Abstract
Sequence analysis upstream of the Rhizobium etli fixLJ homologous genes revealed the presence of three open reading frames homologous to the arcABC genes of Pseudomonas aeruginosa. The P. aeruginosa arcABC genes code for the enzymes of the arginine deiminase pathway: arginine deiminase, catabolic ornithine carbamoyltransferase (cOTCase), and carbamate kinase. OTCase activities were measured in free-living R. etli cells and in bacteroids isolated from bean nodules. OTCase activity in free-living cells was observed at a different pH optimum than OTCase activity in bacteroids, suggesting the presence of two enzymes with different characteristics and different expression patterns of the corresponding genes. The characteristics of the OTCase isolated from the bacteroids were studied in further detail and were shown to be similar to the properties of the cOTCase of P. aeruginosa. The enzyme has a pH optimum of 6.8 and a molecular mass of approximately 450 kDa, is characterized by a sigmoidal carbamoyl phosphate saturation curve, and exhibits a cooperativity for carbamoyl phosphate. R. etli arcA mutants, with polar effects on arcB and arcC, were constructed by insertion mutagenesis. Bean nodules induced by arcA mutants were still able to fix nitrogen but showed a significantly lower acetylene reduction activity than nodules induced by the wild type. No significant differences in nodule dry weight, plant dry weight, and number of nodules were found between the wild type and the mutants. Determination of the OTCase activity in extracts from bacteroids revealed a strong decrease in activity of this enzyme in the arcA mutant compared to the wild-type strain. Finally, we observed that expression of an R. etli arcA-gusA fusion was strongly induced under anaerobic conditions.
Full Text
The Full Text of this article is available as a PDF (208.1 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Baur H., Luethi E., Stalon V., Mercenier A., Haas D. Sequence analysis and expression of the arginine-deiminase and carbamate-kinase genes of Pseudomonas aeruginosa. Eur J Biochem. 1989 Jan 15;179(1):53–60. doi: 10.1111/j.1432-1033.1989.tb14520.x. [DOI] [PubMed] [Google Scholar]
- Baur H., Stalon V., Falmagne P., Luethi E., Haas D. Primary and quaternary structure of the catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa. Extensive sequence homology with the anabolic ornithine carbamoyltransferases of Escherichia coli. Eur J Biochem. 1987 Jul 1;166(1):111–117. doi: 10.1111/j.1432-1033.1987.tb13489.x. [DOI] [PubMed] [Google Scholar]
- Baur H., Tricot C., Stalon V., Haas D. Converting catabolic ornithine carbamoyltransferase to an anabolic enzyme. J Biol Chem. 1990 Sep 5;265(25):14728–14731. [PubMed] [Google Scholar]
- Colonna-Romano S., Arnold W., Schlüter A., Boistard P., Pühler A., Priefer U. B. An Fnr-like protein encoded in Rhizobium leguminosarum biovar viciae shows structural and functional homology to Rhizobium meliloti FixK. Mol Gen Genet. 1990 Aug;223(1):138–147. doi: 10.1007/BF00315806. [DOI] [PubMed] [Google Scholar]
- Cunin R., Glansdorff N., Piérard A., Stalon V. Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev. 1986 Sep;50(3):314–352. doi: 10.1128/mr.50.3.314-352.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- D'hooghe I., Michiels J., Vlassak K., Verreth C., Waelkens F., Vanderleyden J. Structural and functional analysis of the fixLJ genes of Rhizobium leguminosarum biovar phaseoli CNPAF512. Mol Gen Genet. 1995 Nov 1;249(1):117–126. doi: 10.1007/BF00290243. [DOI] [PubMed] [Google Scholar]
- Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
- Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
- Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
- Galimand M., Gamper M., Zimmermann A., Haas D. Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa. J Bacteriol. 1991 Mar;173(5):1598–1606. doi: 10.1128/jb.173.5.1598-1606.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gamper M., Zimmermann A., Haas D. Anaerobic regulation of transcription initiation in the arcDABC operon of Pseudomonas aeruginosa. J Bacteriol. 1991 Aug;173(15):4742–4750. doi: 10.1128/jb.173.15.4742-4750.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldsmith J. O., Kuo L. C. Protonation of arginine 57 of Escherichia coli ornithine transcarbamoylase regulates substrate binding and turnover. J Biol Chem. 1993 Sep 5;268(25):18485–18490. [PubMed] [Google Scholar]
- Goldsmith J. O., Lee S., Zambidis I., Kuo L. C. Control of L-ornithine specificity in Escherichia coli ornithine transcarbamoylase. Site-directed mutagenic and pH studies. J Biol Chem. 1991 Oct 5;266(28):18626–18634. [PubMed] [Google Scholar]
- Harasawa R., Koshimizu K., Kitagawa M., Asada K., Kato I. Nucleotide sequence of the arginine deiminase gene of Mycoplasma hominis. Microbiol Immunol. 1992;36(6):661–665. doi: 10.1111/j.1348-0421.1992.tb02068.x. [DOI] [PubMed] [Google Scholar]
- Honzatko R. B., Lipscomb W. N. Interactions of phosphate ligands with Escherichia coli aspartate carbamoyltransferase in the crystalline state. J Mol Biol. 1982 Sep 15;160(2):265–286. doi: 10.1016/0022-2836(82)90176-0. [DOI] [PubMed] [Google Scholar]
- Houghton J. E., Bencini D. A., O'Donovan G. A., Wild J. R. Protein differentiation: a comparison of aspartate transcarbamoylase and ornithine transcarbamoylase from Escherichia coli K-12. Proc Natl Acad Sci U S A. 1984 Aug;81(15):4864–4868. doi: 10.1073/pnas.81.15.4864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Itoh Y., Soldati L., Stalon V., Falmagne P., Terawaki Y., Leisinger T., Haas D. Anabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa: nucleotide sequence and transcriptional control of the argF structural gene. J Bacteriol. 1988 Jun;170(6):2725–2734. doi: 10.1128/jb.170.6.2725-2734.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jiménez-Zurdo J. I., van Dillewijn P., Soto M. J., de Felipe M. R., Olivares J., Toro N. Characterization of a Rhizobium meliloti proline dehydrogenase mutant altered in nodulation efficiency and competitiveness on alfalfa roots. Mol Plant Microbe Interact. 1995 Jul-Aug;8(4):492–498. doi: 10.1094/mpmi-8-0492. [DOI] [PubMed] [Google Scholar]
- Kuo L. C., Miller A. W., Lee S., Kozuma C. Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis. Biochemistry. 1988 Nov 29;27(24):8823–8832. doi: 10.1021/bi00424a021. [DOI] [PubMed] [Google Scholar]
- Leyva A., Palacios J. M., Murillo J., Ruiz-Argüeso T. Genetic organization of the hydrogen uptake (hup) cluster from Rhizobium leguminosarum. J Bacteriol. 1990 Mar;172(3):1647–1655. doi: 10.1128/jb.172.3.1647-1655.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lüthi E., Baur H., Gamper M., Brunner F., Villeval D., Mercenier A., Haas D. The arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa contains an additional gene, arcD, encoding a membrane protein. Gene. 1990 Mar 1;87(1):37–43. doi: 10.1016/0378-1119(90)90493-b. [DOI] [PubMed] [Google Scholar]
- Marshall M., Cohen P. P. Ornithine transcarbamylases. Ordering of S-cyano peptides and location of characteristically reactive cysteinyl residues within the sequence. J Biol Chem. 1980 Aug 10;255(15):7287–7290. [PubMed] [Google Scholar]
- Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mercenier A., Simon J. P., Vander Wauven C., Haas D., Stalon V. Regulation of enzyme synthesis in the arginine deiminase pathway of Pseudomonas aeruginosa. J Bacteriol. 1980 Oct;144(1):159–163. doi: 10.1128/jb.144.1.159-163.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Michiels J., D'hooghe I., Verreth C., Pelemans H., Vanderleyden J. Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene, a positive regulator of nif gene expression. Arch Microbiol. 1994;161(5):404–408. doi: 10.1007/BF00288950. [DOI] [PubMed] [Google Scholar]
- Nguyen V. T., Baker D. P., Tricot C., Baur H., Villeret V., Dideberg O., Gigot D., Stalon V., Haas D. Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity. Eur J Biochem. 1996 Feb 15;236(1):283–293. doi: 10.1111/j.1432-1033.1996.00283.x. [DOI] [PubMed] [Google Scholar]
- Nguyen V. T., Tricot C., Stalon V., Dideberg O., Villeret V., Haas D. Methionine-321 in the C-terminal alpha-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity. FEMS Microbiol Lett. 1994 Dec 15;124(3):411–417. doi: 10.1111/j.1574-6968.1994.tb07317.x. [DOI] [PubMed] [Google Scholar]
- Noel K. D., Sanchez A., Fernandez L., Leemans J., Cevallos M. A. Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions. J Bacteriol. 1984 Apr;158(1):148–155. doi: 10.1128/jb.158.1.148-155.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Norrander J., Kempe T., Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. doi: 10.1016/0378-1119(83)90040-9. [DOI] [PubMed] [Google Scholar]
- O'Brian M. R., Maier R. J. Molecular aspects of the energetics of nitrogen fixation in Rhizobium-legume symbioses. Biochim Biophys Acta. 1989 May 30;974(3):229–246. doi: 10.1016/s0005-2728(89)80239-7. [DOI] [PubMed] [Google Scholar]
- Ohno T., Ando O., Sugimura K., Taniai M., Suzuki M., Fukuda S., Nagase Y., Yamamoto K., Azuma I. Cloning and nucleotide sequence of the gene encoding arginine deiminase of Mycoplasma arginini. Infect Immun. 1990 Nov;58(11):3788–3795. doi: 10.1128/iai.58.11.3788-3795.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prescott L. M., Jones M. E. Modified methods for the determination of carbamyl aspartate. Anal Biochem. 1969 Dec;32(3):408–419. doi: 10.1016/s0003-2697(69)80008-4. [DOI] [PubMed] [Google Scholar]
- Quandt J., Hynes M. F. Versatile suicide vectors which allow direct selection for gene replacement in gram-negative bacteria. Gene. 1993 May 15;127(1):15–21. doi: 10.1016/0378-1119(93)90611-6. [DOI] [PubMed] [Google Scholar]
- Rella M., Mercenier A., Haas D. Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster. Gene. 1985;33(3):293–303. doi: 10.1016/0378-1119(85)90237-9. [DOI] [PubMed] [Google Scholar]
- Ronson C. W., Lyttleton P., Robertson J. G. C(4)-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4284–4288. doi: 10.1073/pnas.78.7.4284. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruepp A., Müller H. N., Lottspeich F., Soppa J. Catabolic ornithine transcarbamylase of Halobacterium halobium (salinarium): purification, characterization, sequence determination, and evolution. J Bacteriol. 1995 Mar;177(5):1129–1136. doi: 10.1128/jb.177.5.1129-1136.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruepp A., Soppa J. Fermentative arginine degradation in Halobacterium salinarium (formerly Halobacterium halobium): genes, gene products, and transcripts of the arcRACB gene cluster. J Bacteriol. 1996 Aug;178(16):4942–4947. doi: 10.1128/jb.178.16.4942-4947.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Soto M. J., Zorzano A., García-Rodriguez F. M., Mercado-Blanco J., López-Lara I. M., Olivares J., Toro N. Identification of a novel Rhizobium meliloti nodulation efficiency nfe gene homolog of Agrobacterium ornithine cyclodeaminase. Mol Plant Microbe Interact. 1994 Nov-Dec;7(6):703–707. doi: 10.1094/mpmi-7-0703. [DOI] [PubMed] [Google Scholar]
- Spiro S. The FNR family of transcriptional regulators. Antonie Van Leeuwenhoek. 1994;66(1-3):23–36. doi: 10.1007/BF00871630. [DOI] [PubMed] [Google Scholar]
- Staskawicz B., Dahlbeck D., Keen N., Napoli C. Molecular characterization of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea. J Bacteriol. 1987 Dec;169(12):5789–5794. doi: 10.1128/jb.169.12.5789-5794.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tricot C., Nguyen V. T., Stalon V. Steady-state kinetics and analysis of pH dependence on wild-type and a modified allosteric Pseudomonas aeruginosa ornithine carbamoyltransferase containing the replacement of glutamate 105 by alanine. Eur J Biochem. 1993 Aug 1;215(3):833–839. doi: 10.1111/j.1432-1033.1993.tb18099.x. [DOI] [PubMed] [Google Scholar]
- Tricot C., Schmid S., Baur H., Villeret V., Dideberg O., Haas D., Stalon V. Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Changes of allosteric properties resulting from modifications at the C-terminus. Eur J Biochem. 1994 Apr 1;221(1):555–561. doi: 10.1111/j.1432-1033.1994.tb18768.x. [DOI] [PubMed] [Google Scholar]
- Van Vliet F., Cunin R., Jacobs A., Piette J., Gigot D., Lauwereys M., Piérard A., Glansdorff N. Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB. Nucleic Acids Res. 1984 Aug 10;12(15):6277–6289. doi: 10.1093/nar/12.15.6277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vander Wauven C., Piérard A., Kley-Raymann M., Haas D. Pseudomonas aeruginosa mutants affected in anaerobic growth on arginine: evidence for a four-gene cluster encoding the arginine deiminase pathway. J Bacteriol. 1984 Dec;160(3):928–934. doi: 10.1128/jb.160.3.928-934.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Verhoogt H. J., Smit H., Abee T., Gamper M., Driessen A. J., Haas D., Konings W. N. arcD, the first gene of the arc operon for anaerobic arginine catabolism in Pseudomonas aeruginosa, encodes an arginine-ornithine exchanger. J Bacteriol. 1992 Mar;174(5):1568–1573. doi: 10.1128/jb.174.5.1568-1573.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Villeret V., Tricot C., Stalon V., Dideberg O. Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10762–10766. doi: 10.1073/pnas.92.23.10762. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilson K. J., Sessitsch A., Corbo J. C., Giller K. E., Akkermans A. D., Jefferson R. A. beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria. Microbiology. 1995 Jul;141(Pt 7):1691–1705. doi: 10.1099/13500872-141-7-1691. [DOI] [PubMed] [Google Scholar]
- Zhu Y., Shearer G., Kohl D. H. Proline fed to intact soybean plants influences acetylene reducing activity and content and metabolism of proline in bacteroids. Plant Physiol. 1992 Mar;98(3):1020–1028. doi: 10.1104/pp.98.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zimmermann A., Reimmann C., Galimand M., Haas D. Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli. Mol Microbiol. 1991 Jun;5(6):1483–1490. doi: 10.1111/j.1365-2958.1991.tb00794.x. [DOI] [PubMed] [Google Scholar]