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. 1995 Nov;177(22):6432–6439. doi: 10.1128/jb.177.22.6432-6439.1995

Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein.

B Keuntje 1, B Masepohl 1, W Klipp 1
PMCID: PMC177492  PMID: 7592417

Abstract

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments. DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA. The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli. The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms. Therefore, it seems likely that in R. capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA). The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC. The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself. Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression. The open reading frame located downstream of R. capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism. R. capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.

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Selected References

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  1. Allen S. W., Senti-Willis A., Maloy S. R. DNA sequence of the putA gene from Salmonella typhimurium: a bifunctional membrane-associated dehydrogenase that binds DNA. Nucleic Acids Res. 1993 Apr 11;21(7):1676–1676. doi: 10.1093/nar/21.7.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Arnold W., Pühler A. A family of high-copy-number plasmid vectors with single end-label sites for rapid nucleotide sequencing. Gene. 1988 Oct 15;70(1):171–179. doi: 10.1016/0378-1119(88)90115-1. [DOI] [PubMed] [Google Scholar]
  4. Bender R. A. The role of the NAC protein in the nitrogen regulation of Klebsiella aerogenes. Mol Microbiol. 1991 Nov;5(11):2575–2580. doi: 10.1111/j.1365-2958.1991.tb01965.x. [DOI] [PubMed] [Google Scholar]
  5. Brown E. D., Wood J. M. Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline. J Biol Chem. 1993 Apr 25;268(12):8972–8979. [PubMed] [Google Scholar]
  6. Calvo J. M., Matthews R. G. The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol Rev. 1994 Sep;58(3):466–490. doi: 10.1128/mr.58.3.466-490.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen L. M., Maloy S. Regulation of proline utilization in enteric bacteria: cloning and characterization of the Klebsiella put control region. J Bacteriol. 1991 Jan;173(2):783–790. doi: 10.1128/jb.173.2.783-790.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Foster-Hartnett D., Cullen P. J., Gabbert K. K., Kranz R. G. Sequence, genetic, and lacZ fusion analyses of a nifR3-ntrB-ntrC operon in Rhodobacter capsulatus. Mol Microbiol. 1993 May;8(5):903–914. doi: 10.1111/j.1365-2958.1993.tb01636.x. [DOI] [PubMed] [Google Scholar]
  10. Hahn D. R., Myers R. S., Kent C. R., Maloy S. R. Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region. Mol Gen Genet. 1988 Jul;213(1):125–133. doi: 10.1007/BF00333408. [DOI] [PubMed] [Google Scholar]
  11. Harrison S. C., Aggarwal A. K. DNA recognition by proteins with the helix-turn-helix motif. Annu Rev Biochem. 1990;59:933–969. doi: 10.1146/annurev.bi.59.070190.004441. [DOI] [PubMed] [Google Scholar]
  12. Hayward D. C., Delaney S. J., Campbell H. D., Ghysen A., Benzer S., Kasprzak A. B., Cotsell J. N., Young I. G., Miklos G. L. The sluggish-A gene of Drosophila melanogaster is expressed in the nervous system and encodes proline oxidase, a mitochondrial enzyme involved in glutamate biosynthesis. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2979–2983. doi: 10.1073/pnas.90.7.2979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hempel J., Nicholas H., Lindahl R. Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework. Protein Sci. 1993 Nov;2(11):1890–1900. doi: 10.1002/pro.5560021111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Higgins D. G., Bleasby A. J., Fuchs R. CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci. 1992 Apr;8(2):189–191. doi: 10.1093/bioinformatics/8.2.189. [DOI] [PubMed] [Google Scholar]
  15. Hirsch P. R., Beringer J. E. A physical map of pPH1JI and pJB4JI. Plasmid. 1984 Sep;12(2):139–141. doi: 10.1016/0147-619x(84)90059-3. [DOI] [PubMed] [Google Scholar]
  16. Hübner P., Willison J. C., Vignais P. M., Bickle T. A. Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol. 1991 May;173(9):2993–2999. doi: 10.1128/jb.173.9.2993-2999.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones R., Haselkorn R. The DNA sequence of the Rhodobacter capsulatus ntrA, ntrB and ntrC gene analogues required for nitrogen fixation. Mol Gen Genet. 1989 Feb;215(3):507–516. doi: 10.1007/BF00427050. [DOI] [PubMed] [Google Scholar]
  18. Klipp W., Masepohl B., Pühler A. Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol. 1988 Feb;170(2):693–699. doi: 10.1128/jb.170.2.693-699.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kranz R. G., Foster-Hartnett D. Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors. Mol Microbiol. 1990 Nov;4(11):1793–1800. doi: 10.1111/j.1365-2958.1990.tb02027.x. [DOI] [PubMed] [Google Scholar]
  20. Kranz R. G., Pace V. M., Caldicott I. M. Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus. J Bacteriol. 1990 Jan;172(1):53–62. doi: 10.1128/jb.172.1.53-62.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Krzywicki K. A., Brandriss M. C. Primary structure of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Dec;4(12):2837–2842. doi: 10.1128/mcb.4.12.2837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kölling R., Lother H. AsnC: an autogenously regulated activator of asparagine synthetase A transcription in Escherichia coli. J Bacteriol. 1985 Oct;164(1):310–315. doi: 10.1128/jb.164.1.310-315.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Leinfelder W., Forchhammer K., Veprek B., Zehelein E., Böck A. In vitro synthesis of selenocysteinyl-tRNA(UCA) from seryl-tRNA(UCA): involvement and characterization of the selD gene product. Proc Natl Acad Sci U S A. 1990 Jan;87(2):543–547. doi: 10.1073/pnas.87.2.543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ling M., Allen S. W., Wood J. M. Sequence analysis identifies the proline dehydrogenase and delta 1-pyrroline-5-carboxylate dehydrogenase domains of the multifunctional Escherichia coli PutA protein. J Mol Biol. 1994 Nov 11;243(5):950–956. doi: 10.1006/jmbi.1994.1696. [DOI] [PubMed] [Google Scholar]
  25. Macaluso A., Best E. A., Bender R. A. Role of the nac gene product in the nitrogen regulation of some NTR-regulated operons of Klebsiella aerogenes. J Bacteriol. 1990 Dec;172(12):7249–7255. doi: 10.1128/jb.172.12.7249-7255.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Madhusudhan K. T., Lorenz D., Sokatch J. R. The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli. J Bacteriol. 1993 Jul;175(13):3934–3940. doi: 10.1128/jb.175.13.3934-3940.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Magasanik B. Genetic control of nitrogen assimilation in bacteria. Annu Rev Genet. 1982;16:135–168. doi: 10.1146/annurev.ge.16.120182.001031. [DOI] [PubMed] [Google Scholar]
  28. Maloy S. R., Roth J. R. Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions. J Bacteriol. 1983 May;154(2):561–568. doi: 10.1128/jb.154.2.561-568.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Maloy S., Stewart V. Autogenous regulation of gene expression. J Bacteriol. 1993 Jan;175(2):307–316. doi: 10.1128/jb.175.2.307-316.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Masepohl B., Klipp W., Pühler A. Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet. 1988 Apr;212(1):27–37. doi: 10.1007/BF00322441. [DOI] [PubMed] [Google Scholar]
  31. Menzel R., Roth J. Enzymatic properties of the purified putA protein from Salmonella typhimurium. J Biol Chem. 1981 Sep 25;256(18):9762–9766. [PubMed] [Google Scholar]
  32. Menzel R., Roth J. Purification of the putA gene product. A bifunctional membrane-bound protein from Salmonella typhimurium responsible for the two-step oxidation of proline to glutamate. J Biol Chem. 1981 Sep 25;256(18):9755–9761. [PubMed] [Google Scholar]
  33. Moreno-Vivián C., Soler G., Castillo F. Arginine catabolism in the phototrophic bacterium Rhodobacter capsulatus E1F1. Purification and properties of arginase. Eur J Biochem. 1992 Mar 1;204(2):531–537. doi: 10.1111/j.1432-1033.1992.tb16664.x. [DOI] [PubMed] [Google Scholar]
  34. Nakao T., Yamato I., Anraku Y. Nucleotide sequence of putC, the regulatory region for the put regulon of Escherichia coli K12. Mol Gen Genet. 1987 Dec;210(2):364–368. doi: 10.1007/BF00325707. [DOI] [PubMed] [Google Scholar]
  35. Nakao T., Yamato I., Anraku Y. Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12. Mol Gen Genet. 1987 Jun;208(1-2):70–75. doi: 10.1007/BF00330424. [DOI] [PubMed] [Google Scholar]
  36. Ostrovsky de Spicer P., Maloy S. PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator. Proc Natl Acad Sci U S A. 1993 May 1;90(9):4295–4298. doi: 10.1073/pnas.90.9.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ostrovsky de Spicer P., O'Brien K., Maloy S. Regulation of proline utilization in Salmonella typhimurium: a membrane-associated dehydrogenase binds DNA in vitro. J Bacteriol. 1991 Jan;173(1):211–219. doi: 10.1128/jb.173.1.211-219.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Platko J. V., Calvo J. M. Mutations affecting the ability of Escherichia coli Lrp to bind DNA, activate transcription, or respond to leucine. J Bacteriol. 1993 Feb;175(4):1110–1117. doi: 10.1128/jb.175.4.1110-1117.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Prentki P., Krisch H. M. In vitro insertional mutagenesis with a selectable DNA fragment. Gene. 1984 Sep;29(3):303–313. doi: 10.1016/0378-1119(84)90059-3. [DOI] [PubMed] [Google Scholar]
  40. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Staden R. The current status and portability of our sequence handling software. Nucleic Acids Res. 1986 Jan 10;14(1):217–231. doi: 10.1093/nar/14.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Suhr M., Kleiner D. Genetic analysis of the regulatory putP region (coding for proline permease) in Klebsiella pneumoniae M5a1: evidence for regulation by the nac system. FEMS Microbiol Lett. 1993 Dec 1;114(2):191–194. doi: 10.1111/j.1574-6968.1993.tb06572.x. [DOI] [PubMed] [Google Scholar]
  43. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  44. Wall J. D., Braddock K. Mapping of Rhodopseudomonas capsulata nif genes. J Bacteriol. 1984 May;158(2):404–410. doi: 10.1128/jb.158.2.404-410.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang S. S., Brandriss M. C. Proline utilization in Saccharomyces cerevisiae: sequence, regulation, and mitochondrial localization of the PUT1 gene product. Mol Cell Biol. 1987 Dec;7(12):4431–4440. doi: 10.1128/mcb.7.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Willins D. A., Ryan C. W., Platko J. V., Calvo J. M. Characterization of Lrp, and Escherichia coli regulatory protein that mediates a global response to leucine. J Biol Chem. 1991 Jun 15;266(17):10768–10774. [PubMed] [Google Scholar]

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