Engineering the transcriptional activator NifA for the construction of Rhodobacter sphaeroides strains that produce hydrogen gas constitutively

  • Tetsu Shimizu
  • Haruhiko Teramoto
  • Masayuki InuiEmail author
Bioenergy and biofuels


Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas palustris produce hydrogen gas (H2) via proton reduction, which is catalyzed by nitrogenase. Although the expression of nitrogenase is usually repressed under nitrogen-sufficient conditions, a partial deletion of nifA, which encodes a transcriptional activator of nitrogen-fixation genes, has been reported to enable the constitutive expression of nitrogenase in R. palustris. In this study, we evaluated the effects of a similar mutation (nifA* mutation) on H2 production during the photoheterotrophic growth of R. sphaeroides, based on the notion that H2 production by nitrogenase compensates for the loss of CO2 fixation via the Calvin cycle, thereby restoring the redox balance. The chromosomal nifA* mutation resulted in the slight restoration of the photoheterotrophic growth of a mutant strain lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin cycle, when the strain was cultured in van Niel’s yeast medium. In addition, the strain with the nifA* mutation produced detectable levels of H2 during photoheterotrophic growth with acetate and ammonium; however, the H2 production was considerably lower than that observed during the photoheterotrophic growth of the strain with acetate and l-glutamate, where l-glutamate serves as a poor nitrogen source, thereby causing nitrogenase derepression. On the other hand, introduction of a multicopy plasmid harboring nifA* markedly restored the photoheterotrophic growth of the RubisCO-deletion mutant in van Niel’s yeast medium and resulted in efficient H2 production during the photoheterotrophic growth with acetate and ammonium.


Rhodobacter sphaeroides Nitrogenase Hydrogen production NifA Calvin cycle 


Funding information

This work was supported by grants from the Ministry of Economy, Trade and Industry (METI), Japan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or vertebrates performed by any of the authors.

Supplementary material

253_2019_10199_MOESM1_ESM.pdf (236 kb)
ESM 1 (PDF 236 kb)


  1. Alber BE, Spanheimer R, Ebenau-Jehle C, Fuchs G (2006) Study of an alternate glyoxylate cycle for acetate assimilation by Rhodobacter sphaeroides. Mol Microbiol 61(2):297–309. CrossRefPubMedGoogle Scholar
  2. Barbosa MJ, Rocha JM, Tramper J, Wijffels RH (2001) Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 85(1):25–33. CrossRefPubMedGoogle Scholar
  3. Dixon R, Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2(8):621–631. CrossRefPubMedGoogle Scholar
  4. Drepper T, Gross S, Yakunin AF, Hallenbeck PC, 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(Pt 8):2203–2212. CrossRefPubMedGoogle Scholar
  5. Erb TJ, Berg IA, Brecht V, Muller M, Fuchs G, Alber BE (2007) Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proc Natl Acad Sci U S A 104(25):10631–10636. CrossRefPubMedPubMedCentralGoogle Scholar
  6. Erb TJ, Fuchs G, Alber BE (2009) (2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. Mol Microbiol 73(6):992–1008. CrossRefPubMedGoogle Scholar
  7. Falcone DL, Tabita FR (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(6):2099–2108. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Fang HHP, Zhu H, Zhang T (2006) Phototrophic hydrogen production from glucose by pure and co-culture of Clostridium butyricum and Rhodobacter sphaeroides. Int J Hydrogen Energy 31:2223–2230. CrossRefGoogle Scholar
  9. Farmer RM, Laguna R, Panescu J, McCoy A, Logsdon B, Zianni M, Moskvin OV, Gomelsky M, Tabita FR (2014) Altered residues in key proteins influence the expression and activity of the nitrogenase complex in an adaptive CO2 fixation-deficient mutant strain of Rhodobacter sphaeroides. Microbiology 160(1):198–208. CrossRefPubMedGoogle Scholar
  10. Gest H, Kamen MD (1949) Photoproduction of molecular hydrogen by Rhodospirillum rubrum. Science 109(2840):558–559. CrossRefPubMedGoogle Scholar
  11. Heiniger EK, Oda Y, Samanta SK, Harwood CS (2012) How posttranslational modification of nitrogenase is circumvented in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively. Appl Environ Microbiol 78(4):1023–1032. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Hillmer P, Gest H (1977) H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: H2 production by growing cultures. J Bacteriol 129(2):724–731PubMedPubMedCentralGoogle Scholar
  13. Hubner P, Willison JC, Vignais PM, Bickle TA (1991) Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol 173(9):2993–2999. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Inui M, Nakata K, Roh JH, Vertès AA, Yukawa H (2003) Isolation and molecular characterization of pMG160, a mobilizable cryptic plasmid from Rhodobacter blasticus. Appl Environ Microbiol 69(2):725–733. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Joshi HM, Tabita FR (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(25):14515–14520. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Keskin T, Abo-Hashesh M, Hallenbeck PC (2011) Photofermentative hydrogen production from wastes. Bioresour Technol 102(18):8557–8568. CrossRefPubMedGoogle Scholar
  17. Khadka N, Milton RD, Shaw S, Lukoyanov D, Dean DR, Minteer SD, Raugei S, Hoffman BM, Seefeldt LC (2017) Mechanism of nitrogenase H2 formation by metal-hydride protonation probed by mediated electrocatalysis and H/D isotope effects. J Am Chem Soc 139(38):13518–13524. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Kim E-J, Lee M-K, Kim M-S, Lee JK (2008) Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum. Int J Hydrogen Energy 33(5):1516–1521. CrossRefGoogle Scholar
  19. Laguna R, Tabita FR, Alber BE (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(2):151–154. CrossRefPubMedGoogle Scholar
  20. Macler BA, Pelroy RA, Bassham JA (1979) Hydrogen formation in nearly stoichiometric amounts from glucose by a Rhodopseudomonas sphaeroides mutant. J Bacteriol 138(2):446–452PubMedPubMedCentralGoogle Scholar
  21. McKinlay JB, Harwood CS (2010) Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proc Natl Acad Sci U S A 107(26):11669–11675. CrossRefPubMedPubMedCentralGoogle Scholar
  22. McKinlay JB, Oda Y, Rühl M, Posto AL, Sauer U, Harwood CS (2013) Non-growing Rhodopseudomonas palustris increases the hydrogen gas yield from acetate by shifting from the glyoxylate shunt to the tricarboxylic acid cycle. J Biol Chem 289(4):1960–1970. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ren N, Guo W, Liu B, Cao G, Ding J (2011) Biological hydrogen production by dark fermentation: challenges and prospects towards scaled-up production. Curr Opin Biotechnol 22(3):365–370. CrossRefGoogle Scholar
  24. Rey FE, Heiniger EK, Harwood CS (2007) Redirection of metabolism for biological hydrogen production. Appl Environ Microbiol 73(5):1665–1671. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Schafer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Puhler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145(1):69–73. CrossRefPubMedGoogle Scholar
  26. Shimizu T, Teramoto H, Inui M (2019) Introduction of glyoxylate bypass increases hydrogen gas yield from acetate and l-glutamate in Rhodobacter sphaeroides. Appl Environ Microbiol 85(2):e01873–e01818. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Wang D, Zhang Y, Welch E, Li J, Roberts GP (2010) Elimination of Rubisco alters the regulation of nitrogenase activity and increases hydrogen production in Rhodospirillum rubrum. Int J Hydrogen Energy 35(14):7377–7385. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Yakunin AF, Fedorov AS, Laurinavichene TV, Glaser VM, Egorov NS, Tsygankov AA, Zinchenko VV, Hallenbeck PC (2001) Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus. Can J Microbiol 47(3):206–212CrossRefGoogle Scholar
  29. Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H (2005) Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing Escherichia coli strains. Appl Environ Microbiol 71(11):6762–6768. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Zhang Y, Pohlmann EL, Ludden PW, Roberts GP (2001) Functional characterization of three glnB Homologs in the photosynthetic bacterium Rhodospirillum rubrum: Roles in sensing ammonium and energy status. J Bacteriol 183(21):6159–6168. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Zhang Y, Pohlmann EL, Roberts GP (2004) Identification of critical residues in GlnB for its activation of NifA activity in the photosynthetic bacterium Rhodospirillum rubrum. Proc Natl Acad Sci U S A 101(9):2782–2787. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Research Institute of Innovative Technology for the EarthKyotoJapan
  2. 2.Division of Biological SciencesNara Institute of Science and TechnologyNaraJapan

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