Raymond Cunin

The evolutionary history of carbamoyltransferases: A complex set of paralogous genes was already present in the last universal common ancestor.

Abstract. Forty-four sequences of ornithine carbamoyltransferases (OTCases) and 33 sequences of aspartate carbamoyltransferases (ATCases) representing the three domains of life were multiply aligned and a phylogenetic tree was inferred from this multiple alignment. The global topology of the composite rooted tree (each enzyme family being used as an outgroup to root the other one) suggests that present-day genes are derived from paralogous ancestral genes which were already of the same size and argues against a mechanism of fusion of independent modules. A closer observation of the detailed topology shows that this tree could not be used to assess the actual order of organismal descent. Indeed, this tree displays a complex topology for many prokaryotic sequences, with polyphyly for Bacteria in both enzyme trees and for the Archaea in the OTCase tree. Moreover, representatives of the two prokaryotic Domains are found to be interspersed in various combinations in both enzyme trees. This complexity may be explained by assuming the occurrence of two subfamilies in the OTCase tree (OTC α and OTC β) and two other ones in the ATCase tree (ATC I and ATC II). These subfamilies could have arisen from duplication and selective losses of some differentiated copies during the successive speciations. We suggest that Archaea and Eukaryotes share a common ancestor in which the ancestral copies giving the present-day ATC II/OTC β combinations were present, whereas Bacteria comprise two classes: one containing the ATC II/OTC α combination and the other harboring the ATC I/OTC β combination. Moreover, multiple horizontal gene transfers could have occurred rather recently amongst prokaryotes. Whichever the actual history of carbamoyltransferases, our data suggest that the last common ancestor to all extant life possessed differentiated copies of genes coding for both carbamoyltransferases, indicating it as a rather sophisticated organism.

Cloning and endonuclease restriction analysis of argF and of the control region of the argECBH bipolar operon in Escherichia coli.

A 1.8 kb DNA fragment, liberated by endonuclease HindIII, contains the control region of the argECBH bipolar operon near one end and the weak secondary promoter of argH at the other extremity; it has been cloned in plasmid pBR322. The same plasmid vector has been used to clone the argF gene liberated from the chromosome by endonuclease BamHI. Restriction patterns for the two hybrid plasmids have been determined, using enzymes AluI, BglI, EcoRI, HaeIII, HincII, HindIII, HpaI and II, PstI and SalI. Two AluI sites situated on either side of and close to a HincII target delineate two short fragments covering the whole of the argECBH control region. The argF control elements are located in a region accessible to further dissection by BamHI, EcoRI, PstI and HindIII. Carriers of the argF plasmid produce extremely high amounts of ornithine carbamoyltransferase, a feature useful for purification of this enzyme.

Control Regions within the argECBH Gene Cluster of Escherichia coli K12

SummaryIn Escherichia coli K12, four of the nine structural genes involved in the biosynthesis of arginine (argE, C, B and H) form a tight cluster within which a clockwise-polarized unit of expression (argCBH) had previously been identified. From a mutant carrying an argCB deletion that greatly lowers the rate of expression of argE but falls short of known argE markers, we have isolated several derivatives in which the expression of argE is partly restored. In about a third of these strains repression of both E and H enzymes by arginine is almost abolished. The mutations responsible appear to be cis-dominant and to map to the right of argE, probably between argE and C. One mutant in which control of argE alone is affected has also been found; it is shown to carry a duplication of argE in addition to the argCB deletion of the parental strain. We discuss the hypothesis that argE and argCBH form two operons transcribed in opposite directions from an internal promoter-operator complex.It is also suggested that a secondary promoter exists at or near the argB-H boundary.

Arginine Biosynthesis in Escherichia coli EXPERIMENTAL PERTURBATION AND MATHEMATICAL MODELING

A basic challenge in cell biology is to understand how interconnected metabolic pathways are regulated to provide the adequate cellular outcome when changing levels of metabolites and enzyme expression. In Escherichia coli, the arginine and pyrimidine biosynthetic pathways are connected through a common metabolite provided by a single enzyme. The different elements of the arginine biosynthetic system of Escherichia coli, including the connection with pyrimidine biosynthesis, and the principal regulatory mechanisms operating at genetic and enzymatic levels were integrated in a mathematical model using a molecular kinetic approach combined with a modular description of the system. The model was then used to simulate a set of perturbed conditions as follows: genetic derepression, feedback resistance of the first enzymatic step, and low constitutive synthesis of the intermediate carbamyl phosphate. In all cases, an excellent quantitative agreement between simulations and experimental results was found. The model was used to gain further insight into the function of the system, including the synergy between the different regulations. The outcome of combinations of perturbations on cellular arginine concentration was predicted accurately, establishing the model as a powerful tool for the design of arginine-overproducing strains.

Aspartate Transcarbamylase from the Hyperthermophilic Archaeon Pyrococcus abyssi: Thermostability and 1.8A Resolution Crystal Structure of the Catalytic Subunit Complexed With the Bisubstrate Analogue N-Phosphonacetyl-L-aspartate.

The Pyrococcus abyssi aspartate transcarbamylase (ATCase) shows a high degree of structural conservation with respect to the well-studied mesophilic Escherichia coli ATCase, including the association of catalytic and regulatory subunits. The adaptation of its catalytic function to high temperature was investigated, using enzyme purified from recombinant E.coli cells. At 90 degrees C, the activity of the trimeric catalytic subunit was shown to be intrinsically thermostable. Significant extrinsic stabilization by phosphate, a product of the reaction, was observed when the temperature was raised to 98 degrees C. Comparison with the holoenzyme showed that association with regulatory subunits further increases thermostability. To provide further insight into the mechanisms of its adaptation to high temperature, the crystal structure of the catalytic subunit liganded with the analogue N-phosphonacetyl-L-aspartate (PALA) was solved to 1.8A resolution and compared to that of the PALA-liganded catalytic subunit from E.coli. Interactions with PALA are strictly conserved. This, together with the similar activation energies calculated for the two proteins, suggests that the reaction mechanism of the P.abyssi catalytic subunit is similar to that of the E.coli subunit. Several structural elements potentially contributing to thermostability were identified: (i) a marked decrease in the number of thermolabile residues; (ii) an increased number of charged residues and a concomitant increase of salt links at the interface between the monomers, as well as the formation of an ion-pair network at the protein surface; (iii) the shortening of three loops and the shortening of the N and C termini. Other known thermostabilizing devices such as increased packing density or reduction of cavity volumes do not appear to contribute to the high thermostability of the P.abyssi enzyme.

Arginine gene duplications in recombination proficient and deficient strains of Escherichia coli K 12

SummaryDuplications of arg genes selected as suppressors of polar effects occur in recA strains. Some of them are tandem duplications of short chromosomal segments. An “illegitimate” crossing-over between the two arms of a replication fork is envisioned as a possible mode of formation. The evolutionary interest of duplicated genes which have escaped their normal regulation is stressed.

Heterotropic interactions in Escherichia coli aspartate transcarbamylase : subunit interfaces involved in CTP inhibition and ATP activation

In Escherichia coli aspartate transcarbamylase, each regulatory chain is involved in two kinds of interfaces with the catalytic chains, one with the neighbour catalytic chain which belongs to the same half of the molecule (R1-C1 type of interaction), the other one with a catalytic chain belonging to the other half of the molecule (R1-C4 type of interaction). In the present work, site-directed mutagenesis was used to investigate the involvement of the C-terminal region of the regulatory chain in the process of feed-back inhibition by CTP. Removal of the two last C-terminal residues of the regulatory chains is sufficient to abolish entirely the sensitivity of the enzyme to CTP. Thus, it appears that the contact between this region and the 240s loop of the catalytic chain (R1-C4 type of interaction) is essential for the transmission of the regulatory signal which results from CTP binding to the regulatory site. None of the modifications made in the R1-C4 interface altered the sensitivity of the enzyme to the activator ATP, suggesting that the effect of this nucleotide rather involves the R1-C1 type of interface. These results are in agreement with the previously proposed interpretation that CTP and ATP do not simply act in inverse ways on the same equilibrium.

On the functional organization of the arg ECBH cluster of genes in Escherichia coli K-12

SummaryAmong the four seemingly adjacent loci of the argECBH cluster of E. coli K-12, the last three are shown to belong to the same unit of coordinated expression; the latter exhibits a clockwise polarity in contrast to all other known E. coli operons, except the cluster governing the synthesis of the pyruvate dehydrogenase complex.The analysis of several deletion and nonsense mutants suggests that argE (the expression of which is not strictly correlated with the functioning of the argCBH group) has the same polarity but is not integrated with the three other genes into one operon.Between polar argC B and B mutants the coefficient of repressibility of enzyme H synthesis varies widely. This feature resembles the reduced repressibility of distal gene activity found in polar mutants in the tryptophan operons of E. coli and S. typhimurium but not in the lac, gal (E. coli) and his (S. typhimurium) operons.Possible implications of the present results and some relevant data that have appeared in the recent literature are discussed.

Divergent transcription in the argECBH cluster of genes in Escherichia coli K12

Abstract DNA-RNA hybridization experiments performed with separated strands of a φ80 transducing bacteriophage carrying the argECBH cluster from Escherichia coli show that the transcription of the latter is bidirectional and under repression control by arginine. This is consistent with previous genetic evidence concluding that the transcription of argE and argCBH proceeds in divergent directions from an internal operator-promoter complex containing a repressor binding site common to the two wings of the cluster.

Positive and negative regulation of CAR1 expression in Saccharomyces cerevisiae

SummaryWe localized the chromosomal targets of several of the regulatory controls of expression of theCAR1 gene. Fusion tolacZ of several fragments of the 5′ non-coding region showed that induction ofCAR1 by arginine is positively regulated by the products of theARGR genes. The target lies upstream of another site where repression by the CARGRI molecule occurs. The latter control is not specific to arginine catabolism since it also affectsCYC-1 and indeed does not appear to involve arginine. The primary target of the two other regulatory allelesCARGRII andCARGRIII is not situated in the 5′ non-coding region. Deletion analysis supports the fusion data and confirms the order of the regulatory regions: 5′—nitrogen catabolite repression—activation by arginine—CARGRI-mediated repression—CAR1.