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. 2017;40(1 suppl 1):261-275.
doi: 10.1590/1678-4685-GMB-2016-0050. Epub 2017 Mar 20.

Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions

Alberto A Esteves-Ferreira  1   2 , João Henrique Frota Cavalcanti  1   2 , Marcelo Gomes Marçal Vieira Vaz  1   2 , Luna V Alvarenga  1   2 , Adriano Nunes-Nesi  1   2 , Wagner L Araújo  1   2
Affiliations

Cyanobacterial nitrogenases: phylogenetic diversity, regulation and functional predictions

Alberto A Esteves-Ferreira et al. Genet Mol Biol. 2017.

Abstract

Cyanobacteria is a remarkable group of prokaryotic photosynthetic microorganisms, with several genera capable of fixing atmospheric nitrogen (N2) and presenting a wide range of morphologies. Although the nitrogenase complex is not present in all cyanobacterial taxa, it is spread across several cyanobacterial strains. The nitrogenase complex has also a high theoretical potential for biofuel production, since H2 is a by-product produced during N2 fixation. In this review we discuss the significance of a relatively wide variety of cell morphologies and metabolic strategies that allow spatial and temporal separation of N2 fixation from photosynthesis in cyanobacteria. Phylogenetic reconstructions based on 16S rRNA and nifD gene sequences shed light on the evolutionary history of the two genes. Our results demonstrated that (i) sequences of genes involved in nitrogen fixation (nifD) from several morphologically distinct strains of cyanobacteria are grouped in similarity with their morphology classification and phylogeny, and (ii) nifD genes from heterocytous strains share a common ancestor. By using this data we also discuss the evolutionary importance of processes such as horizontal gene transfer and genetic duplication for nitrogenase evolution and diversification. Finally, we discuss the importance of H2 synthesis in cyanobacteria, as well as strategies and challenges to improve cyanobacterial H2 production.

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Figures

Figure 1
Figure 1. Natural morphological variation within cyanobacterial genera. Unicellular strains: (a) Synechocystis sp. PCC6803 and (b) Synechococcus elongatus PCC4972. Non-heterocytous strains: (c) Arthrospira maxima, (d) Trichodesmium sp. and (e) Phormidium sp. CCM-UFV034. False branching or non-branching heterocytous strains: (f) Nostoc sp. CCM-UFV028 and (g) Brasilonema octagenarum CCM-UFVE1. True-branching heterocytous strain: (h) Stigonema sp. CCM-UFV036. Synechocystis sp. (a) and Arthrospira maxima (c) are unable to perform N2 fixation, whereas the strains shown in b and e present a temporal separation of metabolism: photosynthetic CO2 fixation is performed in the light while N2 fixation occurs during darkness. Trichodesmium sp. (d) is the unique non-heterocytous cyanobacterium that shown N2 fixation under light conditions. Conversely, in other strains (f, g, and h), there is a spatial separation of metabolism, with N2 fixation occurring in heterocytes (ht). Abbreviations: (ak) akinetes; (fb) false branching; (tb) true branching. The picture of Arthrospira maxima (c) was kindly provided by the Culture Collection of Autotrophic Organisms (CCALA), http://ccala.butbn.cas.cz and the picture of Trichodesmium sp. (d) by Prof. Ondøej Práil, Institute of Microbiology, Czech Academy of Sciences, Czech Republic. The other pictures are from strains kept at Collection of Cyanobacteria and Microalgae from Universidade Federal de Viçosa (CCM-UFV).
Figure 2
Figure 2. Nitrogenase gene structure. (A) Map illustrating two of the nif gene clusters present in Anabaena variabilis ATCC29413. The operons are represented using different colours: yellow (nifB-fdxN-nifSU), green (nifHDK), blue (nifENXW) and red (nifVZT). White arrows indicate genes which encode proteins with unknown functions. The 11-kb excision element observed in the nifHDK operon is not present in the chromosomes of heterocytes. nifVZT is part of a second nif gene cluster. (B) Nitrogenase structure and biochemical activity. Dinitrogenase reductase is a homodimer (2×30 kDa) with one [4Fe-4S]-cluster (Fe protein) encoded by nifH, whereas dinitrogenase is a FeMo-protein encoded by the genes nifD (α-subunit) and nifK (β-subunit) and organized in a α2β2 tetramer of 240 kDa associated with two FeMo-cofactors (FeMo-co) and two P-clusters. Notably, there are three types of dinitrogenases normally found in cyanobacterial nitrogenases, which vary depending on the metal content. Thus, type 1 contains molybdenum (Mo), type 2 contains vanadium (V), and type 3 iron (Fe). The reduction of nitrogen (N2) to ammonia (NH3) requires metabolic energy in the form of ATP, and in this context two ATP molecules are used for each electron transferred from the dinitrogenase reductase to dinitrogenase. Consequently, the reaction requires a total of 16 ATP molecules until the dinitrogenase has accumulated enough electrons to reduce N2 to NH3. In addition, this reaction is accompanied by the reduction of two protons (H+) to one hydrogen molecule (H2).
Figure 3
Figure 3. Maximum Likelihood (ML) phylogenetic reconstruction based on partial nifD gene sequences. A total of 39 nucleotide sequences of α-subunit of dinitrogenase were used. A matrix with 6,904 base pair lenght was obtained after alingment. The general time reversible evolutionary model of substitution with gamma distribution and with an estimate of proportion of invariable sites (GTR + G + I) was selected as the fittest for the alignment by MrModelTest 2.3 (Nylander, 2004). Phylogenetic trees were reconstructed using the ML and Bayesian methods. For Bayesian analysis, the trees were searched using the software MrBayes 3.2.6 (Ronquist et al., 2012) and the Bayesian analysis consisted of two independent runs, with four Markov chains each, of 50 million generations sampled every 5,000 generations. Posterior probabilities (PP) were calculated at the conclusion of the Markov-Chain-Monte-Carlo analysis and a traditional burn-in on the first 25% of the trees was performed. The ML trees were reconstructed using the MEGA program package, version 5 (Tamura et al., 2011). The robustness of the phylogenetic trees was estimated via bootstrap analysis using 1,000 replications. ML and Bayesian methods resulted in nearly identical topologies, with indications of bootstrap values (ML) and Bayesian PPs in the relevant nodes. The cyanobacterial morphologies are highlighted with different colours: yellow for unicellular strains, blue for filamentous non-heterocytous strains, green for filamentous heterocytous strains without branching, and red for filamentous heterocytous strains with true branching. Sequence data from this article can be found in the NCBI database under the accession numbers, which are presented together with the strain name.
Figure 4
Figure 4. Maximum Likelihood (ML) phylogenetic reconstruction based on partial 16S rRNA sequences. A total of 54 sequences were used. A matrix with 1,463 base pair lenght was obtained after alingment. The general time reversible evolutionary model of substitution with gamma distribution and with an estimate of proportion of invariable sites (GTR + G + I) was selected as the fittest for the alignment by MrModelTest 2.3 (Nylander, 2004). Phylogenetic trees were reconstructed using the ML and Bayesian methods. For Bayesian analysis, the trees were searched using the software MrBayes 3.2.6 (Ronquist et al., 2012) and the Bayesian analysis consisted of two independent runs, with four Markov chains each, of 50 million generations sampled every 5,000 generations. Posterior probabilities (PP) were calculated at the conclusion of the Markov-Chain-Monte-Carlo analysis and a traditional burn-in on the first 25% of the trees was performed. The ML trees were reconstructed using the MEGA program package, version 5 (Tamura et al., 2011). The robustness of the phylogenetic trees was estimated via bootstrap analysis using 1000 replications. ML and Bayesian methods resulted in nearly identical topologies, with indications of bootstrap values (ML) and Bayesian PPs in the relevant nodes. The cyanobacterial morphologies are highlighted with different colours: yellow for unicellular strains, blue for filamentous non-heterocytous strains, green for filamentous heterocytous strains without branching, and red for filamentous heterocytous strains with true branching. Sequence data from this article can be found in the NCBI database under the accession numbers, which are presented together with the strain name.
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References

    1. Akkerman I, Janssen M, Rocha J, Wijffels RH. Photobiological hydrogen production: Photochemical efficiency and bioreactor design. Int J Hydrogen Energ. 2002;27:1195–1208.
    1. Alfonso M, Perewoska I, Kirilovsky D. Redox control of ntcA gene expression in Synechocystis sp. PCC 6803. Nitrogen availability and electron transport regulate the levels of the NtcA protein. Plant Physiol. 2001;125:969–981. - PMC - PubMed
    1. Anbar AD, Knoll AH. Proterozoic Ocean Chemistry and Evolution: A Bioinorganic Bridge? Science. 2002;297:1137–1142. - PubMed
    1. Andreote APD, Vaz MGMV, Genuário DB, Barbiero L, Rezende-Filho AT, Fiore MF. Nonheterocytous cyanobacteria from Brazilian saline-alkaline lakes. J Phycol. 2014;50:675–684. - PubMed
    1. Baebprasert W, Jantaro S, Khetkorn W, Lindblad P, Incharoensakdi A. Increased H2 production in the cyanobacterium Synechocystis sp. strain PCC 6803 by redirecting the electron supply via genetic engineering of the nitrate assimilation pathway. Metab Eng. 2011;13:610–616. - PubMed

Internet Resources

    1. Rambaut A. FigTree v1. 3.1: Tree figure drawing tool. 2009. http://tree.bio.ed.ac.uk/software/figtree/
-