Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene

Abstract

In Pseudomonas aeruginosa PAO the anabolic ornithine carbamoyltransferase (OTCase, EC 2.1.3.3) is the product of the argF gene and the only arginine biosynthetic enzyme whose synthesis is repressible by arginine. We have determined the complete nucleotide sequence of the argF gene including its promoter-control region. The deduced amino acid sequence of the anabolic OTCase consists of 305 residues (Mr 33,924), and this was confirmed by the N-terminal amino acid sequence, the total amino acid composition, and the subunit Mr of the purified enzyme. The native anabolic OTCase (Mr 110,000 to 125,000) was found to be a trimer by cross-linking experiments. P. aeruginosa also has a catabolic OTCase (the arcB gene product), which catalyzes the reverse reaction of the anabolic conversion. At the nucleotide sequence level, the P. aeruginosa argF gene had 52.4% identity with the arcB gene. The Escherichia coli argF and argI genes, which code for anabolic OTCase isoenzymes, had 47.3 and 44.9% identity, respectively, with the P. aeruginosa argF sequence. This suggests that these four genes have evolved from a common ancestral gene. The arcB gene appears to be more closely related to the E. coli argF gene than to the P. aeruginosa argF gene. Two transcripts (mRNA-1, mRNA-2) of the P. aeruginosa argF gene were identified by S1 mapping. The transcription initiation site for mRNA-1 was preceded by sequences having partial homology with the E. coli -35 and -10 consensus promoter sequences. No sequence similar to consensus promoters of enteric bacteria was found upstream of the 5' end of mRNA-2. E. coli carrying a P. aeruginosa argF+ recombinant plasmid produced mRNA-1 with low efficiency but no (or very little) mRNA-2. Arginine repressed argF transcription in P. aeruginosa. In the argF promoter region no sequence homologous to the "arg box" (arginine operator module) of E. coli was found. The mechanism of arginine repression in P. aeruginosa thus appears to be different from that in E. coli.Pseudomonas aeruginosa PAO has two ornithine carbamoyltransferases (OTCases; EC 2.1.3.3). The anabolic OTCase, which is the product of the argF gene [designated argF (P) quarternary structure of the purified anabolic OTCase from the same organism. This allowed us to make comparisons with the known sequences and structures of other OTCases, notably the catabolic arcB enzyme from P. aeruginosa (2) and the two anabolic argF [designated argF(E) herein] and argI isoenzymes from Escherichia coli K-12 (3, 48). We found that all of these enzymes had substantial sequence similarities.In P. aeruginosa, the anabolic OTCase is the only arginine biosynthetic enzyme whose synthesis is repressed by exogenous arginine (25,49); the arginine biosynthetic pathway is controlled essentially by feedback inhibition of the first two enzymes (17,18). Little is known about the mechanism of argF(P) repression because no regulatory mutants are available. By contrast, regulation of arginine biosynthesis has been studied extensively in E. coli K-12, in which the argR rep...

Transcriptome Analysis of the ArgR Regulon inPseudomonas aeruginosa

“…Exogenous agmatine but not arginine induced these genes (14,29). Very little is known about the enzymes or genes of the ADH pathway; only the gbuA gene and the bifunctional kauB gene have been characterized (32).For arginine biosynthesis, only the argF gene and the carAB operon, which encode ornithine carbamoyltransferase and carbamoylphosphate synthetase, respectively, have been reported to be repressible by arginine (1,17,19,49).With the completion of the Pseudomonas Genome Project and the use of innovative DNA microarray technology (42), it has become feasible to identify and characterize genes of metabolic pathways in a very efficient and systematic way. Considering the relatively large size of the P. aeruginosa genome and its reputation as a metabolically versatile organism, it is very likely that many "hypothetical" or "unknown" genes encode catabolic enzymes for the utilization of different nutrients that this organism encounters in its varied habitats.…”

“…For arginine biosynthesis, only the argF gene and the carAB operon, which encode ornithine carbamoyltransferase and carbamoylphosphate synthetase, respectively, have been reported to be repressible by arginine (1,17,19,49).…”

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis

“…The presence of a possible promoter region (TGTAAG and GGGTGT in Fig. 6) similar to the Ϫ10 and Ϫ35 regions (TATAAG and TTGTGT) of an argF promoter identified in P. aeruginosa (15) suggests that the dapB gene is transcribed independently from upstream genes.…”

Novel intragenic deletions and point mutations of the ornithine transcarbamylase gene in congenital hyperammonemia

“…Interestingly both the G39C mutation reported here and the R40H or R40C mutations at the following codon accounted for late- (Itoh et al, 1988;Van Huffel et al, 1992). The C303R mutation occurred in a highly conserved domain, which is believed to play a role in the binding of omithine.…”