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. 1997 Apr;179(7):2181–2188. doi: 10.1128/jb.179.7.2181-2188.1997

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

B A Chopade 1, S Shankar 1, G W Sundin 1, S Mukhopadhyay 1, A M Chakrabarty 1
PMCID: PMC178953  PMID: 9079902

Abstract

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.

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Selected References

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  1. Almaula N., Lu Q., Delgado J., Belkin S., Inouye M. Nucleoside diphosphate kinase from Escherichia coli. J Bacteriol. 1995 May;177(9):2524–2529. doi: 10.1128/jb.177.9.2524-2529.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  3. Bowman B. J., Blasco F., Slayman C. W. Purification and characterization of the plasma membrane ATPase of Neurospora crassa. J Biol Chem. 1981 Dec 10;256(23):12343–12349. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Brock H. W. Sequence and genomic structure of ras homologues Dmras85D and Dmras64B of Drosophila melanogaster. Gene. 1987;51(2-3):129–137. doi: 10.1016/0378-1119(87)90301-5. [DOI] [PubMed] [Google Scholar]
  6. Carlier M. F. Guanosine-5'-triphosphate hydrolysis and tubulin polymerization. Review article. Mol Cell Biochem. 1982 Sep 3;47(2):97–113. doi: 10.1007/BF00234410. [DOI] [PubMed] [Google Scholar]
  7. Cassel D., Selinger Z. Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3307–3311. doi: 10.1073/pnas.74.8.3307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen S. M., Takiff H. E., Barber A. M., Dubois G. C., Bardwell J. C., Court D. L. Expression and characterization of RNase III and Era proteins. Products of the rnc operon of Escherichia coli. J Biol Chem. 1990 Feb 15;265(5):2888–2895. [PubMed] [Google Scholar]
  9. Darzins A., Chakrabarty A. M. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol. 1984 Jul;159(1):9–18. doi: 10.1128/jb.159.1.9-18.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deschenes R. J., Broach J. R. Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function. Mol Cell Biol. 1987 Jul;7(7):2344–2351. doi: 10.1128/mcb.7.7.2344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fukui Y., Kaziro Y., Yamamoto M. Mating pheromone-like diffusible factor released by Schizosaccharomyces pombe. EMBO J. 1986 Aug;5(8):1991–1993. doi: 10.1002/j.1460-2075.1986.tb04454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  13. Gentry D. R., Hernandez V. J., Nguyen L. H., Jensen D. B., Cashel M. Synthesis of the stationary-phase sigma factor sigma s is positively regulated by ppGpp. J Bacteriol. 1993 Dec;175(24):7982–7989. doi: 10.1128/jb.175.24.7982-7989.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gibbs J. B., Marshall M. S. The ras oncogene--an important regulatory element in lower eucaryotic organisms. Microbiol Rev. 1989 Jun;53(2):171–185. doi: 10.1128/mr.53.2.171-185.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  16. Hughes S. M. Are guanine nucleotide binding proteins a distinct class of regulatory proteins? FEBS Lett. 1983 Nov 28;164(1):1–8. doi: 10.1016/0014-5793(83)80006-4. [DOI] [PubMed] [Google Scholar]
  17. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  18. Lerner C. G., Inouye M. Pleiotropic changes resulting from depletion of Era, an essential GTP-binding protein in Escherichia coli. Mol Microbiol. 1991 Apr;5(4):951–957. doi: 10.1111/j.1365-2958.1991.tb00770.x. [DOI] [PubMed] [Google Scholar]
  19. Lin Y. P., Sharer J. D., March P. E. GTPase-dependent signaling in bacteria: characterization of a membrane-binding site for era in Escherichia coli. J Bacteriol. 1994 Jan;176(1):44–49. doi: 10.1128/jb.176.1.44-49.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lowy D. R., Willumsen B. M. Function and regulation of ras. Annu Rev Biochem. 1993;62:851–891. doi: 10.1146/annurev.bi.62.070193.004223. [DOI] [PubMed] [Google Scholar]
  21. March P. E., Lerner C. G., Ahnn J., Cui X., Inouye M. The Escherichia coli Ras-like protein (Era) has GTPase activity and is essential for cell growth. Oncogene. 1988 Jun;2(6):539–544. [PubMed] [Google Scholar]
  22. March P. E. Membrane-associated GTPases in bacteria. Mol Microbiol. 1992 May;6(10):1253–1257. doi: 10.1111/j.1365-2958.1992.tb00845.x. [DOI] [PubMed] [Google Scholar]
  23. May T. B., Chakrabarty A. M. Pseudomonas aeruginosa: genes and enzymes of alginate synthesis. Trends Microbiol. 1994 May;2(5):151–157. doi: 10.1016/0966-842x(94)90664-5. [DOI] [PubMed] [Google Scholar]
  24. Moore P. B. Molecular mimicry in protein synthesis? Science. 1995 Dec 1;270(5241):1453–1454. doi: 10.1126/science.270.5241.1453. [DOI] [PubMed] [Google Scholar]
  25. Mozer B., Marlor R., Parkhurst S., Corces V. Characterization and developmental expression of a Drosophila ras oncogene. Mol Cell Biol. 1985 Apr;5(4):885–889. doi: 10.1128/mcb.5.4.885. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pillutla R. C., Sharer J. D., Gulati P. S., Wu E., Yamashita Y., Lerner C. G., Inouye M., March P. E. Cross-species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity. J Bacteriol. 1995 Apr;177(8):2194–2196. doi: 10.1128/jb.177.8.2194-2196.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Shankar S., Kamath S., Chakrabarty A. M. Two forms of the nucleoside diphosphate kinase of Pseudomonas aeruginosa 8830: altered specificity of nucleoside triphosphate synthesis by the cell membrane-associated form of the truncated enzyme. J Bacteriol. 1996 Apr;178(7):1777–1781. doi: 10.1128/jb.178.7.1777-1781.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sundin G. W., Shankar S., Chakrabarty A. M. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis. J Bacteriol. 1996 Dec;178(24):7120–7128. doi: 10.1128/jb.178.24.7120-7128.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sundin G. W., Shankar S., Chugani S. A., Chopade B. A., Kavanaugh-Black A., Chakrabarty A. M. Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis. Mol Microbiol. 1996 Jun;20(5):965–979. doi: 10.1111/j.1365-2958.1996.tb02538.x. [DOI] [PubMed] [Google Scholar]
  30. Trach K., Hoch J. A. The Bacillus subtilis spo0B stage 0 sporulation operon encodes an essential GTP-binding protein. J Bacteriol. 1989 Mar;171(3):1362–1371. doi: 10.1128/jb.171.3.1362-1371.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Haastert P. J., Kesbeke F., Reymond C. D., Firtel R. A., Luderus E., Van Driel R. Aberrant transmembrane signal transduction in Dictyostelium cells expressing a mutated ras gene. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4905–4909. doi: 10.1073/pnas.84.14.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wang X., Lutkenhaus J. The FtsZ protein of Bacillus subtilis is localized at the division site and has GTPase activity that is dependent upon FtsZ concentration. Mol Microbiol. 1993 Aug;9(3):435–442. doi: 10.1111/j.1365-2958.1993.tb01705.x. [DOI] [PubMed] [Google Scholar]
  33. Welsh K. M., Trach K. A., Folger C., Hoch J. A. Biochemical characterization of the essential GTP-binding protein Obg of Bacillus subtilis. J Bacteriol. 1994 Dec;176(23):7161–7168. doi: 10.1128/jb.176.23.7161-7168.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Xie Z. D., Hershberger C. D., Shankar S., Ye R. W., Chakrabarty A. M. Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA. J Bacteriol. 1996 Aug;178(16):4990–4996. doi: 10.1128/jb.178.16.4990-4996.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Zuber M., Hoover T. A., Powell B. S., Court D. L. Analysis of the rnc locus of Coxiella burnetii. Mol Microbiol. 1994 Oct;14(2):291–300. doi: 10.1111/j.1365-2958.1994.tb01290.x. [DOI] [PubMed] [Google Scholar]

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