13
factors, for example, the V and Fe nitrogenases only being expressed if Mo
availability is low (Newton 2015).
The nitrogenase gene protein family, including the nifHDKEN, vnfHDGKEN and
anfHDGK genes are closely related to other metal-binding proteins, the protochlorophyllide and chlorophyllide reductases (Boyd et al. 2011; Mus et al. 2018;
Raymond et al. 2004) (Fig. 2.2). The phylogeny of nitrogenases differs somewhat
among the subunits (particularly nifH), and concatenated gene trees are now used to
investigate N 2 fixation evolution and diversity (Fig. 2.2a). The phylogeny of nifH
forms several clusters, annotated as Clusters 1–4 (sometimes 4 is divided into 5
(Raymond et al. 2004) (Fig. 2.2b). Cluster 1 contains nifH genes from Mo nitrogenases, with Clusters 2 containing only the anfH genes (Zehr et al. 2003). Cluster 2
and 3 nomenclature is opposite to the scheme of (Raymond et al. 2004) (see annotation in Fig.2.2b). Cluster 4 contains evolutionarily related proteins which generally
have been believed not to be active in N 2 fixation and Cluster 5 the evolutionarily
related chlorophyllide reductases (Boyd et al. 2011; Mus et al. 2018). Recently,
there has been a report of a Cluster 4 nitrogenase that is active in N 2 fixation
(McRose et al. 2017; Zhang et al. 2016). Phylogenies of the other subunits are similar with a major difference being that the V nitrogenase subunits form a separate
cluster (Boyd and Peters 2013; Raymond et al. 2004) (Fig. 2.2). The current phylogenies for determining evolutionary relationships are based on concatenated gene
trees (Boyd and Peters 2013; Mus et al. 2018) that are not sensitive to the different
evolutionary history of nifH as the nifH phylogeny is (Fig. 2.2a).
The evolutionary history of the nif genes is still the subject of some controversy.
Postgate (1998) suggested that the high degree of similarity of nif genes across taxa,
and the fact that many closely related taxa do not have nif genes, could be explained
either by early evolution of the genes with subsequent losses during vertical transmission, or by horizontal gene transfer. Some of the distribution of nif genes can be
explained by vertical transmission (Zehr et al. 2003), but there is evidence of horizontal transmission early in evolution, but after Archaea diverged from Bacteria
(Boyd and Peters 2013; Mus et al. 2018; Raymond et al. 2004). Although initially
thought that the alternative nitrogenases might have evolved first, leading to the
evolution of Mo nitrogenase, more recent analyses suggest Mo nitrogenase came
first (Garcia et al. 2020). The nif genes may have evolved from a protonitrogenase
>3 bya, with the Mo nitrogenase evolving first and the vnf and anf operons arising
from duplications. The Mo nitrogenase operon also arose from gene duplications
giving rise to nifEN (Mus et al. 2018). Symbiotic microorganisms, which have a
larger set of genes involved in symbiosis and N 2 fixation, evolved more recently and
have a more complex pattern of horizontal as well as vertical transmission
(Lindstrom et al. 2015).
Much is known about the structure and biochemistry of nitrogenase from decades
of research inspired by the potential applications in agriculture (De Bruijn 2015;
Howard and Rees 1996; Postgate 1998). Applications of genetics (cloning and
mutant analysis, genomics, molecular biology), X-ray crystallography, spectroscopy, and electronic structure calculations have provided information on the genes,
regulation, and structure of nitrogenase (Dixon and Kahn 2004; Rutledge and
2.2 Nitrogen Fixation Reaction and Nitrogenases
factors, for example, the V and Fe nitrogenases only being expressed if Mo
availability is low (Newton 2015).
The nitrogenase gene protein family, including the nifHDKEN, vnfHDGKEN and
anfHDGK genes are closely related to other metal-binding proteins, the protochlorophyllide and chlorophyllide reductases (Boyd et al. 2011; Mus et al. 2018;
Raymond et al. 2004) (Fig. 2.2). The phylogeny of nitrogenases differs somewhat
among the subunits (particularly nifH), and concatenated gene trees are now used to
investigate N 2 fixation evolution and diversity (Fig. 2.2a). The phylogeny of nifH
forms several clusters, annotated as Clusters 1–4 (sometimes 4 is divided into 5
(Raymond et al. 2004) (Fig. 2.2b). Cluster 1 contains nifH genes from Mo nitrogenases, with Clusters 2 containing only the anfH genes (Zehr et al. 2003). Cluster 2
and 3 nomenclature is opposite to the scheme of (Raymond et al. 2004) (see annotation in Fig.2.2b). Cluster 4 contains evolutionarily related proteins which generally
have been believed not to be active in N 2 fixation and Cluster 5 the evolutionarily
related chlorophyllide reductases (Boyd et al. 2011; Mus et al. 2018). Recently,
there has been a report of a Cluster 4 nitrogenase that is active in N 2 fixation
(McRose et al. 2017; Zhang et al. 2016). Phylogenies of the other subunits are similar with a major difference being that the V nitrogenase subunits form a separate
cluster (Boyd and Peters 2013; Raymond et al. 2004) (Fig. 2.2). The current phylogenies for determining evolutionary relationships are based on concatenated gene
trees (Boyd and Peters 2013; Mus et al. 2018) that are not sensitive to the different
evolutionary history of nifH as the nifH phylogeny is (Fig. 2.2a).
The evolutionary history of the nif genes is still the subject of some controversy.
Postgate (1998) suggested that the high degree of similarity of nif genes across taxa,
and the fact that many closely related taxa do not have nif genes, could be explained
either by early evolution of the genes with subsequent losses during vertical transmission, or by horizontal gene transfer. Some of the distribution of nif genes can be
explained by vertical transmission (Zehr et al. 2003), but there is evidence of horizontal transmission early in evolution, but after Archaea diverged from Bacteria
(Boyd and Peters 2013; Mus et al. 2018; Raymond et al. 2004). Although initially
thought that the alternative nitrogenases might have evolved first, leading to the
evolution of Mo nitrogenase, more recent analyses suggest Mo nitrogenase came
first (Garcia et al. 2020). The nif genes may have evolved from a protonitrogenase
>3 bya, with the Mo nitrogenase evolving first and the vnf and anf operons arising
from duplications. The Mo nitrogenase operon also arose from gene duplications
giving rise to nifEN (Mus et al. 2018). Symbiotic microorganisms, which have a
larger set of genes involved in symbiosis and N 2 fixation, evolved more recently and
have a more complex pattern of horizontal as well as vertical transmission
(Lindstrom et al. 2015).
Much is known about the structure and biochemistry of nitrogenase from decades
of research inspired by the potential applications in agriculture (De Bruijn 2015;
Howard and Rees 1996; Postgate 1998). Applications of genetics (cloning and
mutant analysis, genomics, molecular biology), X-ray crystallography, spectroscopy, and electronic structure calculations have provided information on the genes,
regulation, and structure of nitrogenase (Dixon and Kahn 2004; Rutledge and
2.2 Nitrogen Fixation Reaction and Nitrogenases