André Pierard

Control of the biosynthesis of carbamoyl phosphate in Escherichia coli

Eight one-step mutations leading to a double auxotrophy for arginine and the pyrimidines map in a small region of the Escherichia coli chromosome. Mutants bearing these mutations lack glutamino-carbamoyl phosphate synthetase. A mutation leading to sensitivity towards uracil is located within the locus responsible for the double auxotrophy. The joint evidences from genetic and enzymo-logical studies show that these mutations affect a single glutamino-carbamoyl phosphate synthetase which supplies carbamoyl phosphate for both the arginine and pyrimidine pathways. This enzyme is submitted to a double end-product control: (a) cumulative repression of its synthesis by arginine and uracil; (b) partial feedback inhibition of its activity by uridine-5′-monophosphate. The uracil-sensitive mutant has a hyper-retro-inhibited glutamino-carbamoyl phosphate synthetase.

Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation.

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria

Summary: The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus aquaticus and Thermotoga maritima, and in two thermophilic archaeobacteria, Sulfolobus solfataricus and Pyrococcus furiosus. In the first three organisms, arginine biosynthesis proceeds via N-acetylated intermediates as in mesophilic microorganisms. Only the enzymes catalysing the three last steps of the pathway could be detected in P. furiosus. The two eubacterial strains possess an ornithine acetyltransferase and are thus able to recycle the acetyl group from acetylornithine to glutamate. The archaeobacterium, S. solfataricus, uses the linear pathway in which the formation of ornithine is mediated by the hydrolytic enzyme acetylornithinase. Repression of enzyme synthesis by arginine was observed for most of the enzymes tested in T. aquaticus and S. solfataricus. Feedback inhibition by arginine was shown only on the ornithine acetyltransferase from T. aquaticus. This inhibition pattern is of interest since it would be the first example of control of arginine biosynthesis at this particular step. Data concerning the thermal stability of the arginine biosynthetic enzymes are presented.

The occurrence of a catabolic and an anabolic ornithine carbamoyltransferase in Pseudomonas

Abstract The occurrence of two ornithine carbamoyltransferases (carbamoylphosphate: l -ornithine carbamoyltransferase, EC 2.1.3.3) in Pseudomonas is demonstrated by their separation with ammonium sulfate. The two enzymes are distinguished by their activities as a function of pH. On the basis of the regulation of their synthesis, an anabolic function is assigned to one of these enzymes, a catabolic function to the other.

A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species

Summary: The expression of Bacillus stearothermophilus genes complementing arginine auxotrophs of Escherichia coli was studied. The activity responsible for the formation of ornithine in B. stearothermophilus was identified as a repressible ornithine acetyltransferase (genetic symbol argJ) encoded by the same DNA fragment as the argC, orgA and argB genes. Bacillus subtilis and Bacillus licheniformis displayed the same pattern of enzyme activities as B. stearothermophilus. In contrast to previous reports, these organisms consequently use the cyclic pathway of ornithine biosynthesis. B. stearothermophilus also possesses a broad specificity aminoacylase which exhibits low affinity towards N 2-acetyl-l-ornithine.

Primary Structure, Partial Purification and Regulation of Key Enzymes of the Acetyl Cycle of Arginine Biosynthesis in Bacillus Stearothermophilus: Dual Function of Ornithine Acetyltransferase.

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.

Regulation of the carbamoylphosphate synthetase belonging to the arginine biosynthetic pathway of Saccharomyces cerevisiae

Abstract Two classes of regulatory mutations affecting the synthesis of the carbamoylphosphate synthetase belonging to the arginine biosynthetic pathway have been selected in Saccharomyces cerevisiae. Together, they delineate a negative type of control. The cpaI0 mutations, closely linked with one of the two genes coding for the enzyme and cis dominant, meet properties of operator mutations. The cpaR mutations can be interpreted as mutations impairing the formation of an active repressor of carbamoylphosphate synthetase which is distinct from the one acting on the synthesis of the other enzymes of the arginine biosynthetic pathway.

Evolution of enzyme activities and microbial populations during composting of cattle manure

SummaryPopulation changes in the levels of mesophilic and thermophilic fungi and actinomycetes were studied during composting of cattle manure.Activities of urease, phosphatase, and cellulase were also followed. The results obtained in a bench-scale composter were compared with those obtained during pile-composting. The consequences of disturbances in the composting process were studied during a bench-scale operation. Our results indicate the potential usefulness of enzyme measures as indexes of accuracy of real composting.

The specialization of the two ornithine carbamoyltransferases of Pseudomonas

Abstract The two ornithine carbamoyltransferases (carbamoylphosphate: l -ornithine carbamoyltransferase, EC 2.1.3.3) of Pseudomonas are integrated into different metabolic sequences. This integration appears both in their regulation and in their activities. In Pseudomonas IRC 204, the catabolic ornithine carbamoyltransferase is subject to catabolite repression in the same way as arginine deiminase ( l -arginine iminohydrolase, EC 3.5.3.6) and carbamate kinase (ATP: carbamate phosphotransferase, EC 2.7.2.2). The catabolic ornithine carbamoyltransferase in vivo, has little or no biosynthetic function. The anabolic ornithine carbamoyltransferase, unlike other similar enzymes, is unable to perform the catabolic phosphorolysis.

Physiology and genetics of carbamoylphosphate synthesis in Escherichia coli K12

Summary76 mutants have been isolated in which the function of the single carbamoylphosphate synthetase of Escherichia coli K 12 is affected. A wide variety of phenotypes have been observed among these mutants, the most typical ones being: requirement for arginine and uracil, arginineless behaviour, sensitivity towards arginine and sensitivity towards uracil. The mutations have been localized by reciprocal transduction and deletion mapping; all are clustered in the same locus, car. The study of carbamoylphosphate synthesizing activities of these mutants and the combination of car mutations in various in vivo as well as in vitro complementation tests lead to the conclusion that car contains two genes: carA, covering the left part of the locus and coding for the “glutamine subunit” of the enzyme; carB, to the right, governing the synthesis of the heavy subunit of the enzyme.