same authors were unable to detect DNA binding of mixed multimers consisting of intact and truncated C-terminal ArgR subunits, unless four intact subunits were present ( 479). This result seems to exclude the binding of an E. coli ArgR trimer to operator DNA and suggests that the effective minimal functional unit is one pair of subunits belonging to opposite trimers. Obviously, further experimentation is required to settle this delicate point and to unravel the molecular mechanism of the allosteric activation of ArgR.
The regulation of arginine biosynthesis presents a paradox: while E. coli K-12 exhibits potentially high and extensively repressible levels of enzyme, E. coli B displays lower levels slightly inducible by arginine. Nevertheless, fully derepressed mutants with comparable specific activities could be obtained from both strains by selection for resistance to canavanine ( 157). Further work ( 214, 215, 228) disclosed that argR K and argR B were alleles and specified repressor molecules with different properties. E. coli B is slightly derepressed at low intracellular arginine concentrations; it is repressed at intermediate concentrations but becomes slightly derepressed again beyond a certain threshold. The strain B repressor was therefore postulated to exist in different interconvertible forms ( 215, 228). The genetic aspects of this unifying account were confirmed by the extensive complementation studies of Kadner and Maas ( 224). In each combination of alleles (K-12–K-12 or K-12–B), the allele giving the lowest enzyme level in the presence of arginine was found to be dominant. Sequence studies showed that the B and K-12 repressors differ by one amino acid residue only: a proline (residue 70) in strain K-12 is substituted by a leucine residue in strain B. The strain B protein is also a hexamer and exhibits physicochemical properties which are slightly different from those of its K-12 counterpart ( 499). Most significantly, however, DNA footprinting experiments revealed that in the presence of arginine, the K-12 repressor has greater affinity for argF operator DNA than that of B does, whereas in the absence of arginine, the reverse obtains ( 499). It therefore appears that free B repressor (but not free K-12 repressor) is able to repress the transcription of arg genes under physiological conditions. When arginine is present, the B strain becomes only weakly repressed because (i) the affinity of the repressor bound to arginine for operator DNA is lower in strain B than in strain K-12 and (ii) autoregulation of the synthesis of B repressor reduces the intracellular concentration of the free protein.
Could such different patterns of regulation as seen in strains K-12 and B reflect adaptation to different natural habitats? The K-12 strain has a much greater potential for enzyme synthesis and may therefore be better armed for survival under conditions of arginine starvation ( 499). To test this possibility, the argR gene of E. coli B has been introduced in the strain K-12 background and the fitness of both strains has been compared using chemostat competition experiments ( 475). The results indicate that the argR K allele is favored in the presence of arginine or at high growth rates in the absence of arginine, whereas the argR B allele is favored only at low growth rates in the absence of arginine. Therefore, the K-12 strain appears to be favored during rapid growth in nutrient-rich environments whereas the argR B-carrying derivative would be favored during slow growth in nutrient-poor environments. The results of cycling environments indicate that the outcome of competition depends on the cycling rate between arginine-free and arginine-supplemented media, with strain K-12 being favored at high switching rates and strain B at low cycling rates. Interestingly, these results suggest that in variable environments a weak constitutive expression may be an adaptive strategy that constitutes a selective advantage over strong regulation.
Regarding feedback inhibition of N-acetylglutamate synthetase, the intrinsically low level of enzyme present in strain B ensures efficient regulation.
Since transcription of the carAB operon is cumulatively repressed by arginine and the pyrimidines and to some extent also by purines, the cognate control region is expected to be considerably more complex than those of other genes of the arg regulon. Indeed, both in E. coli and in S. enterica serovar Typhimurium, transcription of carAB is initiated at two tandem promoters ( Fig. 2 and Fig. 5) ( 40, 247, 403) which differ in their regulatory properties, as detailed below. The full-range regulation by signal molecules from three different biosyntheses requires the participation of at least five multifunctional proteins: the arginine repressor (ArgR), integration host factor (IHF), aminopeptidase A (PepA), UMP-kinase (PyrH), and the purine repressor (PurR), some of which combine catalytic activity and regulatory properties.
P2, the downstream promoter, is regulated specifically by arginine. A repression coefficient (ratio of activities in argR –/ argR +) of 50 was established with a low-copy carp2-lacZ fusion ( 83). P2 overlaps a tandem of ARG boxes as do all other arg promoters and is regulated by the argR product ( Fig. 2). Binding of RNA polymerase and binding of ArgR at carp2 were shown to be mutually exclusive in E. coli ( 85), and single-round in vitro transcription assays with supercoiled template DNA demonstrate that repression is based on an arginine-dependent exclusion of RNA-polymerase binding at carp2 (Charlier, unpublished data).
P1, the upstream promoter, is specifically regulated by pyrimidines through two different mechanisms: UTP-sensitive reiterative transcription initiation, which accounts for about 30% of the regulation and is essentially operative in pyrimidine-limited cells ( 182), and a major, architectural and regulatory protein-dependent mechanism which is still not fully understood but involves several multifunctional proteins (IHF, PepA, PyrH; discussed below). In addition, in excess purines the carp1 activity is two- to threefold downregulated by PurR, the master regulator of the purine biosynthesis ( 121, 296), and, moreover, carP1 is subject to stringent control ( 40). In both species, it is clear that ArgR is not involved in P1 control ( 85, 294) and, most importantly, that transcription initiated at P1 is not blocked by P2-bound repressor in vivo ( 247, 403) or in vitro (Charlier, unpublished data). P1 does not present the attenuation features found in the control region of the pyrBI operon ( 431, 508).
The bulk of data available on P1 control give a rather complex and as yet still incomplete vision of how pyrimidines exert their control on carAB expression. In vivo studies with single-copy carp1- lacZ operon fusions indicated that excess pyrimidines result in an approximately 20-fold downregulation of promoter activity ( 121, 182). Three multifunctional proteins, IHF (integration host factor), PepA (aminopeptidase A), and PyrH (UMP-kinase) ( 78, 82, 243, 432), are directly involved in this process in E. coli and likely in S. enterica serovar Typhimurium as well. Old observations on "escape" synthesis of CPSase in E. coli suggested the existence of a pyrimidine-specific regulatory molecule able to repress carAB ( 152). Studies of mutants obtained later on revealed the existence of a car-specific regulatory gene, carP, a trans-dominant allele of which leads to constitutive P1 expression ( 432). carP was subsequently found to be identical to pepA (also xerB) ( 76), encoding the major aminopeptidase of E. coli, a multifunctional protein that is also involved in the Xer/ cer site-specific recombination reaction ( 469), where it ensures, in conjunction with ArgR, that the reaction is preferentially intramolecular ( 96, 165). Further mutant analyses of pepA established its negative effect on P1 transcription ( 80). PepA is a homohexameric protein of 55-kDa subunits; its three-dimensional structure (dimer of trimers) has recently been determined ( 473). Each subunit consists of two domains; a smaller N-terminal domain is connected through a 24-amino-acids-long α-helix to the larger C-terminal domain that carries the active site and forms the oligomeric core. The aminopeptidase activity of the protein is not required for its function in transcriptional control and in the resolution reaction ( 78, 80, 320). Purified PepA was shown to bind the carAB control region of E. coli and S. enterica serovar Typhimurium ( 78). The complex DNase I footprint suggested the direct interaction with two approximately 26-bp-long targets (PEPA boxes) in the carAB operator ( Fig. 5) and wrapping of the several hundred-base-pair-long control region around the bulky protein ( 78). PepA was also shown to bind its own control region, where it exerts negative autoregulation ( 78, 80), and to the ColE1 cer site ( 8). In the latter case, binding of PepA and ArgR is cooperative. PepA has no recognizable DNA-binding motif, but it shows deep grooves, running over the surface of the hexamer, that could accomodate a piece of DNA ( 473). Mutant analyses have indicated the importance of the N-terminal domain of PepA in DNA binding ( 80). Mutations in the PEPA boxes of the carAB control region or alterations of the distance separating these sites result in partial constitutive initiation at P1 ( 77, 121).
Binding of IHF to a target site 300 bp upstream of the initiation site of P1 transcription in the E. coli and S. enterica serovar Typhimurium carAB control regions ( Fig. 5) exerts antagonistic effects: stimulation of carp1 promoter activity in minimal medium and enhanced repression in the presence of pyrimidines ( 82). Moreover, the addition of pyrimidines significantly enhances the amount of IHF present in the cell, a phenomenon which is probably of more general significance ( 82). The involvement of IHF and PepA in various cellular activities; the mode of action of IHF in other regulatory systems; the observation that binding of IHF and PepA, in vitro at least, occurs in the absence of any pyrimidine-specific cofactor; and the profound structural alterations imposed on the control region upon binding of these proteins suggest a major structural role for PepA and IHF in the elaboration of the regulatory nucleoprotein complex.
The sensor of the pyrimidine-specific regulatory response modulating carp1 activity appears to be the hexameric UMP-kinase, the pyrH gene product ( 243). This statement is inferred from the behavior of single amino acid substitution mutants that do not significantly affect the phosphorylation rate but yet are impaired in the pyrimidine-specific repression of carp1 activity. This regulatory deficiency could not be complemented by overexpression of the recessive mutant allele, nor by the introduction in E. coli of the heterologous UMP-CMP-kinase gene of Dictyostelium discoideum, in spite of an elevated UMP-kinase catalytic activity observed in such transformants ( 243). The regulatory pyrH mutations have only a minor and likely indirect effect on the expression of the other pyrimidine biosynthetic genes.
Curiously, E. coli UMP-kinase does not display significant sequence similarity to other NMP-kinases but is a member of the aspartokinase family ( 460) that was shown by comparative modeling to exhibit structural similarity with carbamate kinase ( 269). The hexameric protein is subject to complex allosteric regulation by GTP (activator) and UTP (inhibitor) ( 55, 460). In contrast to the ArgR and PepA hexamers, which are composed of two trimers, one on top of the other, PyrH appears to exist as an in-plane assembly of three dimers ( 269).
Specific binding of purified PyrH to the carAB control region has not been demonstrated, but the characteristics of particular pepA mutants, not affected in the DNA binding capacity but yet deficient for the regulatory process, suggest that protein-protein contacts, maybe with PyrH, might play an important role in the establishment of the regulatory response ( 80).
In E. coli, pyrH is an essential gene; it has also (under the denomination smbA) been described as being involved in chromosome partitioning ( 560). Whether this requires a property of the protein different from its enzymatic activity remains, however, to be elucidated. At first sight, at least, this appears unlikely since the pleiotropic phenotype of the smbA mutant could be suppressed by overexpression of the cmk gene encoding CMP-kinase ( 134, 559).
In S. enterica serovar Typhimurium the carp1 activity was shown to be about threefold downregulated in the presence of excess purines by binding of liganded dimeric purine repressor (PurR) to a PUR box sequence, 120 bp upstream of the initiation site of P1 transcription ( 296). A similar effect was recently demonstrated in E. coli as well ( Fig. 5), and the molecular mechanism of this control was investigated in depth ( 121). In vivo repressibility studies in various genetic backgrounds ( ihfA, pepA, pyrH, purR) affecting the trans-acting elements involved in pyrimidine-specific and in purine-specific regulatory elements and in cis-acting O c type mutants, coupled with in vitro PurR binding studies and single-round in vitro transcription assays, clearly established that liganded PurR is by itself unable to inhibit P1 activity ( 121). Instead, PurR action was shown to rely on the complex nucleoprotein structure that also imposes pyrimidine-specific repression of carp1. This tight coupling between the purine- and pyrimidine-specific regulatory mechanisms is unprecedented for PurR-mediated control and is likely due to the unique position of the PUR box in the carAB control region ( Fig. 5), too far upstream of the promoter to allow a direct inhibition of RNA polymerase binding. The structural modifications of the control region brought about by binding of the architectural proteins PepA and IHF might bring PurR into a position from where it could directly interact with polymerase binding. Alternatively, liganded PurR could become part of the pyrimidine-specific complex, thus extending its regulatory range in the presence of excess purines.
P1 activity was shown to be reduced in a relA + strain in conditions of amino acid starvation, indicating that the carAB operon belongs to the class of stringently controlled genes ( 40), as is the pyrBI operon ( 506, 566) encoding ATCase, catalyzing the first committed step in pyrimidine biosynthesis. In keeping with this observation, the carP1 promoters of both E. coli and S. enterica serovar Typhimurium bear a potential discriminator box sequence (GCCGCCG), in between the –10 promoter element and the start point of transcription, that might function as a high-energy barrier and reduce the isomerization rate. Noteworthy, RNA polymerase binding to carp1, but not carp2, was found to be very sensitive to heparin when present on linear templates ( 85), but not on supercoiled DNA (Charlier, unpublished data). Similar observations have been made with other stringent promoters ( 566), and the sensitivity to salt, heparin, and superhelicity appears to be a common feature of this class of promoters, which reflects their unique interaction with the RNA polymerase. Indeed, the rate-limiting step in transcription initiated at stringent promoters appears to be the stability of the RNA polymerase-promoter complex ( 566).
To add to the complexity but also to the interest of the carAB system, it should be mentioned that in Salmonella species, mutations affecting the RNA polymerase itself may lead either to enhanced levels of CPSase or to reduced levels and hyperrepressibility by uracil and/or arginine, with the rest of the arginine and pyrimidine regulons remaining unaffected ( 219, 362). In Salmonella species also, the use-1 mutation ( 58) which results in superrepression of P1 by pyrimidines was shown to affect the gene for a minor arginyl-tRNA gene, an observation which remains unexplained ( 292).
Even if no complete molecular picture is as yet available for pyrimidine- and purine-specific modulation of carp1 activity, the results at least converge in suggesting that several proteins (IHF, PepA, PurR, and maybe PyrH) interact with the carAB control region and, directly or indirectly, with RNA polymerase to modulate transcription initiation at P1. IHF, PepA, and PyrH act in concert in the elaboration of a complex nucleoprotein structure that is required for pyrimidine-specific modulation of carp1 activity. This could be concluded from in vivo studies on the methylation status of a GATC site 106 bp upstream of the start point of P1 transcription (in between the PurR and downstream PepA binding sites; Fig. 5), whose protection against Dam methylase is correlated with downregulation of promoter activity, an effect that requires intact genes encoding the trans-acting factors IHF, PepA, and PyrH and an intact carAB control region ( 77, 79, 80, 243). Moreover, the analysis of the purine-specific repression indicates that PurR action relies on the same nucleoprotein assembly and structural deformations of the control region ( 121).
In broad outline, therefore, the carAB operon functions as follows. Both promoters are active under conditions of derepression. Excess pyrimidines inactivate P1, an effect that is enhanced in the presence of excess purines, and excess arginine represses P2. In the latter circumstance, P1 is accessible but, to express the operon, the polymerase has to displace the arginine repressor bound at P2. For this, two mechanisms appear possible a priori: either the formation of an active RNA polymerase-DNA complex at P1 actually destabilizes P2-bound repressor by a direct protein-protein interaction, or the very initiation of transcription at P1 renders the polymerase relatively insensitive to a downstream-bound repressor, as in the case of the trp-lac fusion studied by Reznikoff et al. ( 422) or the argEp2 and IS 3 outward promoters mentioned in Structure of Control Regions, above ( 81, 103). Whatever may be the mechanism, the recent observation of the phenomenon in a pure in vitro transcription assay with supercoiled template DNA (Charlier, unpublished data) eliminates the need for an additional (pyrimidine-specific) protein. Since it is clear that transcription initiated at P1 overrides arginine-mediated repression at P2, it may be not surprising that under strong pyrimidine limitation, carAB expression is only weakly repressed by arginine ( 85, 392). Moreover, at least in E. coli, repression by arginine at P2 is more intense when P1 is also repressed by pyrimidines ( 85); this can be easily understood if initiation at P1 facilitates expression at P2, leading to an apparent increase of repression at P2 when P1 becomes inhibited. In keeping with this interpretation, the enhancement of P2 repression by pyrimidines is not observed in an E. coli mutant in which P1 does not operate ( 85). The data are, however, not completely concordant in E. coli and S. enterica serovar Typhimurium. In the latter organism, some P2 derepression is observed when CTP is limiting, even in a P1-deficient mutant ( 297); taking into account that pyrimidine nucleotides exert no effect on ArgR binding to the carAB operator, the authors advocate a possible interaction between the arginine repressor and a specific pyrimidine regulatory protein ( 294).
The physiological significance of a dual-promoter structure is, in the case of carAB, particularly clear. At the level of gene expression, it complements the regulatory effects exerted on the enzyme itself. It also constitutes an elegant alternative to the existence of independently controlled CPSase isoenzymes as in fungi and B. subtilis ( 106, 113).
The two genes of the carAB operon are separated by a short intercistronic space ( 369, 403), and translation of carA is initiated at the atypical UUG codon ( 544). UUG is a weak initiating codon; carAB is, nevertheless, efficiently translated thanks to the presence of a strong ribosome-binding site ( 543).
The molecular description of the arginine regulon is now well advanced, but a number of basic physiological questions remain intriguing. The biological significance, if any, of the scattering of arginine genes in several units of expression which are not strictly coordinated is one of them. Operons may have been primordial structures ( 148) but, in principle, controlling genes individually offers greater flexibility than coordinated operon-type control. However, in Bacillus spp. ( 22, 106), most of the arginine biosynthetic genes form a tight cluster. It is true that in the latter case, there are two CPSases, one that is arginine controlled and encoded by a gene clustered with the arg loci and one that is pyrimidine controlled. It may well be that the pattern of gene organization encountered in E. coli and S. enterica serovar Typhimurium is, both qualitatively and quantitatively, related to the existence of only one CPSase in those organisms. At such a branching point, the system must be rather delicately poised; we have seen above (see Introduction) that even in the wild type, interference between the two tributary pathways does occur. Optimal functioning of such a system may be more readily achieved by individual fine tuning of the genes directly concerned ( carAB, argF, argI, and pyrBI) than by operon control. This necessity for fine tuning of genetic regulatory interactions was emphasized by the finding that expression of the argR gene from the strong tac promoter brings about the rather paradoxical result that formation of OTCase becomes derepressed in the absence of arginine because of increased repression of CPSase ( 499). This experiment is also a good example of the experimental approaches that have been made possible as a result of the cloning and sequence analysis of all the genes of the regulon.
At the protein level it would probably be misleading to try to interpret quantitatively the functioning of the pathway by viewing it exclusively as a succession of free enzymes. It is indeed likely that to function efficiently, the pathway is organized as a channel, even if it is a leaky or an unstable one. Early reports ( 5, 6) indicated that Salmonella OTCase was involved in the proper assembly and function, perhaps even the folding, of CPSase, therefore suggesting that specific contacts occur between the two enzymes. In Saccharomyces cerevisiae, acetylglutamate synthetase and acetylglutamate kinase were shown to associate in a complex allowing the coordination of the catalytic activities and feedback regulation of the two enzymes ( 1, 384). It is certainly not excluded that this complex also interacts with the reductase ( 1, 384; see also references 191 and 382 for N. crassa). In extreme thermophiles, in which carbamoylphosphate would be too thermolabile to resist exposure to the aqueous phase and is moreover decomposed into cyanate, a nonspecific carbamoylating agent, channeling of this metabolite was predicted to be a necessity and has indeed been shown to occur ( 275, 313, 314, 413, 516); in Pyrococcus furiosus it was shown that this involves actual contact between the consecutive enzymes ( 313, 314; J. Massant, Ph.D. thesis, Vrije Universiteit Brussel, Brussels, Belgium, 2004). It would be of considerable interest to investigate the occurrence of biosynthetic metabolic channels in mesophilic prokaryotes as well, considering, furthermore, that the structural basis for the specificity that would be expected to underlie such enzyme associations is a virtually unexplored domain. In psychrophilic microorganisms, where the catalytic efficiency of key metabolic enzymes, such as OTCase and dihydrofolate reductase, appears severely limited by a marked trade-off between catalytic activity and affinity ( 554, 555), metabolic channeling could compensate for these kinetic shortcomings.
In amino acid metabolism, arginine biosynthesis is one of the rare cases where, according to the organism considered, one and the same step (here the synthesis of ornithine) can be catalyzed by two completely nonhomologous enzymes: ArgE and the much more widespread ArgJ. Comparisons between gamma-proteobacteria suggest that ArgJ was lost in an ancestor common to the Vibrionaceae and the Enterobacteriaceae and was replaced by a deacetylase ( 556).
It is tempting to consider regulation of arginine biosynthesis in enteric bacteria as a striking example of recruitment since the same protein—ArgR (or XerA)—functions both in metabolic regulation and in a totally unrelated phenomenon, the resolution of ColE1 dimers, in association with the XerB protein (alias CarP or PepA). Similarly, ArgP, also a transcriptional regulator, proved identical with IciA, an inhibitor of initiation of chromosomal replication in vitro (see Arginine Transport, below). DNA-protein interactions involved in such primordial processes as chromosomal and plasmid replication control could have been recruited to implement transcriptional control of genes involved in metabolism.
Arginyl-tRNA synthetase shares an unusual property with glutamyl- and glutaminyl-tRNA synthetases, the two other class I (subclass C) tRNA-synthetases: it does not catalyze ATP-PP i exchange in the absence of tRNA Arg ( 118, 323, 324; also see reference 143 and references therein). Furthermore, no arginyl-adenylate intermediate accumulates, whether tRNA Arg is present or not ( 100). It is not clear whether ATP, arginine, and tRNA Arg coreact in one step by a concerted mechanism ( 291) or whether binding with tRNA Arg is a prerequisite to activate the enzyme and make the formation of arginyl-adenylate possible ( 323). If the latter were the case, the aminoacyl-AMP would react immediately with the tRNA Arg. It was shown ( 39) that in E. coli, arginyl-tRNA synthetase catalyzes ATP-PP i exchange in the presence of the analog tRNA Argc-c-2' dA (i.e., tRNA with 2'-deoxyadenosine at the 3' end), which cannot be acetylated. A classical two-step mechanism was therefore suggested.
The enzyme itself ( α protein) is a monomer with a molecular mass of 60 to 70 kDa ( 87, 287), in keeping with the nucleotide sequence of the cognate gene ( 130); it is copurified with a 40-kDa β protein whose function remains unknown. The two molecules can best be separated by two successive affinity elutions with a tRNA gradient after absorption of the protein preparation on a column of phosphocellulose ( 87). This procedure provides a 1,000-fold-purified, 95% homogeneous enzyme which is more active than previously reported preparations. A somewhat improved purification procedure was published subsequently ( 287).
Apparent K m values for arginine in the aminoacylation reaction are in the micromolar range ( 86, 87, 287, 288), and there is agreement on a random substrate addition mechanism ( 87, 288, 378). The K m for tRNA Arg, also in the micromolar range, appears higher than for other activating enzymes, even arginine-specific enzymes from other organisms ( 287, 288). If significant, this difference suggests that the actual concentration of tRNA Arg may be more critical in E. coli than in other systems. Canavanine is also esterified by the synthase. The K m for canavanine is 4.10 -4 M, and the V max is twice that found with arginine. Both canavanine and homoarginine inhibit the binding of arginine competitively, but homoarginine is not esterified ( 323, 337). Whether the enzyme is also inhibited by arginine precursors has been the subject of some debate. The synthetase of E. coli strains W and K-12 was claimed to be inhibited by argininosuccinate, ornithine, and citrulline ( 49). However, subsequent studies on partially purified extracts of strains W and K-12 (including the strain used by Brenchley and Williams [ 49]) showed that the enzyme remained insensitive to ornithine and citrulline up to 2 mM ( 86). At relatively high concentrations of argininosuccinate, the apparent inhibition exerted by this compound could be ascribed to isotopic dilution of the labeled arginine used in the assay by nonradioactive arginine produced from argininosuccinate by argininosuccinase present in the extract ( 86). When lower concentrations of argininosuccinate (1 μM) were used, the observations made by the two groups remained conflicting, and the discrepancy remains unresolved.
Mutants affected in arginyl-tRNA synthetase have been described. Canavanine-resistant (Can r) isolates of E. coli with both altered arginyl-tRNA synthetase activities and nonrepressible arginine biosynthetic enzymes have been reported ( 49). However, these observations could not be confirmed; double mutants may have been involved. In contrast, other Can r synthase mutants, with increased K m values for arginine or for ATP, were found to be fully repressible by arginine ( 192, 193); moreover, mutants with a block drastic enough to accumulate endogenous arginine showed enhanced repression. Hence, it was assumed that arginine and not arginyl-tRNA was the corepressor of the arginine regulon, an assumption validated by the results reported in the previous section. The mutants isolated by Hirschfield et al. ( 192, 193) provide evidence for a single arginine-activating enzyme in E. coli, encoded by the argS gene, which was cloned and sequenced ( 130). One argS mutation conferring resistance to canavanine and increasing the K m for ATP by five- to sixfold was shown to substitute a serine for an arginine, which is therefore suggested to be critical both for ATP binding and for the amino acid activation step ( 131).
Little is known about regulation of the synthesis of arginyl-tRNA synthase, but the upstream region does not show ARG box sequences. Like isoleucyl- and phenylalanyl-tRNA synthetases, the enzyme becomes permanently derepressed during cognate amino acid starvation ( 343, 361). In the case of phenylalanine-tRNA synthetase, this phenomenon was correlated with the existence of an attenuation mechanism dependent on the concentrations of tRNA Phe ( 505). A positive correlation between growth rate and the steady-state concentration of several synthetases has been observed ( 360). The approximate number of molecules of arginyl-tRNA synthetase per genome shifts from 192 when the cells use acetate as carbon source to 510 on glucose-supplemented medium and to 867 on rich medium. These values may be compared with the 1,500 molecules of OTCase and 2,000 molecules of CPSase that the cell makes on minimal medium supplemented with glucose. These levels of the different synthetases remain in approximate balance with those of tRNA, elongation factors, and ribosomes.
Transport of arginine into E. coli and S. enterica serovar Typhimurium is of the periplasmic type, common in gram-negative bacteria. Osmotic shock of wild-type E. coli abolishes transport activities and liberates three different periplasmic proteins binding arginine ( 548). One of them binds arginine, ornithine, and lysine (the argT-encoded LAO protein [ 72, 433, 434, 549]) and is part of the so-called high-affinity system for arginine transport ( K m, l0 -9 M for arginine); another binds arginine and ornithine (the AO protein coded for by gene abpS [ 65]) and belongs to a lower-affinity system ( K m, l0 -7 M for arginine); and the third appears to be specific for arginine and displays high affinity for it ( 433) (see below). The LAO system is repressible by lysine and the AO system is repressible by arginine and ornithine ( 72). It is not known whether the LAO system actually transports arginine. At any rate, at arginine concentrations of 10 -6 M or higher, the main route of entry appears to be through the lower-affinity system ( 179). In addition, an arginine-repressible acetylornithine permease was reported by Vogel ( 530); its relationships with the components involved in arginine transport are not known. Studies on arginine uptake and accumulation have taken considerable advantage of the fact that decarboxylation of this amino acid into agmatine is inhibited by aminooxyacetic acid ( 547).
The AO system has been thoroughly studied by Celis and coworkers ( 64, 65, 66, 67, 68, 512). Large quantities of the AO-binding protein are produced by strains obtained by selecting for d-arginine utilization ( 64). These mutants display increased uptake of arginine and ornithine (no longer repressible by these amino acids) and are oversensitive to canavanine ( 53). The corresponding regulatory mutation was called abpR, whereas the structural gene, identified by nonsense mutations producing truncated versions of the AO-binding protein ( 63), was called abpS. Both types of mutations were mapped close to argA ( 64). It is not known whether the abpR mutations act in trans or in cis.
Celis and Urban also characterized a membrane protein which has an intrinsic ATPase activity and is able to phosphorylate the AO- and LAO-binding proteins. The corresponding structural gene ( argK) was located close to serA by using an argK-defective, canavanine-resistant mutant ( 67, 68, 512). Normal transport was shown to require ATP hydrolysis by the argK protein but not the phosphorylation of the LAO- and AO-binding proteins, as revealed by the analysis of canavanine-sensitive revertants of argK mutants ( 70). Despite its functional similarity with the ATP-binding subunit of ABC transporters, ArgK does not display significant sequence identity with proteins of this class. Several other transport-deficient and canavanine-resistant strains proved to harbor mutations located in the same region ( 63, 72, 301, 302, 434, 454). One of them—called argP—was shown to result in reduced transport of arginine, ornithine, and lysine; the cognate mutant also displayed reduced amounts of the arginine transport elements—including ArgK—although the K d for arginine binding of the AO protein and the K m for ATP of ArgK remained unmodified ( 68). Celis ( 69) showed that the argP gene codes for a regulatory protein of the lysR family, which negatively regulates its own synthesis but activates the synthesis of ArgK. Curiously, ArgP proved to be identical with and share the same DNA target as IciA, a protein which inhibits initiation of chromosomal replication in vitro.
The characterization of ArgP clearly demonstrated that a major component of the arginine transport system was regulated independently from the arginine regulon, as indicated previously by the behavior of argR mutants ( 64). However, the cluster of genes called artPIQMJ ( art meaning arginine transport), which was mapped around min 16 ( 549; also identified in the Salmonella genome), displays putative ARG boxes in front of artP and artJ. artJ encodes a protein binding l-arginine specifically and with high affinity; it could be the arginine-binding protein first described by Rosen ( 433). Considering their sequence similarity with the genes involved in the transport of lysine, arginine, ornithine, and histidine in S. enterica serovar Typhimurium (see below), artQ and artM might encode transmembranous proteins and artP might encode a membrane-associated ATPase ( 549).
The situation in S. enterica serovar Typhimurium differs somewhat from that in E. coli. The existence of a high-affinity, arginine-specific transport system and of another one common to lysine, arginine, and ornithine (LAO) was documented long ago ( 414). Homoserine and transhydroxyproline proved to be good inhibitors but not substrates of the arginine-specific system; homoserine was without effect in E. coli ( 63). The effect of these substances and the lack of effect of ε- N-methylhomoarginine in S. enterica serovar Typhimurium suggest that the secondary nitrogen of arginine may act as a donor in hydrogen bond formation during the recognition process ( 414).
The LAO-binding protein of S. enterica serovar Typhimurium was found to interact with the membrane-bound proteins involved in the histidine transport system, as originally brought to light by the study of hisP mutants; the latter, however, retain normal high-affinity, arginine-specific transport ( 264). The relationship between the LAO protein and the components of the histidine transport system has been analyzed in detail in S. enterica serovar Typhimurium but remains to be determined in E. coli. The argT gene, encoding the Salmonella LAO protein, lies immediately upstream from the hisJQMP operon, which encodes, in order, a histidine-binding protein (clearly homologous to the argT protein), two transmembranous proteins, and an ATP-binding protein ( 189, 257). A similar gene complex exists in E. coli ( 257, 367). The Salmonella LAO protein has been purified ( 365) and crystallized in the liganded and unliganded forms ( 227), and the three-dimensional structure has been determined ( 370).
From this concise survey, it appears that the situations in E. coli and S. enterica serovar Typhimurium are similar but the equivalent, if any, of the E. coli AO system remains to be identified in serovar Typhimurium.
The biosynthesis of putrescine, spermidine, and cadaverine in E. coli is well known inasmuch as the cognate enzymes and structural genes have been characterized ( 46, 95, 260, 302, 486, 552; see also below), but the cognate regulatory mechanisms have not been studied in much detail. Genes homologous to those of E. coli (also for transport functions) have been identified in the Salmonella genome sequence ( 319). It is clear that polyamines are required for optimal growth, although large variations of the polyamine pool can be present without exerting a pronounced effect on growth ( 483, 484). It is, however, very difficult to evaluate which cellular functions are most directly affected by their presence or their absence. The reader is referred to reviews by Davis et al. ( 114) and by P. Cohen ( 95) for a thorough discussion of the physiological and structural role of polyamines in bacteria, fungi, and mammals. More recent reports have involved polyamines in SOS induction of genes recA and uvrA ( 373) in enteroinvasion ( 61, 254), in protection against oxidative stress ( 88, 173, 242), and in porin-mediated outer membrane permeability ( 445, 446). Decrease in cell viability by excessive accumulation of polyamines can be prevented by covalent modifications of spermidine (acetylation or linkage to glutathione [ 135, 265]). Volume 94 of Methods in Enzymology contains detailed information concerning polyamine biosynthetic and biodegradative enzymes, as well as mutant screening techniques ( 485). Table 1 gives the gene-enzyme relationship for the steps involved in polyamine biosynthesis. A critical phylogenetic analysis of polyamine biosynthetic enzymes has been carrried out ( 457). In Euryarchaea the polyamine biosynthetic pathway uses an arginine decarboxylase which is not homologous to the bacterial one ( 163).
Both the biodegradative and the biosynthetic arginine decarboxylases have been well characterized. The biodegradative enzyme ( 137) was purified from E. coli B ( 35, 345) and shown to be a decamer with a molecular mass of 820 kDa. It is encoded by the adiA gene ( 467) and has been clearly involved in acid resistance in E. coli (the so-called AR system, not observed in Salmonella [ 286]; see reference 316 for a discussion of the regulatory network of acid resistance genes in E. coli). The adiA gene is positively regulated by AdiY, a transcriptional regulator of the XylS/AraC family ( 468). AdiA appears homologous to CadA, which codes for a lysine decarboxylase that is inducible by the activator CadC, a membrane protein, but repressible by LysP, a lysine permease also known as CadR ( 119, 359). Enteroinvasiveness of certain E. coli strains was found to be correlated with mutations inactivating cadC ( 60). AdiA is also homologous to SpeC (biosynthetic ornithine decarboxylase) and SpeF (biodegradative ornithine decarboxylase, absent in certain E. coli and Salmonella strains) but not to SpeA (biosynthetic arginine decarboxylase) ( 467).
The biosynthetic arginine decarboxylase (SpeA) is a tetramer (296 kDa) of a 70-kDa subunit ( 345, 551). The enzyme is located in the periplasm; the subunit is processed from a 74-kDa precursor ( 54). Because of this periplasmic location, the substrate arginine is channeled into putrescine before becoming mixed with the endogenous pool ( 488). The enzyme is inhibited by putrescine and spermidine, but, since the cognate K i values are in the millimolar range and the enzyme is extracellular, these effects probably have no physiological significance. Arginine decarboxylase is inhibited by difluoromethylarginine ( 225) and by aminooxyacetic acid ( 547), a property that has been particularly useful for studies on arginine transport. A specific antizyme (AtoC), together with ribosomal proteins S20 and L34, inhibits biosynthetic arginine and ornithine decarboxylases posttranslationally but is inactive on their biodegradative counterparts ( 59, 373, 374). After correction of cloning artifacts, the antizyme was found to be identical with the atoC gene product (see Swiss-Prot AccNum Q06065 and A. Sekowska, Ph.D. thesis, Université de Versailles-Saint Quentin-en-Yvelines, 1999). AtoC exerts a dual function in E. coli since it also acts as a positive regulator of the atoDAEB operon, involved in acetoacetate metabolism ( 89, 218).
Agmatine ureohydrolase (or agmatinase), which converts agmatine into putrescine and urea ( 350), has been purified from E. coli ( 450). It is a dimer of 38-kDa subunits. The enzyme is functionally and structurally similar to arginase (see reference 457). Inhibitory effects of ornithine and arginine were noted but are probably not physiologically significant. Together with arginine decarboxylase, this enzyme constitutes the so-called putrescine biosynthetic pathway II ( 346) ( Fig. 1). Mutations in this pathway were isolated by screening for strains defective in the production of urea ( 346) or by looking for strains requiring putrescine for optimal growth in the presence of excess arginine, which curtails ornithine synthesis and therefore prevents formation of putrescine by ornithine decarboxylation ( 194, 302, 305). The first approach gave mutants only partially blocked in speA (the arginine decarboxylase gene) or speB (the ureohydrolase gene) and not requiring putrescine for optimal growth. The second approach delivered tight speA and speB mutants with a reduced growth rate in the absence of putrescine, spermidine, or spermine. speA and speB form a complex operon ( 478) (see below).
Pathway I leads directly from ornithine to putrescine via the biosynthetic ornithine decarboxylase ( 349). Like the biodegradative ornithine decarboxylase, the biosynthetic one is a dimer with a molecular mass of 160 kDa. The two proteins appear evolutionarily related ( 15, 16, 345); they show 65.6% sequence identity (77.8% similarity). The regulation of the biosynthetic enzyme appears at first sight surprisingly complex: it is inhibited by putrescine and spermidine, activated by GTP, and inhibited by ppGpp ( 484) and appears to be regulated by phosphorylation ( 9). In addition, polyamines increase the specific activity of the inhibitory proteins called antizymes ( 187, 188, 266). Moreover, "antiantizymes," which bind to the antizyme and thus release the decarboxylase from the complex, have been described as well ( 266). In mammals, the antizyme would appear to facilitate degradation of the enzyme ( 114). It is not clear that all these regulatory effects are significant in E. coli: the K i values for putrescine and spermidine are again in the millimolar range, and it was even suggested that the antizymes may not be functional in vivo ( 230). Igarashi et al. ( 208), using partially purified preparations, suggested that ornithine decarboxylase had significant lysine decarboxylase activity, but this contradicts an earlier report ( 16). Enzymes decarboxylating both amino acids have been reported, however ( 274).
speC mutants define the gene for biosynthetic ornithine decarboxylase. They were isolated as derivatives of a speA strain requiring putrescine or spermidine in the absence of arginine ( 108). Their behavior suggests that putrescine can partially replace spermidine. speC appears homologous to speF (see below).
S-Adenosylmethionine decarboxylase was purified from E. coli ( 310, 545). It is a homohexamer with a molecular mass of 108 kDa. It requires covalently bound pyruvate for activity but not pyridoxal phosphate, and it is inhibited by decarboxylated S-adenosylmethionine. Unlike its mammalian counterpart, it is activated by Mg 2+ rather than by putrescine ( 484). It is also feedback inhibited by spermidine but at much lower concentrations than ornithine decarboxylase is.
Of all reported regulatory effects on the activity of polyamine biosynthetic enzymes in E. coli, it is this inhibition of S-adenosylmethionine decarboxylase by spermidine which would appear the most significant. It probably limits polyamine biosynthesis when the intracellular concentration of spermidine (most of which is normally bound to nucleic acids and phospholipids) becomes excessive. The concentration of putrescine is regulated largely by excretion of excess putrescine (see the discussion of transport systems, below).
The gene encoding S-adenosylmethionine decarboxylase is speD. Tight speD mutants grow at 75% of the wild-type rate and require spermidine for optimal growth ( 490, 553).
Pure spermidine synthase (or putrescine aminopropyltransferase), a dimer with a molecular mass of 72 kDa, was obtained from E. coli ( 41, 482). The enzyme also transfers the aminopropyl group to spermidine (to form spermine) or to cadaverine but much less efficiently than to putrescine; spermine is not normally found in E. coli, although small amounts were detected in speA speB speC mutants growing in the presence of spermidine ( 177). A putative transition state analog, S-adenosyl-1,8-diamino-3-thiooctane, inhibits the enzyme ( 385). The corresponding gene is speE, which forms an operon with speD ( 552).
Combinations of tight spe mutations have been used to create strains totally unable to synthesize putrescine and spermidine, in order to assess the physiological importance of these substances ( 177). A strain containing deletions affecting speA, speB, speC, and speD still grew at one-third the rate observed in the presence of spermidine. The cells were abnormal with respect to phage λ production, the mating ability of Hfr strains, and the adsorption of phage f2 ( 177). Putrescine was slightly less efficient than spermidine in restoring the growth rate. This partial requirement could be made absolute in a speA speB speC mutant by introducing an rpsL mutation ( 489), which affects the S12 ribosomal protein. Changes in ribosomal structure or conformation could therefore explain the stringent polyamine requirement. Indeed, polyamines are involved in ribosomal structure and in protein synthesis ( 481, 483, 484). The absolute requirement for polyamines displayed by the speA speB speC rpsL strain is also interesting because this strain should at first sight not be impaired in the synthesis of cadaverine, a putative substitute for the other diamines (but see below).
Under conditions of putrescine starvation, mutants unable to synthesize this compound produce detectable amounts of cadaverine (a product of lysine decarboxylation) and of its aminopropyl derivatives ( 177). Furthermore, exogenous cadaverine stimulates the growth of speB mutants depleted of polyamines by the addition of arginine ( 124). It was therefore conceivable ( 124; see also reference 542) that cadaverine would act as a substitute for other diamines. Data from Leifer (Z. Leifer, Ph.D. thesis, New York University Medical School, New York, N.Y., 1972) and Goldenberg ( 155) suggested that under physiological growth conditions, cadaverine would be produced by a lysine decarboxylase different from the inducible one (CadA) studied by Sabo et al. ( 439; see also reference 34). Evidence for such an enzyme was provided by Wertheimer and Leifer ( 542), who showed that E. coli grown in minimal medium at neutral pH displays a lysine decarboxylase activity inhibited by putrescine and spermidine. The gene responsible for this putative biosynthetic lysine decarboxylase ( ldc, homologous to cadA) has now been characterized, also in Salmonella ( 246, 284, 354, 558). Evidence for a substitute role of cadaverine is still inconclusive, however. Tabor et al. ( 480) constructed a speA speB speC speD strain also lacking inducible lysine decarboxylase activity (with a cadA genotype) and found this organism to be phenotypically identical to the cadA + parent, growing at a rate one-third of that found in the presence of polyamines. However, as noted by Wertheimer and Leifer ( 542), the identical growth rate of cadA and cadA + versions of the multiple spe mutants may reflect the fact that in the cadA + parent the cadaverine pool was already unusually low; ldc is indeed expressed only weakly ( 246, 284).
Of the so-called constitutive genes involved in the biosynthesis of polyamines, several have in fact been shown to be subject to metabolic control. However, observations made with different strains are conflicting, and their biological significance therefore remains unclear. The expression of speA and speC is partially repressed by putrescine ( 481, 487) and Panagiotidis et al. ( 373) have reported the existence of repressor proteins binding to the speC promoter. According to Boyle's group ( 449, 550), speA, speB, and speC are negatively and partially controlled by cAMP via the mediation of the cAMP receptor protein. From other data, this effect appears indirect and might involve an additional inhibitory protein ( 478). speD is probably repressed by spermidine ( 230). In contrast, working with strains different from those studied by Boyle and coworkers, Halpern and coworkers ( 180, 461) found that speB expression was subject to catabolite repression whereas speA and speC did not respond to different carbon sources. In addition, speB was stimulated by nitrogen limitation, which could actually override catabolite repression ( 461). speB can be induced by agmatine from a promoter internal to the speA speB operon ( 478); this accounts for previous claims that the two genes were not part of the same operon ( 177). Two- to threefold repression of speA by the purine repressor was reported ( 185).
Several systems are involved in the transport of polyamines ( 95, 206, 207, 353, and references below). The PotE protein actually excretes putrescine and is specific for this substrate ( 232). It functions as a putrescine/ornithine antiporter; its involvement in uptake of putrescine is probably small under standard conditions. A detailed functional analysis of PotE has been carried out and the putrescine recognition site has been identified ( 232). Since accumulation of high internal concentrations of polyamines is detrimental to cell growth ( 114), the potE system exerts a physiologically important function in the adjustment of polyamine content. potE is part of an operon (mapping at min 16) which also includes speF ( 234), encoding an ornithine decarboxylase inducible at low pH. speF appears homologous to speC (65.6% amino acids sequence identity, 77.8% similarity). Many E. coli strains were reported to lack the biodegradative ornithine decarboxylase, however ( 15). A CadB lysine/cadaverine antiporter also exists in E. coli; the protein is expressed from the same operon as CadA, the inducible lysine decarboxylase ( 61, 359, 464).
A complex transport system for putrescine and spermidine (but active mainly with spermidine) is encoded by four genes— potA, potB, potC, and potD—clustered in that order into an operon and mapping at min 15 ( 136). The system belongs to the family of ABC permeases. PotD binds to putrescine and spermidine. It is the periplasmic component of the system; its crystal structure has been determined ( 474). PotA is an ATP-binding protein associated with the membrane; the ATPase activity of PotA is inhibited by spermidine, which is thus a feedback inhibitor for its own transport. PotB and PotC form a transmembrane channel allowing spermidine transport. The interactions between these components have been analyzed in detail and the functional domains of PotA have been well defined ( 231).
Still another periplasmic transport system of the ABC family, whose genetic components map at min 19, has been reported ( 390). The system is encoded by one operon clustering the genes potF, potG, potH, and potI, respectively homologous to potD, potA, potB, and potC. The potF protein is a putrescine-specific periplasmic protein homologous to the potD protein; the crystal structure has been determined ( 523).
Periplasmic polyamine transport is, at least in part, energy linked ( 114, 136, 464), but sequestration of polyamines by binding to intracellular components is expected to favor global unidirectional transfer. Curiously, it would appear that streptomycin enters the E. coli cell via an inducible polyamine transport system ( 198). Further investigations of polyamine transport might help clarify some apparent contradictions: whereas Satishchandran and Boyle ( 449) found that none of their E. coli K-12 strains was able to utilize agmatine as a source of nitrogen, the reverse has been reported by Stalon and Mercenier ( 465) and Shaibe et al. ( 461) for other K-12 strains. Polyamine breakdown in microorganisms has been reviewed ( 173; see also references 295 and 356).
The network of reactions involved in arginine and polyamine biosynthesis is now well established. A previously unsuspected connection with lysine biosynthesis has been disclosed, other regulatory interferences begin to be understood, and the recent analysis of the inducible, cryptic ArgM transaminase has culminated in the identification of a new catabolic pathway. The "linear" logic that prevailed for so long in metabolic studies is yielding increasingly to more integrated analyses of cellular networks. At the genetic level, the interaction between a unique repressor molecule activated by arginine and a family of promoter regions containing similar operators accounts for the quantitative variations observed from gene to gene in the repression response, the molecular details of which are becoming clear. ArgR also appeared very early as a positive regulatory molecule—probably the first instance of regulatory protein with such a dual function—as indicated by the expression of the cryptic inducible argM ( astC) gene.
It was surprising to identify the XerA protein (involved in plasmid recombination) as identical to the ArgR and to see the XerB (alias PepA) protein involved, in conjunction with ArgR and UMP kinase, in the control of carbamoylphosphate synthesis. Here also, molecular studies of gene expression disclosed much more complex interactions than originally suggested by the successive identification of adjacent sites on the promoter. The dual role played by proteins such as XerA, XerB, and ArgP (alias IciA) suggests that proteins originally involved in the control of DNA replication and partition may have been recruited for transcriptional control in the course of evolution.
The physiological significance of the partially uncoordinated repression response of the arg genes remains a matter of debate, but it is clear that in terms of amplitude of expression, the genes for OTCase and ATCase stand out with respect to the other arg and pyr genes. When comparing different bacteria two alternative patterns actually emerge: either a large arg operon integrating an arginine-specific CPSase isoenzyme, or a scattered regulon with a car locus independently and subtly controlled by the two tributary pathways. Studies on possible metabolic channeling of biosynthetic intermediates and interactions between enzymes of the pathway could contribute much to our understanding of cell physiology.
Two peculiar genetic arrangements have been disclosed in the course of these studies: (i) the converging, facing promoters involved in the divergent transcription pattern of the argECBH cluster and (ii) the tandem promoters of the carAB operon. The physiological significance of the latter structure is particularly clear, even if the mechanisms controlling the promoter appear surprisingly complex.
Other observations of general interest made with the arginine system deserve further investigations, such as the influence exerted by mRNA sequences 5' to the Shine-Dalgarno box on the efficiency of translation (the case of mutations affecting argE translation) and the formation of chromosomal rearrangements resulting in tandem or inverted repeats of the argE gene. Several examples of putative enzyme recruitment (homologous enzymes performing analogous functions) have been mentioned in this chapter. They provide excellent material for evolutionary studies on the genetic basis of substrate specificity and functional flexibility in modern enzymes. It is also clear that our knowledge of the arginine and polyamine systems has benefited considerably from studies carried out with other microorganisms: we now understand better the place and the interconnections of these pathways in cellular metabolism, and rich material is at hand for further studies on molecular evolution in the prokaryotic domains.
Thanks are due to J. Charlier for critical reading of the manuscript, to P. Cornelis for suggesting a possible explanation for the effect of arginine on the growth of hemA mutants, and to J. P. ten Have for the artwork. Work pursued in our laboratory was supported over the years by Belgian Research Foundations (FNRS-NFWO, FRFC-FKFO, IRSIA-IWONL), the Research Programme of the Fund for Scientific Research-Flanders (FWO-Vlaanderen), the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen), the Research Council of the Brussels Free University (OZR-VUB), the Flanders Interuniversity Institute for Biotechnology (VIB), Concerted Actions between the Brussels University and the Belgian State (GOA), Grants from the European Communities, and the Vlaamse Gemeenschapscommissie.
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Module3.6.1.10
