Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster

Abstract

Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter. Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium). Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found. They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein. The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides. The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available. Transcripts from H. salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses. It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter. The data indicate that expression of the arc gene cluster and its regulation differ in H. salinarium and P. aeruginosa.The arginine deiminase pathway of fermentative arginine utilization consists of the three enzymes arginine deiminase (ADI, EC 3.5.3.6), catabolic ornithine transcarbamylase (cOTCase, EC 2.1.3.3), and carbamate kinase (CK, EC 2.7.2.2) and a membrane-bound arginine-ornithine antiporter. The degradation of arginine is coupled to the equimolar generation of ATP by substrate level phosphorylation. This pathway is found in a variety of phylogenetic groups within the domain Bacteria, e.g., Pseudomonas spp., Mycoplasma spp., Bacillus spp., and lactic bacteria (for a review, see reference 8). The pathway has been most extensively studied in Pseudomonas aeruginosa (21), the only species for which the sequences of the four genes have been determined (1,2,29). The genes are organized in an operon, and a polycistronic mRNA is transcribed from a single promoter and subsequently processed into a variety of smaller transcripts (17) in an RNase E-dependent reaction (18). The transcription of the arcDABC operon is under the positive control of the ANR protein (15, 22), a member of the FNR protein family.Within the domain Archaea, arginine-dependent anaerobic growth has only been reported for some halobacterial species (25,37). The consumption of arginine was coupled to the equimolar production o...

The arginine deiminase pathway in the wine lactic acid bacterium Lactobacillus hilgardii X 1 B: structural and functional study of the arcABC genes

“…It encodes a 418-amino acid residue, 47,2-kDa protein, 70% identical to the putative Oenococcus oeni arginine deiminase (accession no. AF124851) and 66% identical to that of L. sakei (Zúñiga et al, 1998 (Ruepp and Soppa, 1996) and ahrC in Clostridium prefringens (Ohtani et al, 1997) and in L. lactis (Bolotin et al 2001); in contrast, regulatory genes have not been described in the L. sakei arc operon (Zúñiga et al, 13 1998 …”

“…Cell extracts from E. coli JM109 (DE3) harboring the recombinant plasmid pRM7 showed OTC activity (7 x 10 3 U/mg protein), whereas extracts prepared from control cells containing the vector plasmid alone did not. CK activity was measured by an indirect assay described by Ruepp and Soppa (1996). In cell extracts of E. coli JM109 (DE3) harboring pRM8, CK activity, which was absent in control cultures, could be measured.…”

“…OTC activity was determined by measuring the formation of citrulline from ornithine and carbamyl phosphate. CK activity was measured by an indirect assay described by Ruepp and Soppa (1996). Briefly, in a first step, cell extract of E. coli JM109 (DE3) harboring the arcC gene cloned into plasmid pT7-7 and cell extract with pT7-7 as control were incubated for different times at 37ºC in the presence of 40 mM ornithine and 10 mM carbamyl phosphate.…”

Molecular Biology and Pathogenicity of Mycoplasmas

“…The degradation of arginine is coupled to equimolar generation of ATP by substrate-level phosphorylation. The arginine dihydrolase pathway is found in a variety of phylogenetic bacterial groups, e.g., Pseudomonas, Bacillus, and lactic acid bacteria (396). The role of this pathway as a sole energy-generating source in nonfermentative mollicutes has been frequently questioned (181,182,356).…”

“…Arginine hydrolysis by this pathway results in the production of ornithine, ATP, CO 2 , and ammonia, raising the pH of the culture medium (356). The pathway uses three enzymes: arginine deiminase, ornithine carbamoyl transferase, and carbamate kinase (396). The degradation of arginine is coupled to equimolar generation of ATP by substrate-level phosphorylation.…”