1887

Abstract

Previously, the RubisCO-compromised spontaneous adaptive mutant, strain 16PHC, was shown to derepress the expression of genes that encode the nitrogenase complex under normal repressive conditions. As a result of this adaptation, the active nitrogenase complex restored redox balance, thus allowing strain 16PHC to grow under photoheterotrophic conditions in the absence of an exogenous electron acceptor. A combination of whole genome pyrosequencing and whole genome microarray analyses was employed to identify possible loci responsible for the observed phenotype. Mutations were found in two genes, and , whose products are involved in the regulatory cascade that controls nitrogenase complex gene expression. In addition, a nucleotide reversion within the gene, which encodes a subunit of the nitrogenase complex, was also identified. Subsequent genetic, physiological and biochemical studies revealed alterations that led to derepression of the synthesis of an active nitrogenase complex in strain 16PHC.

Funding
This study was supported by the:
  • Chemical Sciences, Geosciences, and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (Award DE-FG02-08ER15976)
  • University of Wyoming School of Energy Resources
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.073031-0
2014-01-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/160/1/198.html?itemId=/content/journal/micro/10.1099/mic.0.073031-0&mimeType=html&fmt=ahah

References

  1. Almassy R. J., Janson C. A., Hamlin R., Xuong N. H., Eisenberg D. ( 1986). Novel subunit–subunit interactions in the structure of glutamine synthetase. Nature 323:304–309 [View Article][PubMed]
    [Google Scholar]
  2. Anderson L. E., Fuller R. C. ( 1969). Photosynthesis in Rhodospirillum rubrum. IV. Isolation and characterization of ribulose 1,5-diphosphate carboxylase. J Biol Chem 244:3105–3109[PubMed]
    [Google Scholar]
  3. Ausubel F. M., Brent R., Kingston R. E., Moore D., Seidman J. G., Smith J. A., Struhl K. (editors) ( 2001). Current Protocols in Molecular Biology New York: Wiley; [View Article]
    [Google Scholar]
  4. Barbosa M. J., Rocha J. M., Tramper J., Wijffels R. H. ( 2001). Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 85:25–33 [View Article][PubMed]
    [Google Scholar]
  5. Brigle K. E., Setterquist R. A., Dean D. R., Cantwell J. S., Weiss M. C., Newton W. E. ( 1987). Site-directed mutagenesis of the nitrogenase MoFe protein of Azotobacter vinelandii. Proc Natl Acad Sci U S A 84:7066–7069 [View Article][PubMed]
    [Google Scholar]
  6. Buchanan-Wollaston V., Cannon M. C., Cannon F. C. ( 1981a). The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet 184:102–106 [View Article][PubMed]
    [Google Scholar]
  7. Buchanan-Wollaston V., Cannon M. C., Beynon J. L., Cannon F. C. ( 1981b). Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294:776–778 [View Article][PubMed]
    [Google Scholar]
  8. Crooks G. E., Hon G., Chandonia J. M., Brenner S. E. ( 2004). WebLogo: a sequence logo generator. Genome Res 14:1188–1190 [View Article][PubMed]
    [Google Scholar]
  9. Dixon R., Kahn D. ( 2004). Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2:621–631 [View Article][PubMed]
    [Google Scholar]
  10. Dixon R., Kennedy C., Kondorosi A., Krishnapillai V., Merrick M. ( 1977). Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation. Mol Gen Genet 157:189–198 [View Article][PubMed]
    [Google Scholar]
  11. Drepper T., Gross S., Yakunin A. F., Hallenbeck P. C., Masepohl B., Klipp W. ( 2003). Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiology 149:2203–2212 [View Article][PubMed]
    [Google Scholar]
  12. Edgren T., Nordlund S. ( 2004). The fixABCX genes in Rhodospirillum rubrum encode a putative membrane complex participating in electron transfer to nitrogenase. J Bacteriol 186:2052–2060 [View Article][PubMed]
    [Google Scholar]
  13. Eisenberg D., Gill H. S., Pfluegl G. M., Rotstein S. H. ( 2000). Structure–function relationships of glutamine synthetases. Biochim Biophys Acta 1477:122–145 [View Article][PubMed]
    [Google Scholar]
  14. Falcone D. L., Tabita F. R. ( 1991). Expression of endogenous and foreign ribulose 1,5-bisphosphate carboxylase-oxygenase (RubisCO) genes in a RubisCO deletion mutant of Rhodobacter sphaeroides. J Bacteriol 173:2099–2108[PubMed]
    [Google Scholar]
  15. Falcone D. L., Tabita F. R. ( 1993). Complementation analysis and regulation of CO2 fixation gene expression in a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion strain of Rhodospirillum rubrum. J Bacteriol 175:5066–5077[PubMed]
    [Google Scholar]
  16. Franchi E., Tosi C., Scolla G., Penna G. D., Rodriguez F., Pedroni P. M. ( 2004). Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes. Mar Biotechnol (NY) 6:552–565 [View Article][PubMed]
    [Google Scholar]
  17. Gautier L., Cope L., Bolstad B. M., Irizarry R. A. ( 2004). Affy—analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 20:307–315 [View Article][PubMed]
    [Google Scholar]
  18. Hallenbeck P. L., Lerchen R., Hessler P., Kaplan S. ( 1990a). Roles of CfxA, CfxB, and external electron acceptors in regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase expression in Rhodobacter sphaeroides. J Bacteriol 172:1736–1748[PubMed]
    [Google Scholar]
  19. Hallenbeck P. L., Lerchen R., Hessler P., Kaplan S. ( 1990b). Phosphoribulokinase activity and regulation of CO2 fixation critical for photosynthetic growth of Rhodobacter sphaeroides. J Bacteriol 172:1749–1761[PubMed]
    [Google Scholar]
  20. Heiniger E. K., Oda Y., Samanta S. K., Harwood C. S. ( 2012). How posttranslational modification of nitrogenase is circumvented in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively. Appl Environ Microbiol 78:1023–1032 [View Article][PubMed]
    [Google Scholar]
  21. Hübner P., Masepohl B., Klipp W., Bickle T. A. ( 1993). nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol 10:123–132 [View Article][PubMed]
    [Google Scholar]
  22. Irizarry R. A., Bolstad B. M., Collin F., Cope L. M., Hobbs B., Speed T. P. ( 2003). Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31:e15 [View Article][PubMed]
    [Google Scholar]
  23. Jahn A., Keuntje B., Dörffler M., Klipp W., Oelze J. ( 1994). Optimizing photoheterotrophic H2 production by Rhodobacter capsulatus upon interposon mutagenesis in the hupL gene. Appl Microbiol Biotechnol 40:687–690 [View Article][PubMed]
    [Google Scholar]
  24. Johansson B. C., Gest H. ( 1977). Adenylylation/deadenylylation control of the glutamine synthetase of Rhodopseudomonas capsulata. Eur J Biochem 81:365–371 [View Article][PubMed]
    [Google Scholar]
  25. Jones B. L., Monty K. J. ( 1979). Glutamine as a feedback inhibitor of the Rhodopseudomonas sphaeroides nitrogenase system. J Bacteriol 139:1007–1013[PubMed]
    [Google Scholar]
  26. Jonsson A., Teixeira P. F., Nordlund S. ( 2007). The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by α-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro. FEBS J 274:2449–2460 [View Article][PubMed]
    [Google Scholar]
  27. Joshi H. M., Tabita F. R. ( 1996). A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation. Proc Natl Acad Sci U S A 93:14515–14520 [View Article][PubMed]
    [Google Scholar]
  28. Kern M., Kamp P. B., Paschen A., Masepohl B., Klipp W. ( 1998). Evidence for a regulatory link of nitrogen fixation and photosynthesis in Rhodobacter capsulatus via HvrA. J Bacteriol 180:1965–1969[PubMed]
    [Google Scholar]
  29. Khatipov E., Miyake M., Miyake J., Asada Y. ( 1998). Accumulation of poly-β-hydroxybutyrate by Rhodobacter sphaeroides on various carbon and nitrogen substrates. FEMS Microbiol Lett 162:39–45
    [Google Scholar]
  30. Koku H., Eroğlu İ., Gündüz U., Yücel M., Türker L. ( 2002). Aspects of the metabolism of hydrogen production by Rhodobacter sphaeroides. Int J Hydrogen Energy 27:1315–1329 [View Article]
    [Google Scholar]
  31. Krahn E., Schneider K., Muller A. ( 1996). Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative “iron-only”� nitrogenase of Rhodobacter capsulatus hup mutants. Appl Microbiol Biotechnol 46:285–290 [View Article]
    [Google Scholar]
  32. Laguna R., Tabita F. R., Alber B. E. ( 2011). Acetate-dependent photoheterotrophic growth and the differential requirement for the Calvin–Benson–Bassham reductive pentose phosphate cycle in Rhodobacter sphaeroides and Rhodopseudomonas palustris. Arch Microbiol 193:151–154 [View Article][PubMed]
    [Google Scholar]
  33. Leigh J. A., Dodsworth J. A. ( 2007). Nitrogen regulation in bacteria and archaea. Annu Rev Microbiol 61:349–377 [View Article][PubMed]
    [Google Scholar]
  34. Li X., Liu T., Wu Y., Zhao G., Zhou Z. ( 2010). Derepressive effect of NH4+ on hydrogen production by deleting the glnA1 gene in Rhodobacter sphaeroides. Biotechnol Bioeng 106:564–572 [View Article][PubMed]
    [Google Scholar]
  35. Little R., Dixon R. ( 2003). The amino-terminal GAF domain of Azotobacter vinelandii NifA binds 2-oxoglutarate to resist inhibition by NifL under nitrogen-limiting conditions. J Biol Chem 278:28711–28718 [View Article][PubMed]
    [Google Scholar]
  36. Maniatis T., Fritsch E. F., Sambrook J. ( 1982). Molecular Cloning: a Laboratory Manual Cold Springs Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  37. Masepohl B., Krey R., Klipp W. ( 1993). The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase. J Gen Microbiol 139:2667–2675 [View Article][PubMed]
    [Google Scholar]
  38. Masepohl B., Drepper T., Paschen A., Gross S., Pawlowski A., Raabe K., Riedel K. U., Klipp W. ( 2002). Regulation of nitrogen fixation in the phototrophic purple bacterium Rhodobacter capsulatus. J Mol Microbiol Biotechnol 4:243–248[PubMed]
    [Google Scholar]
  39. Oelze J., Klein G. ( 1996). Control of nitrogen fixation by oxygen in purple nonsulfur bacteria. Arch Microbiol 165:219–225 [View Article][PubMed]
    [Google Scholar]
  40. Ormerod J. G., Ormerod K. S., Gest H. ( 1961). Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys 94:449–463 [View Article][PubMed]
    [Google Scholar]
  41. Pappas C. T., Sram J., Moskvin O. V., Ivanov P. S., Mackenzie R. C., Choudhary M., Land M. L., Larimer F. W., Kaplan S., Gomelsky M. ( 2004). Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes. J Bacteriol 186:4748–4758 [View Article][PubMed]
    [Google Scholar]
  42. Paschen A., Drepper T., Masepohl B., Klipp W. ( 2001). Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium. FEMS Microbiol Lett 200:207–213 [View Article][PubMed]
    [Google Scholar]
  43. Rey F. E., Heiniger E. K., Harwood C. S. ( 2007). Redirection of metabolism for biological hydrogen production. Appl Environ Microbiol 73:1665–1671 [View Article][PubMed]
    [Google Scholar]
  44. Rizk M. L., Laguna R., Smith K. M., Tabita F. R., Liao J. C. ( 2011). Redox homeostasis phenotypes in RubisCO-deficient Rhodobacter sphaeroides via ensemble modeling. Biotechnol Prog 27:15–22 [View Article][PubMed]
    [Google Scholar]
  45. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. ( 1978). Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol 136:267–279[PubMed]
    [Google Scholar]
  46. Robinson J. T., Thorvaldsdóttir H., Winckler W., Guttman M., Lander E. S., Getz G., Mesirov J. P. ( 2011). Integrative genomics viewer. Nat Biotechnol 29:24–26 [View Article][PubMed]
    [Google Scholar]
  47. Schneider T. D., Stephens R. M. ( 1990). Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18:6097–6100 [View Article][PubMed]
    [Google Scholar]
  48. Smith S. A., Tabita F. R. ( 2002). Up-regulated expression of the cbbI and cbbII operons during photoheterotrophic growth of a ribulose 1,5-bisphosphate carboxylase-oxygenase deletion mutant of Rhodobacter sphaeroides. J Bacteriol 184:6721–6724 [View Article][PubMed]
    [Google Scholar]
  49. Thorvaldsdóttir H., Robinson J. T., Mesirov J. P. ( 2013). Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192 [View Article][PubMed]
    [Google Scholar]
  50. Tichi M. A., Tabita F. R. ( 2000). Maintenance and control of redox poise in Rhodobacter capsulatus strains deficient in the Calvin–Benson–Bassham pathway. Arch Microbiol 174:322–333 [View Article][PubMed]
    [Google Scholar]
  51. Vasilyeva L., Miyake M., Khatipov E., Wakayama T., Sekine M., Hara M., Nakada E., Asada Y., Miyake J. ( 1999). Enhanced hydrogen production by a mutant of Rhodobacter sphaeroides having an altered light-harvesting system. J Biosci Bioeng 87:619–624 [View Article][PubMed]
    [Google Scholar]
  52. Wall J. D., Gest H. ( 1979). Derepression of nitrogenase activity in glutamine auxotrophs of Rhodopseudomonas capsulata. J Bacteriol 137:1459–1463[PubMed]
    [Google Scholar]
  53. Wang X., Falcone D. L., Tabita F. R. ( 1993). Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell. J Bacteriol 175:3372–3379[PubMed]
    [Google Scholar]
  54. Wang D., Zhang Y., Pohlmann E. L., Li J., Roberts G. P. ( 2011). The poor growth of Rhodospirillum rubrum mutants lacking RubisCO is due to the accumulation of ribulose-1,5-bisphosphate. J Bacteriol 193:3293–3303 [View Article][PubMed]
    [Google Scholar]
  55. Weaver K. E., Tabita F. R. ( 1983). Isolation and partial characterization of Rhodopseudomonas sphaeroides mutants defective in the regulation of ribulose bisphosphate carboxylase/oxygenase. J Bacteriol 156:507–515[PubMed]
    [Google Scholar]
  56. Willison J. C., Madern D., Vignais P. M. ( 1984). Increased photoproduction of hydrogen by non-autotrophic mutants of Rhodopseudomonas capsulata. Biochem J 219:593–600[PubMed]
    [Google Scholar]
  57. Zhang Y., Cummings A. D., Burris R. H., Ludden P. W., Roberts G. P. ( 1995). Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. J Bacteriol 177:5322–5326[PubMed]
    [Google Scholar]
  58. Zinchenko V., Babykin M., Glaser V., Mekhedov S., Shestakov S. ( 1997). Mutation in ntrC gene leading to the derepression of nitrogenase synthesis in Rhodobacter sphaeroides. FEMS Microbiol Lett 147:57–61 [View Article][PubMed]
    [Google Scholar]
  59. Zou X., Zhu Y., Pohlmann E. L., Li J., Zhang Y., Roberts G. P. ( 2008). Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum. Microbiology 154:2689–2699 [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.073031-0
Loading
/content/journal/micro/10.1099/mic.0.073031-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error