18
time and place. Cyanobacteria are diverse, occurring as individuals or clumps of
unicells, filaments, or filaments containing differentiated cells (Fay 1992; Gallon
1992). Cyanobacteria species generally avoid O 2 inhibition by fixing N 2 at night, or
by making differentiated, specialized cells for N 2 fixation called heterocysts
(Berman-Frank et al. 2003; Fay 1992; Stewart et al. 1969). Heterocysts are cells that
differentiate from vegetative cells in a complex genetically programmed process,
and are interspersed, usually at regular intervals, in some filamentous species. They
have a thick cell envelope that slows diffusion of O 2 into the cell, and lack O 2 -
evolving photosystem II activity (Fay 1992). Tolerance of O 2 in non-heterocystforming cyanobacteria is less well-understood (Fay 1992; Gallon and Stal 1992).
2.4 Non-structural nif Genes and Protein Assembly
The structural enzyme proteins are not the only genes or protein products required
for N 2 fixation (and not all genes involved in nitrogen fixation are nif genes)
(Fig. 2.4). The nif genes are found as large conserved clusters in many organisms
including the dominant marine N 2 -fixers (Fig. 2.4). N 2 fixation gene clusters are
primarily found on the chromosome (Fig. 2.4) although they are found on a plasmid
in rhizobia, along with nod and fix genes (Noel 2009). There are many nif genes that
encode metal transporters, encode cofactors, or are involved in protein assembly
and activation, first described in Klebsiella pneumoniae (Dixon 2004; Postgate
1998) (Table 2.3). The main cluster of N 2 fixation genes in A. vinelandii include
nifHDK and 32 other genes in 9 transcriptional units (Burén et al. 2020). One set of
genes, that includes nifH itself, is involved in FeMo-co synthesis (nifB, nifV, nifENX,
nifQ) (Kennedy and Bishop 2004). The nifEN genes have similarity to the DK
genes, and probably arose from gene duplication (Fani et al. 2000). Formation of the
FeS clusters involves the products of nifU and nifS (Kennedy and Bishop 2004).
Some of these genes work as scaffolds for assembly (nifU, nifB, nifQ and nifEN)
(Burén et al. 2020). Others transport cofactors to the final location in proteins (nifX,
nafY) or are involved in final processing or maturation of the associated metal clusters (nifM, nifH, nifW, nifZ). Some are enzymes for synthesizing components of the
proteins (nifS, nifV, nifB) (Burén et al. 2020). Note that nifH itself plays multiple
roles in assembly as well as the active enzyme (Table 2.3).
nifM and nifW are involved in maturation of the active enzyme (Kennedy and
Bishop 2004) (Table 2.3). Other nif genes encode regulatory proteins (nifLA), or
flavodoxin (nifF). In recent years genome sequencing has provided much information on the comparative genetics of nif clusters among diverse organisms (Fischer
1994; Palacios and Newton 2005). A bioinformatic analysis of genomes suggests
that there may be a minimum set of nif genes required to identify a microorganism
as a diazotroph are just six (nifHDK, EN and B) (Santos et al. 2012), however, this
has not been proven to be conclusive for identifying diazotrophs in metagenomes.
Other genes are involved in N 2 fixation in different organisms. These include nod
(nodulation) and fix (genes that did not have homologues in K. pneumoniae) genes.
2 Fundamentals of N 2 Fixation
time and place. Cyanobacteria are diverse, occurring as individuals or clumps of
unicells, filaments, or filaments containing differentiated cells (Fay 1992; Gallon
1992). Cyanobacteria species generally avoid O 2 inhibition by fixing N 2 at night, or
by making differentiated, specialized cells for N 2 fixation called heterocysts
(Berman-Frank et al. 2003; Fay 1992; Stewart et al. 1969). Heterocysts are cells that
differentiate from vegetative cells in a complex genetically programmed process,
and are interspersed, usually at regular intervals, in some filamentous species. They
have a thick cell envelope that slows diffusion of O 2 into the cell, and lack O 2 -
evolving photosystem II activity (Fay 1992). Tolerance of O 2 in non-heterocystforming cyanobacteria is less well-understood (Fay 1992; Gallon and Stal 1992).
2.4 Non-structural nif Genes and Protein Assembly
The structural enzyme proteins are not the only genes or protein products required
for N 2 fixation (and not all genes involved in nitrogen fixation are nif genes)
(Fig. 2.4). The nif genes are found as large conserved clusters in many organisms
including the dominant marine N 2 -fixers (Fig. 2.4). N 2 fixation gene clusters are
primarily found on the chromosome (Fig. 2.4) although they are found on a plasmid
in rhizobia, along with nod and fix genes (Noel 2009). There are many nif genes that
encode metal transporters, encode cofactors, or are involved in protein assembly
and activation, first described in Klebsiella pneumoniae (Dixon 2004; Postgate
1998) (Table 2.3). The main cluster of N 2 fixation genes in A. vinelandii include
nifHDK and 32 other genes in 9 transcriptional units (Burén et al. 2020). One set of
genes, that includes nifH itself, is involved in FeMo-co synthesis (nifB, nifV, nifENX,
nifQ) (Kennedy and Bishop 2004). The nifEN genes have similarity to the DK
genes, and probably arose from gene duplication (Fani et al. 2000). Formation of the
FeS clusters involves the products of nifU and nifS (Kennedy and Bishop 2004).
Some of these genes work as scaffolds for assembly (nifU, nifB, nifQ and nifEN)
(Burén et al. 2020). Others transport cofactors to the final location in proteins (nifX,
nafY) or are involved in final processing or maturation of the associated metal clusters (nifM, nifH, nifW, nifZ). Some are enzymes for synthesizing components of the
proteins (nifS, nifV, nifB) (Burén et al. 2020). Note that nifH itself plays multiple
roles in assembly as well as the active enzyme (Table 2.3).
nifM and nifW are involved in maturation of the active enzyme (Kennedy and
Bishop 2004) (Table 2.3). Other nif genes encode regulatory proteins (nifLA), or
flavodoxin (nifF). In recent years genome sequencing has provided much information on the comparative genetics of nif clusters among diverse organisms (Fischer
1994; Palacios and Newton 2005). A bioinformatic analysis of genomes suggests
that there may be a minimum set of nif genes required to identify a microorganism
as a diazotroph are just six (nifHDK, EN and B) (Santos et al. 2012), however, this
has not been proven to be conclusive for identifying diazotrophs in metagenomes.
Other genes are involved in N 2 fixation in different organisms. These include nod
(nodulation) and fix (genes that did not have homologues in K. pneumoniae) genes.
2 Fundamentals of N 2 Fixation