1887

Abstract

The conversion of nicotinamide to nicotinic acid by nicotinamidase enzymes is a critical step in maintaining NAD homeostasis and contributes to numerous important biological processes in diverse organisms. In , the nicotinamidase enzyme, PncA, is required for spirochaete survival throughout the infectious cycle. Mammals lack nicotinamidases and therefore PncA may serve as a therapeutic target for Lyme disease. Contrary to the importance of PncA, the current annotation for the ORF suggests that the encoded protein may be inactive due to the absence of an N-terminal aspartic acid residue that is a conserved member of the catalytic triad of characterized PncA proteins. Herein, we have used genetic and biochemical strategies to determine the N-terminal sequence of PncA. Our data demonstrate that the PncA protein is 24 aa longer than the currently annotated sequence and that translation is initiated from the rare, non-canonical initiation codon AUU. These findings are an important first step in understanding the catalytic function of this -essential protein.

Funding
This study was supported by the:
  • NIH, NIAID (Award 5K22AI081730)
  • Intramural Research Program of the NIH, NIAID
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2011-10-01
2024-10-06
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References

  1. Anderson R. M., Bitterman K. J., Wood J. G., Medvedik O., Sinclair D. A. ( 2003). Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 423:181–185 [View Article][PubMed]
    [Google Scholar]
  2. Balan V., Miller G. S., Kaplun L., Balan K., Chong Z. Z., Li F., Kaplun A., VanBerkum M. F., Arking R. et al. ( 2008). Life span extension and neuronal cell protection by Drosophila nicotinamidase. J Biol Chem 283:27810–27819 [View Article][PubMed]
    [Google Scholar]
  3. Barbour A. G. ( 1984). Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57:521–525[PubMed]
    [Google Scholar]
  4. Bestor A., Stewart P. E., Jewett M. W., Sarkar A., Tilly K., Rosa P. A. ( 2010). Use of the Cre-lox recombination system to investigate the lp54 gene requirement in the infectious cycle of Borrelia burgdorferi. Infect Immun 78:2397–2407 [View Article][PubMed]
    [Google Scholar]
  5. Binns N., Masters M. ( 2002). Expression of the Escherichia coli pcnB gene is translationally limited using an inefficient start codon: a second chromosomal example of translation initiated at AUU. Mol Microbiol 44:1287–1298 [View Article][PubMed]
    [Google Scholar]
  6. Bono J. L., Tilly K., Stevenson B., Hogan D., Rosa P. ( 1998). Oligopeptide permease in Borrelia burgdorferi: putative peptide-binding components encoded by both chromosomal and plasmid loci. Microbiology 144:1033–1044 [View Article][PubMed]
    [Google Scholar]
  7. Byram R., Stewart P. E., Rosa P. ( 2004). The essential nature of the ubiquitous 26-kilobase circular replicon of Borrelia burgdorferi. J Bacteriol 186:3561–3569 [View Article][PubMed]
    [Google Scholar]
  8. Cao G. J., Sarkar N. ( 1992a). Identification of the gene for an Escherichia coli poly(A) polymerase. Proc Natl Acad Sci U S A 89:10380–10384 [View Article][PubMed]
    [Google Scholar]
  9. Cao G. J., Sarkar N. ( 1992b). Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety. Proc Natl Acad Sci U S A 89:7546–7550 [View Article][PubMed]
    [Google Scholar]
  10. Casjens S., Palmer N., van Vugt R., Huang W. M., Stevenson B., Rosa P., Lathigra R., Sutton G., Peterson J. et al. ( 2000). A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol Microbiol 35:490–516 [View Article][PubMed]
    [Google Scholar]
  11. Elias A. F., Stewart P. E., Grimm D., Caimano M. J., Eggers C. H., Tilly K., Bono J. L., Akins D. R., Radolf J. D. et al. ( 2002). Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect Immun 70:2139–2150 [View Article][PubMed]
    [Google Scholar]
  12. Elias A. F., Bono J. L., Kupko J. J. III, Stewart P. E., Krum J. G., Rosa P. A. ( 2003). New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi. J Mol Microbiol Biotechnol 6:29–40 [View Article][PubMed]
    [Google Scholar]
  13. Evans C., Bogan K. L., Song P., Burant C. F., Kennedy R. T., Brenner C. ( 2010). NAD+ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity. BMC Chem Biol 10:2 [View Article][PubMed]
    [Google Scholar]
  14. Fraser C. M., Casjens S., Huang W. M., Sutton G. G., Clayton R., Lathigra R., White O., Ketchum K. A., Dodson R. et al. ( 1997). Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390:580–586 [View Article][PubMed]
    [Google Scholar]
  15. French J. B., Cen Y., Vrablik T. L., Xu P., Allen E., Hanna-Rose W., Sauve A. A. ( 2010). Characterization of nicotinamidases: steady state kinetic parameters, classwide inhibition by nicotinaldehydes, and catalytic mechanism. Biochemistry 49:10421–10439 [View Article][PubMed]
    [Google Scholar]
  16. Frothingham R., Meeker-O’Connell W. A., Talbot E. A., George J. W., Kreuzer K. N. ( 1996). Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. Antimicrob Agents Chemother 40:1426–1431[PubMed]
    [Google Scholar]
  17. Ghislain M., Talla E., François J. M. ( 2002). Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1. Yeast 19:215–224 [View Article][PubMed]
    [Google Scholar]
  18. Grimm D., Tilly K., Byram R., Stewart P. E., Krum J. G., Bueschel D. M., Schwan T. G., Policastro P. F., Elias A. F., Rosa P. A. ( 2004). Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc Natl Acad Sci U S A 101:3142–3147 [View Article][PubMed]
    [Google Scholar]
  19. Grimm D., Tilly K., Bueschel D. M., Fisher M. A., Policastro P. F., Gherardini F. C., Schwan T. G., Rosa P. A. ( 2005). Defining plasmids required by Borrelia burgdorferi for colonization of tick vector Ixodes scapularis (Acari: Ixodidae). J Med Entomol 42:676–684 [View Article][PubMed]
    [Google Scholar]
  20. Hu W. S., Wang R. Y., Shih J. W., Lo S. C. ( 1993). Identification of a putative infC-rpmI-rplT operon flanked by long inverted repeats in Mycoplasma fermentans (incognitus strain). Gene 127:79–85 [View Article][PubMed]
    [Google Scholar]
  21. Hunt L., Holdsworth M. J., Gray J. E. ( 2007). Nicotinamidase activity is important for germination. Plant J 51:341–351 [View Article][PubMed]
    [Google Scholar]
  22. Ivanov I. P., Loughran G., Atkins J. F. ( 2008). uORFs with unusual translational start codons autoregulate expression of eukaryotic ornithine decarboxylase homologs. Proc Natl Acad Sci U S A 105:10079–10084 [View Article][PubMed]
    [Google Scholar]
  23. Jewett M. W., Lawrence K., Bestor A. C., Tilly K., Grimm D., Shaw P., VanRaden M., Gherardini F., Rosa P. A. ( 2007). The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi. Mol Microbiol 64:1358–1374 [View Article][PubMed]
    [Google Scholar]
  24. Jewett M. W., Lawrence K. A., Bestor A., Byram R., Gherardini F., Rosa P. A. ( 2009). GuaA and GuaB are essential for Borrelia burgdorferi survival in the tick-mouse infection cycle. J Bacteriol 191:6231–6241 [View Article][PubMed]
    [Google Scholar]
  25. Kim S., Kurokawa D., Watanabe K., Makino S., Shirahata T., Watarai M. ( 2004). Brucella abortus nicotinamidase (PncA) contributes to its intracellular replication and infectivity in mice. FEMS Microbiol Lett 234:289–295 [View Article][PubMed]
    [Google Scholar]
  26. Lawrence K. A., Jewett M. W., Rosa P. A., Gherardini F. C. ( 2009). Borrelia burgdorferi bb0426 encodes a 2′-deoxyribosyltransferase that plays a central role in purine salvage. Mol Microbiol 72:1517–1529 [View Article][PubMed]
    [Google Scholar]
  27. Lescot M., Audic S., Robert C., Nguyen T. T., Blanc G., Cutler S. J., Wincker P., Couloux A., Claverie J. M. et al. ( 2008). The genome of Borrelia recurrentis, the agent of deadly louse-borne relapsing fever, is a degraded subset of tick-borne Borrelia duttonii. PLoS Genet 4:e1000185 [View Article][PubMed]
    [Google Scholar]
  28. Liveris D., Schwartz J. J., Geertman R., Schwartz I. ( 1993). Molecular cloning and sequencing of infC, the gene encoding translation initiation factor IF3, from four enterobacterial species. FEMS Microbiol Lett 112:211–216 [View Article][PubMed]
    [Google Scholar]
  29. Minard K. I., McAlister-Henn L. ( 2010). Pnc1p supports increases in cellular NAD(H) levels in response to internal or external oxidative stress. Biochemistry 49:6299–6301 [View Article][PubMed]
    [Google Scholar]
  30. Nivinskas R., Vaiskunaite R., Raudonikiene A. ( 1992). An internal AUU codon initiates a smaller peptide encoded by bacteriophage T4 baseplate gene 26. Mol Gen Genet 232:257–261[PubMed]
    [Google Scholar]
  31. Pon C. L., Gualerzi C. O. ( 1986). Mechanism of translational initiation in prokaryotes. IF3 is released from ribosomes during and not before 70 S initiation complex formation. FEBS Lett 195:215–219 [View Article][PubMed]
    [Google Scholar]
  32. Prère M. F., Canal I., Wills N. M., Atkins J. F., Fayet O. ( 2011). The interplay of mRNA stimulatory signals required for AUU-mediated initiation and programmed −1 ribosomal frameshifting in decoding of transposable element IS911. J Bacteriol 193:2735–2744 [View Article][PubMed]
    [Google Scholar]
  33. Purser J. E., Lawrenz M. B., Caimano M. J., Howell J. K., Radolf J. D., Norris S. J. ( 2003). A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host. Mol Microbiol 48:753–764 [View Article][PubMed]
    [Google Scholar]
  34. Ramamoorthy R., McClain N. A., Gautam A., Scholl-Meeker D. ( 2005). Expression of the bmpB gene of Borrelia burgdorferi is modulated by two distinct transcription termination events. J Bacteriol 187:2592–2600 [View Article][PubMed]
    [Google Scholar]
  35. Raynaud C., Etienne G., Peyron P., Lanéelle M. A., Daffé M. ( 1998). Extracellular enzyme activities potentially involved in the pathogenicity of Mycobacterium tuberculosis. Microbiology 144:577–587 [View Article][PubMed]
    [Google Scholar]
  36. Rosa P. A., Hogan D. ( 1992). Colony formation by Borrelia burgdorferi in solid medium: clonal analysis of osp locus variants. In Proceedings of the First International Conference on Tick-Borne Pathogens at the Host–Vector Interface, pp. 95–103. Munderloh U. G., Kurtti T. J. St Paul, MN: University of Minnesota;
    [Google Scholar]
  37. Rosa P. A., Tilly K., Stewart P. E. ( 2005). The burgeoning molecular genetics of the Lyme disease spirochaete. Nat Rev Microbiol 3:129–143 [View Article][PubMed]
    [Google Scholar]
  38. Sacerdot C., Chiaruttini C., Engst K., Graffe M., Milet M., Mathy N., Dondon J., Springer M. ( 1996). The role of the AUU initiation codon in the negative feedback regulation of the gene for translation initiation factor IF3 in Escherichia coli. Mol Microbiol 21:331–346 [View Article][PubMed]
    [Google Scholar]
  39. Sambrook J., Russell D. W. ( 2000). Molecular Cloning: a Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  40. Samuels D. S. ( 1995). Electrotransformation of the spirochete Borrelia burgdorferi. Methods in Molecular Biology253–259 Nickoloff J. A. Totowa, NJ: Humana Press, Inc;
    [Google Scholar]
  41. Sauve A. A. ( 2008). NAD+ and vitamin B3: from metabolism to therapies. J Pharmacol Exp Ther 324:883–893 [View Article][PubMed]
    [Google Scholar]
  42. Silva R. M., Duarte I. C., Paredes J. A., Lima-Costa T., Perrot M., Boucherie H., Goodfellow B. J., Gomes A. C., Mateus D. D. et al. ( 2009). The yeast PNC1 longevity gene is up-regulated by mRNA mistranslation. PLoS One 4:e5212 [View Article][PubMed]
    [Google Scholar]
  43. Stewart P. E., Thalken R., Bono J. L., Rosa P. ( 2001). Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi. Mol Microbiol 39:714–721 [View Article][PubMed]
    [Google Scholar]
  44. Strother K. O., de Silva A. ( 2005). Role of Borrelia burgdorferi linear plasmid 25 in infection of Ixodes scapularis ticks. J Bacteriol 187:5776–5781 [View Article][PubMed]
    [Google Scholar]
  45. Sussman J. K., Simons E. L., Simons R. W. ( 1996). Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo. Mol Microbiol 21:347–360 [View Article][PubMed]
    [Google Scholar]
  46. Tilly K., Grimm D., Bueschel D. M., Krum J. G., Rosa P. ( 2004). Infectious cycle analysis of a Borrelia burgdorferi mutant defective in transport of chitobiose, a tick cuticle component. Vector Borne Zoonotic Dis 4:159–168 [View Article][PubMed]
    [Google Scholar]
  47. Tilly K., Rosa P. A., Stewart P. E. ( 2008). Biology of infection with Borrelia burgdorferi. Infect Dis Clin North Am 22:217–234, v [View Article][PubMed]
    [Google Scholar]
  48. van der Horst A., Schavemaker J. M., Pellis-van Berkel W., Burgering B. M. ( 2007). The Caenorhabditis elegans nicotinamidase PNC-1 enhances survival. Mech Ageing Dev 128:346–349 [View Article][PubMed]
    [Google Scholar]
  49. Vrablik T. L., Huang L., Lange S. E., Hanna-Rose W. ( 2009). Nicotinamidase modulation of NAD+ biosynthesis and nicotinamide levels separately affect reproductive development and cell survival in C. elegans. Development 136:3637–3646 [View Article][PubMed]
    [Google Scholar]
  50. Wang G., Pichersky E. ( 2007). Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis. Plant J 49:1020–1029 [View Article][PubMed]
    [Google Scholar]
  51. Zhu N., Olivera B. M., Roth J. R. ( 1991). Activity of the nicotinamide mononucleotide transport system is regulated in Salmonella typhimurium. J Bacteriol 173:1311–1320[PubMed]
    [Google Scholar]
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