Artificial activation of nif gene expression in nodule bacteria Ex Planta

Cover Page

Cite item

Full Text

Abstract

Background. Rhizobia are the most effective nitrogen-fixing organisms that can fix nitrogen only in symbiosis with leguminous plants. The general transcriptional activator of nitrogen fixation genes in diazotrophic bacteria is NifA. In this work, the possibility of modifying the regulation of nitrogen fixation in the nodule bacteria Mesorhizobium, Ensifer and Rhizobium was studied by introducing an additional copy of the nifA gene into the bacterial genomes during the regulation of induced bacterial promoters.

Materials and methods. A series of expression genetic constructs with NifA genes of nodule bacteria strains under the control of an inducible promoter Pm were created. The resulting constructs were transformed into strains of nodule bacteria. The obtained recombinant strains were investigated for the appearance of their nitrogen-fixing activity in the free-living state.

Results. It was shown that the expression of nifA in recombinant cells of all three genera of bacteria leads to the appearance of insignificant nitrogenase activity. At the same time, the level of nitrogenase activity does not have a correlation with the level of expression of the introduced nifA gene, which, most likely, is a consequence of the multilevel regulation of nitrogen fixation.

Conclusion. The possibility of artificial activation of nitrogenase activity in nodule bacteria in the free-living state by introducing the NifA regulatory protein gene into bacteria was shown.

About the authors

Andrey K. Baymiev

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Author for correspondence.
Email: baymiev@anrb.ru
ORCID iD: 0000-0001-6637-9365
SPIN-code: 1919-5236
ResearcherId: R-9219-2016

PhD, Leading Researcher, Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

Roman S. Gumenko

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: r.gumenko@yandex.ru

Junior Researcher, Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

Anastasiya A. Vladimirova

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: vladimirovaw@bk.ru

Graduate Student, Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

Ekaterina S. Akimova

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: iv.katerina-bio@yandex.ru

PhD, Researcher, Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

Zilya R. Vershinina

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: zilyaver@mail.ru

PhD, Researcher, Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

Aleksey K. Baymiev

Institute of Biochemistry and Genetics – Subdivision of the Ufa Federal Research Centre of the Russian Academy of Sciences

Email: baymiev@mail.ru

PhD, Head of Laboratory of Plant and Microbial Bioengineering

Russian Federation,  71, prospekt Oktyabrya, Ufa, 450054

References

  1. Проворов Н.А., Воробьев Н.И. Генетические основы эволюции растительно-микробного симбиоза. – СПб.: Информ-Навигатор, 2012. [Provorov NA, Vorob’ev NI. Geneticheskie osnovy evolyutsii rastitel’no-mikrobnogo simbioza. Saint Petersburg: Inform-Navigator; 2012. (In Russ.)]
  2. Dludlu MN, Chimphango SBM, Walker G, et al. Horizontal gene transfer among Rhizobia of the Core Cape Subregion of Southern Africa. S Afr J Bot. 2018;118:342-52. https://doi.org/10.1016/j.sajb.2018.02.406.
  3. Andrews M, De Meyer S, James EK, et al. Horizontal transfer of symbiosis genes within and between Rhizobial Genera: Occurrence and Importance. Genes (Basel). 2018;9(7). https://doi.org/10.3390/genes9070321.
  4. Ling J, Wang H, Wu P, et al. Plant nodulation inducers enhance horizontal gene transfer of Azorhizobium caulinodans symbiosis island. Proc Natl Acad Sci USA. 2016;113(48):13875-13880. https://doi.org/10.1073/pnas.1615121113.
  5. Dixon R, Kahn D. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol. 2004;2(8):621-31. https://doi.org/10.1038/nrmicro954.
  6. Kennedy C, Robson RL. Activation of nif gene expression in Azotobacter by the nifA gene product of Klebsiella pneumoniae. Nature. 1983;301(5901):626-628. https://doi.org/10.1038/301626a0.
  7. Kim Y-M, Hidaka M, Masaki H, et al. Constitutive expression of nitrogenase system in Klebsiella oxytoca by gene targeting mutation to the chromosomal nifLA operon. J Biotechnol. 1989;10(3-4):293-301. https://doi.org/10.1016/0168-1656(89)90073-4.
  8. An Q, Dong Y, Wang W, et al. Constitutive expression of the nifA gene activates associative nitrogen fixation of Enterobacter gergoviae 57-7, an opportunistic endophytic diazotroph. J Appl Microbiol. 2007;103(3):613-620. https://doi.org/10.1111/j.1365-2672.2007.03289.x.
  9. Chabot R, Antoun H, Kloepper JW, Beauchamp CJ. Root colonization of maize and lettuce by bioluminescent Rhizobium leguminosarum biovar phaseoli. Appl Environ Microbiol. 1996;62(8):2767-2772.
  10. Chao WL. Antagonistic activity of Rhizobium spp. against beneficial and plant pathogenic fungi. Lett Appl Microbiol. 1990;10(5):213-215. https://doi.org/10.1111/j.1472-765X.1990.tb01336.x.
  11. Blatny JM, Brautaset T, Winther-Larsen HC, et al. Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid. 1997;38(1):35-51. https://doi.org/10.1006/plas.1997.1294.
  12. Sletta H, Nedal A, Aune TE, et al. Broad-host-range plasmid pJB658 can be used for industrial-level production of a secreted host-toxic single-chain antibody fragment in Escherichia coli. Appl Environ Microbiol. 2004;70(12):7033-7039. https://doi.org/10.1128/AEM.70.12.7033-7039.2004.
  13. Lin JJ. Electrotransformation of Agrobacterium. In: Methods in molecular biology. Totowa: Humana Press; 1995. P. 171-178.
  14. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA. 1979;76(9):4350-4354. https://doi.org/10.1073/pnas.76.9.4350.
  15. Mongiardini EJ, Ausmees N, Perez-Gimenez J, et al. The Rhizobial adhesion protein RapA1 is involved in adsorption of Rhizobia to plant roots but not in nodulation. FEMS Microbiol Ecol. 2008;65(2):279-288. https://doi.org/10.1111/j.1574-6941.2008.00467.x.
  16. Tsoy OV, Ravcheev DA, Cuklina J, Gelfand MS. Nitrogen fixation and molecular oxygen: comparative genomic reconstruction of transcription regulation in Alphaproteobacteria. Front Microbiol. 2016;7:1343. https://doi.org/10.3389/fmicb.2016.01343.
  17. González-Pérez MM, Marqués S, Domínguez-Cuevas P, Ramos JL. XylS activator and RNA polymerase binding sites at the Pm promoter overlap. FEBS Letters. 2002;519(1-3):117-122. https://doi.org/10.1016/s0014-5793(02)02730-8.
  18. Brautaset T, Lale R, Valla S. Positively regulated bacterial expression systems. Microb Biotechnol. 2009;2(1):15-30. https://doi.org/10.1111/j.1751-7915.2008.00048.x.
  19. Sullivan JT, Trzebiatowski JR, Cruickshank RW, et al. Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain R7A. J Bacteriol. 2002;184(11):3086-3095. https://doi.org/10.1128/jb.184.11.3086-3095.2002.
  20. Sullivan JT, Brown SD, Ronson CW. The NifA-RpoN regulon of Mesorhizobium loti strain R7A and its symbiotic activation by a novel LacI/GalR-family regulator. PLoS One. 2013;8(1): e53762. https://doi.org/10.1371/journal.pone.0053762.
  21. Gamez-Reyes A, Becerra-Lobato N, Ramírez-Trujillo JA, et al. The Rhizobium leucaenae CFN299 pSym plasmid contains genes expressed in free life and symbiosis, as well as two replication systems. Ann Microbiol. 2017;67(3): 263-73. https://doi.org/10.1007/s13213-017-1257-3.
  22. Nukui N, Minamisawa K, Ayabe S, Aoki T. Expression of the 1-aminocyclopropane-1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in Mesorhizobium loti MAFF303099. Appl Environ Microbiol. 2006;72(7):4964-4969. https://doi.org/10.1128/AEM.02745-05.
  23. Oelze J. Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence? FEMS Microbiol Rev. 2000;24(4):321-333. https://doi.org/10.1111/j.1574-6976.2000.tb00545.x.
  24. Boyd ES, Costas AM, Hamilton TL, et al. Evolution of molybdenum nitrogenase during the transition from anaerobic to aerobic metabolism. J Bacteriol. 2015;197(9):1690-9. https://doi.org/10.1128/JB.02611-14.
  25. Boyd ES, Peters JW. New insights into the evolutionary history of biological nitrogen fixation. Front Microbiol. 2013;4:201. https://doi.org/10.3389/fmicb.2013.00201.
  26. Torres MJ, Rubia MI, de la Pena TC, et al. Genetic basis for denitrification in Ensifer meliloti. BMC Microbiol. 2014;14:142. https://doi.org/10.1186/1471-2180-14-142.
  27. Bobik C, Meilhoc E, Batut J. FixJ: a major regulator of the oxygen limitation response and late symbiotic functions of Sinorhizobium meliloti. J Bacteriol. 2006;188(13): 4890-4902. https://doi.org/10.1128/JB.00251-06.
  28. Cabeza R, Koester B, Liese R, et al. An RNA sequencing transcriptome analysis reveals novel insights into molecular aspects of the nitrate impact on the nodule activity of Medicago truncatula. Plant Physiol. 2014;164(1):400-411. https://doi.org/10.1104/pp.113.228312.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. The strategy of cloning nifA genes into plasmid pJB658

Download (42KB)
Indexing metadata
3. Fig. 2. Analysis of the nitrogenase activity of bacteria. 1 — Rhizobium sp.VSy9PmNifA; 2 — Mesorhizobium sp. LZh7PmNifA; 3 — Ensifer sp. Mlu10PmNifA; 4 — control (Pseudomonas sp. К749 — free-living nitrogen fixer). К1, К2, К3 — wild variants of strains of nodule bacteria

Download (57KB)
Indexing metadata
4. Fig. 3. RT-PCR analysis of the transcriptional activity of the nifA (a) and nifH (б) genes in cells of wild and recombinant rhizobia strains of Rhizobium sp. VSy9, Mesorhizobium sp. LZh7, Ensifer sp. Mlu10. 1 — wild strains of bacteria; 2 — recombinant bacteria strains without induction; 3 — recombinant bacterial strains with induction; 4 — PCR of a total recombinant bacteria tion after DNAse treatment; 5 — negative control

Download (37KB)
Indexing metadata
5. Fig.4. Dot-blot analysis of NifH protein production by recombinant strains of nodule bacteria grown on a nutrient medium without and with the addition of an inducer of m-toluic acid (0.5 mM). 1 — positive control of strain Pseudomonas sp. К749; 2 — non-recombinant strain of bacteria Rhizobium sp. VSy9; 3 — Non-recombinant bacteria strain Ensifer sp. Mlu10; 4 — non-recombinant strain of bacteria Mesorhizobium sp. LZh7; 5 — recombinant strain of bacteria Rhizobium sp. VSy9PmNifA, cultivated on medium without inductor; 6 — recombinant strain of bacteria Ensifer sp. Mlu10PmNifA, cultivated on medium without inductor; 7 — recombinant strain of bacteria Mesorhizobium sp. LZh7PmNifA, cultivated on the medium without inductor; 8 — recombinant strain of bacteria Rhizobium sp. VSy9PmNifA, cultivated on medium with inductor; 9 — recombinant strain of bacteria Ensifer sp. Mlu10PmNifA, cultivated on medium with inducer; 10 — recombinant strain of bacteria Mesorhizobium sp. LZh7PmNifA, cultivated on medium with inductor

Download (13KB)
Indexing metadata

Copyright (c) 2019 Baymiev An.K., Gumenko R.S., Vladimirova A.A., Akimova E.S., Vershinina Z.R., Baymiev Al.K.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.
 


This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies