Molecular Mechanism and Agricultural Application of the NifA–NifL System for Nitrogen Fixation

Abstract

Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, converting inert dinitrogen (N2) in the atmosphere into bioavailable nitrogen. Elaborating the molecular mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural production can alleviate the “nitrogen problem”. Azotobacter vinelandii is a well–established model bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript, we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture, providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation.

Full text

Citation: Zhang, W.; Chen, Y.; Huang,
K.; Wang, F.; Mei, Z. Molecular
Mechanism and Agricultural
Application of the NifA–NifL System
for Nitrogen Fixation. Int. J. Mol. Sci.
2023,24, 907. https://doi.org/
10.3390/ijms24020907
Academic Editor: Lars Matthias Voll
Received: 16 November 2022
Revised: 27 December 2022
Accepted: 30 December 2022
Published: 4 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
International Journal of
Molecular Sciences
Review
Molecular Mechanism and Agricultural Application of the
NifA–NifL System for Nitrogen Fixation
Wenyao Zhang 1, Yihang Chen 1, Keyang Huang 1,2, Feng Wang 1,* and Ziqing Mei 3, *
1
Key Laboratory of Molecular Medicine and Biotherapy, School of Life Science, Beijing Institute of Technology,
Beijing 100081, China
2College of Life Sciences, Yan’an University, Yan’an 716000, China
3School of Chemistry and Biological Engineering, University of Science and Technology Beijing,
Beijing 100083, China
*Correspondence: wfeng@bit.edu.cn (F.W.); marina1977@163.com (Z.M.)
Abstract:
Nitrogen–fixing bacteria execute biological nitrogen fixation through nitrogenase, convert-
ing inert dinitrogen (N
2
) in the atmosphere into bioavailable nitrogen. Elaborating the molecular
mechanisms of orderly and efficient biological nitrogen fixation and applying them to agricultural
production can alleviate the “nitrogen problem”. Azotobacter vinelandii is a well–established model
bacterium for studying nitrogen fixation, utilizing nitrogenase encoded by the nif gene cluster to fix
nitrogen. In Azotobacter vinelandii, the NifA–NifL system fine–tunes the nif gene cluster transcription
by sensing the redox signals and energy status, then modulating nitrogen fixation. In this manuscript,
we investigate the transcriptional regulation mechanism of the nif gene in autogenous nitrogen–fixing
bacteria. We discuss how autogenous nitrogen fixation can better be integrated into agriculture,
providing preliminary comprehensive data for the study of autogenous nitrogen–fixing regulation.
Keywords:
biological nitrogen fixation; nitrogenase; NifA–NifL system; biological nitrogen fertilizer;
agricultural application
1. Introduction
Nitrogen is the core component of biological molecules, such as proteins and nucleic
acids. Nitrogen makes up as much as 78% of the atmosphere, but it is not utilized directly
by most creatures and can be effectively absorbed only when converted into ammonia
or ammonium salts. An increase in available nitrogen content can significantly improve
the yield of crops in poor soils [
1
]. At present, the best way to increase the available
nitrogen content of crops is by applying chemical nitrogen fertilizer. However, there
are two major disadvantages: First, the utilization rate of nitrogen fertilizer is extremely
low, and excessive application of nitrogen fertilizer leads to water eutrophication and
causes an imbalance in the species distribution in the water ecosystem, thus resulting in
the gradual extinction of the whole water ecosystem. Second, for many smallholders in
some developing countries, such as Sub–Saharan Africa, the scarce availability and high
cost of nitrogen fertilizer make it unusable; therefore, these smallholders suffer from low
yields
[2–5]
. Finding clean alternatives to nitrogen fertilizers is essential for sustainable and
safe agricultural development.
The vast majority of nitrogen fixation is executed by nitrogen–fixing microorganisms [
6
].
Biological nitrogen fixation is the process in which nitrogen–fixing microorganisms use ni-
trogenase to directly reduce atmospheric nitrogen to ammonia [
7
]. This process introducing
nitrogen–fixing bacteria or nitrogen–fixing enzymes to the crop provides an opportunity to
increase the available nitrogen content of the crop and improve crop nutrition.
Biofertilizers are cheaper, require less capital to use, and are thus increasingly im-
portant to agriculture. Biofertilizers, also known as biological inoculants, are organic
preparations containing microorganisms [
8
]. When used as a seed treatment or when
Int. J. Mol. Sci. 2023,24, 907. https://doi.org/10.3390/ijms24020907 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023,24, 907 2 of 18
seedling roots are immersed in seeds or soil fertilization, they proliferate rapidly and
form dense populations in the rhizosphere that rapidly fix nitrogen, increasing the avail-
able nitrogen content of the crop. Beneficial biofertilizers for crop production contain
nitrogen–fixing bacteria, azospirals, cyanobacteria, green algae, etc. [
9
–
11
]. However, the
application of most nitrogen–fixing microorganisms as biological nitrogen fertilizers in
agricultural production faces various obstacles, such as harsh growth conditions and low
nitrogen–fixation efficiency. Overcoming these obstacles with biotechnology will facilitate
the wide application of biological nitrogen fertilizers in agricultural production, increasing
the available nitrogen content of the crop for large agricultural returns.
According to the characteristics of nitrogen fixation, nitrogen–fixation microorganisms
can be divided into three types: autogenous nitrogen–fixation bacteria, symbiotic nitrogen–
fixation bacteria, and combined nitrogen–fixation bacteria [
12
]. Autogenous nitrogen–fixing
bacteria are free–living bacteria that can fix nitrogen. Azotobacter vinelandii (A. vinelandii),
with its high expression and nitrogenase activity, can perform efficient nitrogen fixation
under aerobic conditions and is thus becoming a model bacterium for the study of au-
togenous nitrogen–fixing bacteria [
13
]. A. vinelandii contains three types of nitrogenases,
all of which are multi–subunit protein complexes that bind metal ions, namely, ferric–
molybdenum (Fe–Mo) nitrogenase, ferric–vanadium (Fe–V) nitrogenase, and ferric–ferric
(Fe–Fe) nitrogenase [
14
]. The nitrogen–fixing reactions are mainly catalyzed by Fe–Mo
nitrogenase, whose expression is regulated by a NifA–NifL system. In this manuscript,
we summarized the molecular mechanisms of the regulation of the Fe–Mo nitrogenase
expression via NifA–NifL and their application in agricultural development, supplying
preliminary comprehensive data for the study of autogenous nitrogen–fixation regulation.
2. Nitrogenase and Its Transcriptional Regulation
2.1. Nitrogenase
Nitrogenase is a complex oxygen–sensitive metalloenzyme with three isoforms: Fe–
Mo nitrogenase (encoded by nif gene cluster), Fe–V nitrogenase (encoded by vnf gene
cluster), and Fe–Fe nitrogenase (encoded by anf gene cluster) (Figure 1a). All known
diazotrophs contain at least one of the three closely related nitrogenase isoforms. Although
they have different metal contents, these nitrogenase isoforms are related to each other in
terms of their structure, mechanism of action, and phylogeny [15].
The alignment of protein sequences showed that the three nitrogenases share a similar
basic structure. Among them, Mo–Fe nitrogenase plays a dominant role in nitrogen fixation,
while Fe–V nitrogenase and Fe–Fe nitrogenase act as alternative nitrogenases. Alternative
nitrogenases are activated only if the molybdenum in the environment is insufficient. Of
these, the most widely studied subtype is Mo–Fe nitrogenase, which exhibits the highest
nitrogen–fixation efficiency. The catalytic center of Mo–Fe nitrogenase, which is composed
of a Mo–Fe protein (named dinitrogenase) and Fe protein (named nitrogenase reductase),
is encoded by nif H, nif D, and nif K. The Fe protein is a
γ
2–type homodimer encoded
by the nif H gene, and the Mo–Fe protein is an
α
2
β
2–type heterotetramer in which the
nif K gene encodes an
α
subunit and the nif D gene encodes a
β
subunit. Both ends of
the Mo–Fe protein can bind the Fe protein. Recent studies have reported that only the
single–headed complex assembled by one molecule of a Mo–Fe protein and one molecule
of an Fe protein is required for nitrogen fixation, and the double–headed complex exists
in a trapped state [
16
]. Mo–Fe nitrogenase has a unique Mo–Fe cofactor located at the
active site of Mo–Fe protein, which participates in the nitrogen–fixing reaction [
15
]. During
nitrogen fixation, Fe protein couples and hydrolyzes two ATP molecules, while one electron
is transferred to the 8Fe–7S cluster in the Fe protein by the 4Fe–4S cluster. Afterward, the
electrons are transferred to the Mo–Fe cofactor to participate in the reduction of the N
2
molecule. N
2
forms a stable intramolecular triple bond so the reduction of N
2
to NH
3
by
nitrogenase is an extremely difficult reaction that requires overcoming the activation energy
barrier of catalysis (Figure 1b) [
17
]. High–energy electrons stored in the Mo–Fe cofactor
can be used as reducing agents to overcome the activation energy barrier of catalysis in

Int. J. Mol. Sci. 2023,24, 907 3 of 18
the presence of large amounts of ATP and protons [
16
]. This catalytic mechanism is also
applicable to V–Fe nitrogenase and Fe–Fe nitrogenase, but they need more high–energy
electrons, ATP, and protons to overcome the energy barrier. Mo–Fe nitrogenase consumes
at least 16 ATP molecules to immobilize a molecule of N
2
, whereas V–Fe nitrogenase
and Fe–Fe nitrogenase consume 24 to 32 ATP molecules (Figure 1c) [
18
]. The conversion
efficiency of nitrogenase is extra low due to the complexity of the reaction catalyzed by
nitrogenase. In order to satisfy the growth requirements, nitrogen–fixing bacteria express
a large amount of nitrogenase, which generally accounts for 10–20% of the total protein
content [13].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 3 of 19
Fe cofactor located at the active site of Mo–Fe protein, which participates in the nitrogen–
fixing reaction [15]. During nitrogen fixation, Fe protein couples and hydrolyzes two
ATP molecules, while one electron is transferred to the 8Fe–7S cluster in the Fe protein by
the 4Fe–4S cluster. Afterward, the electrons are transferred to the Mo–Fe cofactor to par-
ticipate in the reduction of the N
2
molecule. N
2
forms a stable intramolecular triple bond
so the reduction of N
2
to NH
3
by nitrogenase is an extremely difficult reaction that re-
quires overcoming the activation energy barrier of catalysis (Figure 1b) [17]. High–energy
electrons stored in the Mo–Fe cofactor can be used as reducing agents to overcome the
activation energy barrier of catalysis in the presence of large amounts of ATP and protons
[16]. This catalytic mechanism is also applicable to V–Fe nitrogenase and Fe–Fe nitro-
genase, but they need more high–energy electrons, ATP, and protons to overcome the
energy barrier. Mo–Fe nitrogenase consumes at least 16 ATP molecules to immobilize a
molecule of N
2
, whereas V–Fe nitrogenase and Fe–Fe nitrogenase consume 24 to 32 ATP
molecules (Figure 1c) [18]. The conversion efficiency of nitrogenase is extra low due to
the complexity of the reaction catalyzed by nitrogenase. In order to satisfy the growth
requirements, nitrogen–fixing bacteria express a large amount of nitrogenase, which
generally accounts for 10–20% of the total protein content [13].
Figure 1. Genes encoding the three forms of nitrogenases and their regulatory proteins are re-
quired for nitrogen fixation. (a) The organizations of nif, anf, and vnf clusters encoding the three
forms of nitrogenases in A. vinelandii. Predicted σ54–dependent promoter regions are depicted by
arrows. Shown are genes encoding the regulatory proteins with known or predicted functions
(dark brown), the components involved in the catalytic reduction of N
2
(nifD in blue; nifK in aqua;
nifH in wine red; nifG in yellow), the assembly or stability of nitrogenase (orange), the maturation
of nitrogenase (olive), the maturation of Fe–M cofactor (purple), electron transfer (light red), the
biosynthesis of Fe–M cofactor (light aqua), and an unknown function (gray). (b) A diagram of the
three forms of nitrogenases involved in electron transfer. M is Mo, Fe, or V. The α–subunit is en-
coded by nifK; β–subunit is encoded by nifK; γ–subunit is encoded by nifH; δ–subunit is encoded
by nifG. (c) The nitrogen–fixation reaction catalyzed by the three forms of nitrogenases.
Figure 1.
Genes encoding the three forms of nitrogenases and their regulatory proteins are required
for nitrogen fixation. (
a
) The organizations of nif,anf, and vnf clusters encoding the three forms of
nitrogenases in A. vinelandii. Predicted
σ
54–dependent promoter regions are depicted by arrows.
Shown are genes encoding the regulatory proteins with known or predicted functions (dark brown),
the components involved in the catalytic reduction of N
2
(nif D in blue; nif K in aqua; nif H in wine
red; nif G in yellow), the assembly or stability of nitrogenase (orange), the maturation of nitrogenase
(olive), the maturation of Fe–M cofactor (purple), electron transfer (light red), the biosynthesis of
Fe–M cofactor (light aqua), and an unknown function (gray). (
b
) A diagram of the three forms of
nitrogenases involved in electron transfer. M is Mo, Fe, or V. The
α
–subunit is encoded by nif K;
β
–subunit is encoded by nif K;
γ
–subunit is encoded by nif H;
δ
–subunit is encoded by nif G. (
c
) The
nitrogen–fixation reaction catalyzed by the three forms of nitrogenases.

Int. J. Mol. Sci. 2023,24, 907 4 of 18
V–Fenitrogenase and Fe–Fe nitrogenase are encoded by vnf HDGK genes and anf HKDG
genes, respectively; whereas the vnf H (anf H) gene encodes nitrogen–fixing reductase; and
vnf KDG (anf KDG) genes account for the synthesis of the
α
subunit,
β
subunit, and
δ
subunit to form the
α
2
β
2
δ
2 hexamer (Figure 1b). Although the specific function of the
δ
subunit encoded by vnf G (anf G) remains unclear, it is essential for maintaining nitrogenase
activity. The nitrogen–fixing mechanism of V–Fe nitrogenase and Fe–Fe nitrogenase is
similar to that of Mo–Fe nitrogenase. In regard to the development and evolution of nitro-
genase, some views have proposed that V–Fe nitrogenase and Fe–Fe nitrogenase might
have evolved from Mo–Fe nitrogenase [19].
2.2. Transcriptional Regulation of Nitrogenase
Nitrogenase is extremely sensitive to oxygen, and its catalytic reduction of nitrogen
molecules to ammonia must be carried out in a strictly anaerobic microenvironment. As
result, the expression of nitrogenase requires a specific microenvironment, where strict
anaerobic activity is necessary to maintain the activity of nitrogenase. Additionally, the cell
should be in the peak metabolic stage with a large amount of ATP and sufficient nitrogen
to ensure the precise modulation of nitrogenase expression by nitrogen–fixing bacteria. The
biosynthesis of active nitrogenases relies on a variety of Nif proteins encoded by nif genes
beyond the structural subunits of the catalytic center, including the molecular scaffold
protein gene, the metal cluster carrier protein gene, and the metal cofactor biosynthesis
gene. It was reported that at least nine nif genes are required for the synthesis of bioactive
Mo–Fe nitrogenases: nif H, nif E, nif N, nif S, nif U, nif V, nif Y, nif B and nif Q, which performs
functions including redox provisioning and electron transport [20].
The transcriptional regulation of nitrogenase might differ between nitrogen–fixing
bacteria. For instance, A. vinelandii displays an expression and transcriptional regulation
system of Mo–Fe nitrogenase, encoded by the nif cluster, which includes a major nif
cluster and a minor nif cluster. The major nif cluster contains five major gene clusters
(nif HDKTY, nif ENX, orf5, iscA
nif
nif USV–cysE1nif–nif WZM–clpX2, and nif F) encoding the
nitrogenase complex. The nif HDKTY gene cluster contains the structural genes of Mo–Fe
nitrogenase, which include nif H, nif D, nif K, nif T, and nif Y. Moreover, the nif LA operon,
located at the minor nif cluster (rnf ABCDGEH, nif LAB, fdxN, nif OQ, rhdN, and grx5
nif
),
encodes the NifA protein and NifL protein that regulate and control the transcription of
nitrogenase (Figure 1a) [
21
]. Since the first discovery of the function of the nifLA operon,
the research on the NifL–NifA binary regulatory system has achieved significant progress:
the function of nitrogen fixation is regulated by the nif LA operon in A. vinelandii,nif A
distal, and nif L proximal to the promoter (Figure 1a) [
8
]. Moreover, the expression of the
nif LA gene is rarely affected by environmental factors, and it is continuously expressed
or has little change in various growth stages [
21
,
22
]. NifA is the transcriptional activator
protein of the nif gene cluster, while NifL inhibits the activity of NifA via interacting
with NifA. The NifL–NifA system is affected by the intracellular redox environment and
binding state of the ligand (2–OG, ATP/ADP, FAD). The reversible modification of GlnK
by uridylation also has a regulatory effect on the transcription of the NifL–NifA system.
When there is excess nitrogen, nitrogen metabolism results in low concentrations of 2–OG
and high concentrations of glutamine. Glnk, NifA, and NifL can form the ternary complex
to suppress the activity of NifA, which results in the blockage of nitrogenase expression.
When nitrogen is limited, the floundering nitrogen metabolism results in the accumulation
of 2–OG and the excessive consumption of glutamine, which disrupts the formation of
the ternary complex to activate NifA. The active NifA can promote the expression of
nitrogenase (Figure 2).

Int. J. Mol. Sci. 2023,24, 907 5 of 18
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 5 of 19
Figure 2. The domain structures and functions of NifA and NifL. (a) Schemes of the domain
structures of the NifA and NifL protein; (b) The distinct regulatory modes of NifA–NifL–GlnK
complex in the excess or limitation of nitrogen. In nitrogen excess, GHKL domain of NifL protein
interacts with NifA protein to inhibit NifA. Glnk can interact with NifL, enhancing the inhibition
of NifA activity by NifL. When nitrogen is limited, the uridylylation of Glnk disrupts the interac-
tion between Glnk and NifL. 2–OG can bind to GAF domain of NifA to remove the inhibition of
NifA activity by NifL.
3. The Function and Structure of NifA
NifA is a bacteria enhancer binding protein (bEBP) containing AAA+ domain pro-
teins, which can use the energy released by the hydrolysis of ATP to catalyze the con-
formational change of the σ54 factor, followed by transition of the σ54–RNAP holoen-
zyme from the inactive state to the active state, thus activating the transcription of the nif
gene cluster [23]. Based on their biological function and regulatory patterns, bEBPs are
classified into five categories. NifA is grouped in the third category, which contains the
N–terminal GAF domain, central AAA+ domain, and C–terminal HTH domain (Figure
2a). The GAF domain is a regulatory domain widely found in many proteins, which can
sense and transmit a variety of metabolic signaling molecules, such as cAMP, cGMP,
glutamic acid, α–ketoglutaric acid (2–OG), and porphyrin ring [24]. The binding of 2–
OG to the GAF domain activates NifA in an unknown manner and initiates the tran-
scription of the nif gene cluster in A. vinelandii. The AAA+ domain, a representative
structural element of members of the AAA+ superfamily, can convert chemical energy
into mechanical energy by binding and hydrolyzing ATP. Different from classic AAA+
clade 3 subfamily proteins, the AAA+ domain of NifA possesses two loop structures
named L1 (GAFTGA motif) and L2 [25], which can interact with σ54 factor and recon-
struct its conformation via hydrolyzing ATP [26]. The C–terminus of NifA is a helix–
turn–helix motif of the DNA binding domain, which binds to upstream activator se-
quences (UAS) of the nif gene cluster promoter. Upon bending and cyclizing the DNA
strand of the nif gene cluster, NifA bound to the UAS sequence can approach and inter-
Figure 2.
The domain structures and functions of NifA and NifL. (
a
) Schemes of the domain structures
of the NifA and NifL protein; (
b
) The distinct regulatory modes of NifA–NifL–GlnK complex in the
excess or limitation of nitrogen. In nitrogen excess, GHKL domain of NifL protein interacts with
NifA protein to inhibit NifA. Glnk can interact with NifL, enhancing the inhibition of NifA activity
by NifL. When nitrogen is limited, the uridylylation of Glnk disrupts the interaction between Glnk
and NifL. 2–OG can bind to GAF domain of NifA to remove the inhibition of NifA activity by NifL.
3. The Function and Structure of NifA
NifA is a bacteria enhancer binding protein (bEBP) containing AAA+ domain proteins,
which can use the energy released by the hydrolysis of ATP to catalyze the conformational
change of the
σ
54 factor, followed by transition of the
σ
54–RNAP holoenzyme from the
inactive state to the active state, thus activating the transcription of the nif gene cluster [
23
].
Based on their biological function and regulatory patterns, bEBPs are classified into five
categories. NifA is grouped in the third category, which contains the N–terminal GAF
domain, central AAA+ domain, and C–terminal HTH domain (Figure 2a). The GAF
domain is a regulatory domain widely found in many proteins, which can sense and
transmit a variety of metabolic signaling molecules, such as cAMP, cGMP, glutamic acid,
α
–ketoglutaric acid (2–OG), and porphyrin ring [
24
]. The binding of 2–OG to the GAF
domain activates NifA in an unknown manner and initiates the transcription of the nif
gene cluster in A.vinelandii. The AAA+ domain, a representative structural element of
members of the AAA+ superfamily, can convert chemical energy into mechanical energy
by binding and hydrolyzing ATP. Different from classic AAA+ clade 3 subfamily proteins,
the AAA+ domain of NifA possesses two loop structures named L1 (GAFTGA motif) and
L2 [
25
], which can interact with
σ
54 factor and reconstruct its conformation via hydrolyzing
ATP [
26
]. The C–terminus of NifA is a helix–turn–helix motif of the DNA binding domain,
which binds to upstream activator sequences (UAS) of the nif gene cluster promoter. Upon
bending and cyclizing the DNA strand of the nif gene cluster, NifA bound to the UAS
sequence can approach and interact with the
σ
54–RNAP holoenzyme in the promoter
region, inducing the deformation of
σ
54 and activating the transcriptional activity of the
RNAP–σ54 complex (Figure 3).

Int. J. Mol. Sci. 2023,24, 907 6 of 18
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 19
act with the σ54–RNAP holoenzyme in the promoter region, inducing the deformation
of σ54 and activating the transcriptional activity of the RNAP–σ54 complex (Figure 3).
Despite its key role in the transcriptional activation of the nif gene cluster, a three–
dimensional structure of NifA has not been reported. Fortunately, the partial structure
of its homologous protein has been resolved, which improves the elaboration of the
mechanism of transcriptional activation of NifA at large. NifA–like homolog 1 (Nlh1) is
speculated to function as a σ54 activator in modulating the transcriptional activity of
genes involved in nitrogen assimilation instead of nitrogen fixation [27]. The crystal dif-
fraction and SAXS data of GAF domain revealed that the GAF domain forms a “back–
to–back” dimer, and the dimeric coiled–coil signaling helix that connects the GAF do-
main with the ATPase domains repressed the assembly of the AAA+ domain by holding
them in an inactive face–to–face orientation. The attachment of an unknown ligand to
the ligand binding pocket of the GAF domain can activate Nlh1 by disrupting the di-
meric coiled–coil signaling helix to release the ATPase domains for assembly [27,28].
NifA–like homolog 2 (Nlh2) is reported to act as an NtrC2 homolog in Aquifex aeolicus,
which contains an N–terminal GAF domain rather than the receiver domain, similar to
that of the NifA homolog. Like Nlh1, Nlh2 is also able to stimulate σ54–mediated tran-
scriptional initiation. Studies have shown that the active activator forms a ring oligomer
to facilitate the activation of the σ54–RNAP holoenzyme, such as a hexamer and hep-
tamer. The phage shock protein F (Pspf) protein, a bEBP, is the only protein reported to
form a complex structure with the σ54–RNAP holoenzyme. Recently, the Cryo–EM
structure of the complex between the active PspF (hexamer) and σ54–RNAP holoen-
zyme was also determined, but the ATP binding site, the interaction sites between PspF
and σ54, as well as the interface residues involving a polymer formation, require eluci-
dation [23]. The AAA+ domain of the PspF protein from Escherichia coli (E. coli) exhibits a
48% similar sequence identity to that of NifA, suggesting that NifA is most likely in-
volved in activating the σ54–RNAP holoenzyme in the form of a hexamer ring.
Figure 3. The transcriptional initiation process mediated by nif cluster promoted by NifA. (a) NifA
activation. Active NifA needs to be reconstituted into an oligomeric state (predicted as hexamer).
Figure 3.
The transcriptional initiation process mediated by nif cluster promoted by NifA. (
a
) NifA
activation. Active NifA needs to be reconstituted into an oligomeric state (predicted as hexamer).
The pink ball represents NifA, and green ball represents NifL. The Glnk is represented by brown
ball. (
b
) DNA binding. Oligomeric NifA can bind to the UAS region; (
c
) The formation of the
closed complex. IHF can bend DNA to promote the interaction of oligomeric NifA with RNAP–
σ
54;
(
d
) Open complex formation. Oligomeric NifA provides energy for DNA opening promoted by
RNAP–σ54.
Despite its key role in the transcriptional activation of the nif gene cluster, a three–
dimensional structure of NifA has not been reported. Fortunately, the partial structure of its
homologous protein has been resolved, which improves the elaboration of the mechanism
of transcriptional activation of NifA at large. NifA–like homolog 1 (Nlh1) is speculated to
function as a
σ
54 activator in modulating the transcriptional activity of genes involved in
nitrogen assimilation instead of nitrogen fixation [
27
]. The crystal diffraction and SAXS
data of GAF domain revealed that the GAF domain forms a “back–to–back” dimer, and the
dimeric coiled–coil signaling helix that connects the GAF domain with the ATPase domains
repressed the assembly of the AAA+ domain by holding them in an inactive face–to–face
orientation. The attachment of an unknown ligand to the ligand binding pocket of the GAF
domain can activate Nlh1 by disrupting the dimeric coiled–coil signaling helix to release
the ATPase domains for assembly [
27
,
28
]. NifA–like homolog 2 (Nlh2) is reported to act as
an NtrC2 homolog in Aquifex aeolicus, which contains an N–terminal GAF domain rather
than the receiver domain, similar to that of the NifA homolog. Like Nlh1, Nlh2 is also able
to stimulate
σ
54–mediated transcriptional initiation. Studies have shown that the active
activator forms a ring oligomer to facilitate the activation of the
σ
54–RNAP holoenzyme,
such as a hexamer and heptamer. The phage shock protein F (Pspf) protein, a bEBP, is
the only protein reported to form a complex structure with the
σ
54–RNAP holoenzyme.
Recently, the Cryo–EM structure of the complex between the active PspF (hexamer) and
σ
54–RNAP holoenzyme was also determined, but the ATP binding site, the interaction sites
between PspF and
σ
54, as well as the interface residues involving a polymer formation,
require elucidation [
23
]. The AAA+ domain of the PspF protein from Escherichia coli (E.coli)

Int. J. Mol. Sci. 2023,24, 907 7 of 18
exhibits a 48% similar sequence identity to that of NifA, suggesting that NifA is most likely
involved in activating the σ54–RNAP holoenzyme in the form of a hexamer ring.
4. Structural Characteristics and Functions of the NifL Protein
As an anti–activator, NifL suppresses the transcription of nitrogenase gene activated
by its partner protein NifA in A. vinelandii [
29
]. It is noteworthy that a specific molar
ratio between NifL and NifA is required for this function, indicating that NifL directly
interacts with NifA rather than modifying it. The complex of NifA and NifL has been
found in a variety of microorganisms, including A. vinelandii and Klebsiella pneumoniae
(K. pneumoniae) [
30
,
31
]. NifL suppresses the transcriptional activation of the nitrogenase
gene via prohibiting the interaction of NifA with DNA and the complex formation of
RNAP–σ54–DNA with NifA [31].
The NifL protein consists of four domains: the N–terminus contains two PAS (Per–
ARNT–Sim) domains, named PAS1 and PAS2 [
32
] (shown in Figure 2a). The central region
consists of a glutamine–rich superhelical structure containing a highly conserved histidine
residue, referred as the H–domain; the C–terminal domain contains an ATP/ADP binding
site composed of four highly conserved motifs (N, G1, F, and G2 motif), which is called the
GHKL domain. Of these, the PAS domain is homologous to the GAF domain and can sense
various environmental signals (such as oxygen, redox signaling, and light) via its
α
/
β
fold in response to different cofactors [
33
,
34
]. Both PAS1 and PAS2 are necessary for redox
signal transduction corresponding to oxygen. The PAS1 domain of NifL binds to the flavin
adenine di–nucleotide (FAD) cofactor, while the PAS2 domain can receive the redox signals
from PAS1 and transmit them to the C–terminal H and GHKL domain [
35
]. The PAS2
domain is also identified in the NifL sequences of other aerobic diazotrophs [
36
]. The GHKL
domain and H of NifL and the catalytic domain of histidine protein kinase (H ATPase–C)
are homologous to the ATP binding domain of ATPases from the GHKL superfamily, but
they are only capable of binding ATP/ADP rather than hydrolyzing ATP [33].
Until now, the detailed inhibition mechanism of NifA activity by NifL has remained
elusive, but several studies have provided a basis for elucidating this mechanism.
In vitro
biochemical data proved that deletion of the GAF domain has no significant impact on
the transcriptional activation of the nitrogenase gene. However, the deleted mutant of
NifA required higher concentrations of NifL to inhibit its activity compared with the wild
type [
37
], implying that the GAF domain of NifA is essential for the formation of the
NifL–NifA complex. The activity of the NifA–E356K mutant, a single residue mutant
in the AAA domain, is constitutive and insensitive to NifL, consistent with the fact that
the NifA–E356K mutant can show higher activity than the wild type under anaerobic
nitrogen–limiting conditions [
38
]. Another mutant, NifA–Y254N, can show resistance to
NifL under anaerobic nitrogen–excessive conditions, but NifL is sensitive to the mutant
under aerobic growth conditions [
39
,
40
]. These suggest that the AAA domain of NifA is
also involved in the formation of the NifL–NifA complex. Thus far, no data have reported
that the HTH domain of NifA is related to the formation of the NifL–NifA complex.
The surfaces of NifL that are required for the interaction with NifA are not well
defined, but some experiments have established the fact that the region located between
residues 287 and 360 corresponding to the H–domain might provide the main surface of the
NifL–NifA complex.
In vitro
mutant assays proved that the NifL mutant with N–terminal
PAS1 domain deletion (147~519 residues) can still form the NifL–NifA complex but not
sense the redox signal [
31
]. This suggests that the PAS1 domain is not essential for the
interaction between NifL and NifA. However, biochemical data regarding the mutant
with the deletion of the H domain and GHKL domain showed that both of them were
directly involved in the interaction with NifA [
31
]. The mutant with the replacement of
arginine by cysteine at position 306 (NifL–R306C) can constitutively inhibit NifA, which
further supports the conclusion that the H–domain provides the surface of the NifL–NifA
complex [
38
]. There is no direct information showing that the PAS2 domain is involved
in the interaction with NifA. Some protein structures with the PAS domain show that the

Int. J. Mol. Sci. 2023,24, 907 8 of 18
conformational changes caused by signal sensation initiate the relay of the signal to effector
domains. Oxidation of the FAD moiety results in a quaternary structure change in PAS1,
resulting in a movement of the PAS2 protomers that is proposed to trigger rearrangement
of the H and GHKL domains, promoting access to NifA and the formation of a NifL–NifA
complex that inhibits its activity [33,35].
5. The NifL–NifA System Responds to the Transcriptional Regulation of Nitrogenase
via Environmental Signaling Molecules
The NifL–NifA system of A. vinelandii integrates the intracellular redox and nitrogen
and carbon status to regulate the expression of nitrogenase. The interaction between
NifL and NifA is regulated in response to the intracellular redox environmental, ligand
(2–OG, ATP/ADP, FAD/FADH
2
) binding status, and the signal–transduction protein GlnK.
Under an adverse redox state (excess oxygen) or nitrogen–excess condition, oxidized NifL
and NifA form binary complexes to suppress NifA activity. In addition, non–covalently
modified GlnK can also interact with NifL to promote the formation of a GlnK–NifL–NifA
ternary complex and inhibit NifA activity (Figure 4a). Relatively, in nitrogen–limiting
conditions, 2–OG at a high concentration binds to the GAF domain and leads to uridylation
of Glnk by GlnD. This can ensure NifA dissociation from the NifL–NifA complex so that
free NifA can activate the transcription of the nitrogenase gene (Figure 4b) [29].
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 19
were directly involved in the interaction with NifA [31]. The mutant with the replace-
ment of arginine by cysteine at position 306 (NifL–R306C) can constitutively inhibit Ni-
fA, which further supports the conclusion that the H–domain provides the surface of the
NifL–NifA complex [38]. There is no direct information showing that the PAS2 domain
is involved in the interaction with NifA. Some protein structures with the PAS domain
show that the conformational changes caused by signal sensation initiate the relay of the
signal to effector domains. Oxidation of the FAD moiety results in a quaternary struc-
ture change in PAS1, resulting in a movement of the PAS2 protomers that is proposed to
trigger rearrangement of the H and GHKL domains, promoting access to NifA and the
formation of a NifL–NifA complex that inhibits its activity [33,35].
5. The NifL–NifA System Responds to the Transcriptional Regulation of Nitrogenase
via Environmental Signaling Molecules
The NifL–NifA system of A. vinelandii integrates the intracellular redox and nitro-
gen and carbon status to regulate the expression of nitrogenase. The interaction between
NifL and NifA is regulated in response to the intracellular redox environmental, ligand
(2–OG, ATP/ADP, FAD/FADH
2
) binding status, and the signal–transduction protein
GlnK. Under an adverse redox state (excess oxygen) or nitrogen–excess condition, oxi-
dized NifL and NifA form binary complexes to suppress NifA activity. In addition, non–
covalently modified GlnK can also interact with NifL to promote the formation of a
GlnK–NifL–NifA ternary complex and inhibit NifA activity (Figure 4a). Relatively, in
nitrogen–limiting conditions, 2–OG at a high concentration binds to the GAF domain
and leads to uridylation of Glnk by GlnD. This can ensure NifA dissociation from the
NifL–NifA complex so that free NifA can activate the transcription of the nitrogenase
gene (Figure 4b) [29].
Figure 4. The NifL–NifA system’s responses to environmental and metabolic conditions in A.
vinelandii. (a) In nitrogen–excess conditions, the binding of high concentration of glutamine to
GlnD results in uridylyl removal from Glnk. NifL forms the binary complexes with NifA to inhibit
NifA activation. In addition, the non–covalently modified GlnK can also interact with NifL to
promote the formation of the GlnK–NifL–NifA ternary complex and inhibit NifA activity, leading
to the transcriptional silencing of nif cluster; (b) In nitrogen–limiting conditions, high concentra-
tion of 2–OG can result in the association of NifL and GlnK in the NifA–NifL–Glnk complex. Free
NifA can activate the transcription of nitrogenase gene.
5.1. Regulation of NifA Function by 2–OG
Figure 4.
The NifL–NifA system’s responses to environmental and metabolic conditions in A.
vinelandii. (
a
) In nitrogen–excess conditions, the binding of high concentration of glutamine to GlnD
results in uridylyl removal from Glnk. NifL forms the binary complexes with NifA to inhibit NifA
activation. In addition, the non–covalently modified GlnK can also interact with NifL to promote
the formation of the GlnK–NifL–NifA ternary complex and inhibit NifA activity, leading to the
transcriptional silencing of nif cluster; (
b
) In nitrogen–limiting conditions, high concentration of
2–OG can result in the association of NifL and GlnK in the NifA–NifL–Glnk complex. Free NifA can
activate the transcription of nitrogenase gene.
5.1. Regulation of NifA Function by 2–OG
As an important intermediate product of the tricarboxylic acid cycle (TCA), 2–OG
is considered to be a key signal, which reflects the carbon metabolism status of cells. At
the same time, 2–OG also provides the carbon skeleton for nitrogen assimilation, and its
concentration indirectly corresponds to the status of intracellular nitrogen [
41
].
In vivo
experiments indicated that the physiological concentration of 2–OG increases sharply from
about 100
µ
mol/L to about 1 mmol/L, when the growth condition is changed from a
nitrogen–excess state to a nitrogen–limiting state in E.coli [
42
]. In A. vinelandii, 2–OG

Int. J. Mol. Sci. 2023,24, 907 9 of 18
directly affects the formation of the NifL–NifA complex in a concentration–dependent
manner [36].
The binding of 2–OG to the GAF domain of NifA can regulate the response of NifA to
NifL. The isothermal titration calorimetry (ITC) results showed that both the full–length
NifA protein and GAF domain alone could bind 2–OG, and the affinity for either one of
them is almost 60
µ
mol/L. The deletion of the GAF domain loses the ability to bind to
2–OG [
43
]. Limited protease hydrolysis experiments showed that 2–OG bound to the GAF
domain increases the sensitivity of the GAF domain to trypsin digestion and inhibits the
protection of these digestion sites by NifL [
43
]. This suggests that the binding of 2–OG
probably leads to the allosteric reaction of the GAF domain, interrupting the inhibition of
NifA by NifL. Consistently, the NifA–F119S mutant in the GAF domain is observed to lose
the ability to bind with 2–OG without affecting the ability to bind to NifL, whereas the
complex formed by NifA–F119S and NifL is no longer in control of 2–OG [
36
]. These results
indicate that the binding of 2–OG to the GAF domain in a concentration–dependent manner
induces the conformational changes in the GAF domain, which is followed by dissociation
of the NifL–NifA complex, releasing the activity of NifA to activate transcription of the
nitrogenase gene and promoting nitrogen fixation.
5.2. Effects of ADP and FAD Molecules on NifL Function
The formation of the NifL–NifA complex is associated with ATP/ADP and FAD/FADH
2
.
The results of affinity chromatography proved that NifA forms a stable complex with
NifL at a 1 mmol/L concentration of ADP, whereas the removal of ADP results in complex
dissociation [
44
], suggesting a key role in reinforcing the stability of the NifL–NifA complex.
ADP stabilizes the NifL–NifA complex by binding to the GHKL domain of NifL, enhancing
its inhibitory activity [
33
,
34
,
36
]. The binding of 2–OG to the GAF domain of NifA alters
the NifA conformation to antagonize the inhibitory activity of the ADP–bound NifL [
36
].
The catalysis of open promoter complexes by NifA requires hydrolysis of nucleotide
triphosphate to supply energy, which is usually provided in the form of ATP or GTP.
In vitro
transcriptional experiments of open promoter complexes showed that ATP or
GTP at a saturating concentration, with 4 mM GTP or 3.5 mM of ATP, can improve the
formation of the inhibitory NifL–NifA complex, and the extra–low concentration of ADP
(50
µ
M) can increase inhibition [
45
]. These data support the view that the accumulation of
ADP promotes the formation of the NifA–NifL complex during nitrogen fixation, thereby
regulating the efficiency of nitrogen fixation.
In the NifL–NifA system, FAD and FADH
2
are two signal molecules that manipulate
NifA activity via binding to NifL. FAD and FADH
2
can reflect the intracellular oxida-
tion/reduction status. The PAS1 domain of NifL can sense the redox status of FAD/FADH
2
molecules. In the oxygenated state, the conformational change of NifL produced by the
binding of the PAS1 domain to FAD prohibits the activity of NifA upon interacting with
it. In addition, NifL resembles the oxidized NifL–NifA complex in the FAD spectral char-
acteristics, regardless of the ADP binding state of NifL. This suggests that the FAD signal
rather than ADP determines the inhibition of NifA activity by NifL [
45
]. Relatively, in
the reduced state, the binding of the PAS1 domain to FADH
2
causes NifL to abolish its
ability to interact with NifA [
46
]. Previous studies have reported the crystal structure of
the PAS1 domain bound to FAD. The structure showed that the PAS1 domain exists as a
dimer in an asymmetric unit, and a novel cavity is formed inside each monomer, which
can interact with FAD through salt–bridge, hydrogen–bond, and hydrophobic interactions.
This structure supports the idea that hydrogen peroxide released by the oxidizing reaction
of FAD can mediate the recognition and transmission of the redox signal [
47
,
48
]. The PAS1
domain complexed with FADH
2
is extremely difficult to obtain, since FADH
2
is easily
oxidized to FAD by oxygen. As a result, the structure of NifL has not been reported in a
reduced state.

Int. J. Mol. Sci. 2023,24, 907 10 of 18
5.3. The NifL–NifA System Regulated by GlnK
The PII protein, which is widely distributed in bacteria, archaea, and plants, functions
as a signal–transduction protein in the regulation of nitrogen fixation [
49
]. Several genes
have been verified to encode PII paralogues in proteobacteria, such as glnB, glnK, glnJ, glnY,
and glnZ [
50
]. Current evidence indicates that A. vinelandii carries a single gene encoding a
protein belonging to the PII family, designated glnK [
40
]. The GlnK protein is a PII–like
protein sensing cellular nitrogen signals encoded by the glnK gene, which can participate
in the transcriptional regulation of NifL–NifA system through its covalently modified ury-
lation transition in A. vinelandii [
51
,
52
]. The glnK gene is often clustered and co–transcribed
with amtB genes encoding a membrane–bound NH
4+
channel (AmtB) [
50
]. The expression
of the glnK–amtB operon in A. vinelandii is not affected by a fixed nitrogen supply, which is
in contrast to other bacteria, such as E. coli and K. pneumoniae [
53
]. The GlnK protein exists
in the form of a trimer structurally, which can be reversible and covalently modified by
the uridylyltransferase/uridylyl–removing enzyme (UTase/UR) GlnD encoded by glnD,
a sensor–regulator that responds to the intracellular glutamine concentration. Each GlnK
trimer can be covalently modified by up to three uridine groups. In nitrogen–excess condi-
tions, intracellular nitrogen metabolism is active, and glutamine, as an important nitrogen
metabolic intermediate, is accumulated in the intracellular environment. Subsequently,
GlnD can exert UR activity to catalyze the deuridine acylation of GlnK via binding to
glutamine. The unmodified GlnK is involved in the GlnK–NifL–NifA ternary complex
formation that can suppress NifA activity [
51
]. It is possible that the unmodified GlnK does
not interact directly with NifA but interacts with the C–terminal GHKL domain of NifL to
enhance the inhibition of NifA activity by NifL [
54
]. Under intracellular nitrogen–limiting
conditions, the synthesis of glutamine is probably blocked, leading to a decline in glutamine
concentration. The lower concentration of glutamine reverses GlnD activity, allowing it to
exert UTase activity to catalyze the uridine acylation of GlnK. Consequently, the uridine
acylation of GlnK induces conformational changes, abrogating its capability to interact
with the NifL–NifA complex [53,55].
Mutations in GlnD that decrease its activity in the uridine adenylation of Glnk can
block the synthesis of nitrogenase via stabilizing the formation of the GlnK–NifL–NifA
ternary complex in A. vinelandii [
55
]. In addition,
in vitro
binding assays proved that
unmodified GlnK can promote the formation of the NifA–NifL complex, while uridine–
acylated GlnK lost such an ability. These results supported the function of GlnK in regu-
lating the NifL–NifA complex. The previous crystal structure showed that the working
mechanism of GlnK is reversibly uridylylated at the conserved Try51 by the GlnD protein
in E. coli in the presence of low nitrogen levels. The conserved Try51 is located on the
T–loop interacting with the target protein in E. coli, and the uridine acylation at Try51 can
enhance the flexibility of the T–loop, which is not conducive to the interaction between
Glnk and the target protein [
56
]. This result was further supported by the fact that the GlnK–
Y51F mutation failed proper uridine acylation and constant inhibition of NifA activity by
NifL [
51
]. Except for reversible modification of uridine acylation, GlnK may suffer from
a non–reversible modification in direct response to nitrogen availability. The irreversible
modification was identified as specific cleavage of the first three N–terminal amino acids of
Glnk in Streptomyces coelicolor, which is speculated to be caused by ammonium shock. How-
ever, it has not yet been verified that the specific cleavage of the three N–terminal amino
acids can affect the regulation of NifA activity by NifL by modulating GlnK stability [57].
The interaction of the PII protein with other proteins is also modulated by the binding
of the effectors, including adenylylate energy charge (ADP and ATP) and 2–OG, which
regulates signal–transduction proteins, metabolic energy, and permeases involved in nitro-
gen assimilation [
58
,
59
]. GlnK itself can directly perceive nitrogen limitation in response to
2–OG and ATP/ADP. Moreover, 2–OG, at the appropriate concentration, is a prerequisite
for the interaction between unmodified GlnK and NifL in A. vinelandii, consistent with
the interaction between PII proteins and other targeted proteins in E. coli [
29
,
31
]. One
GlnK trimer in A. vinelandii can bind two to three 2–OG molecules, but 2–OG at high

Int. J. Mol. Sci. 2023,24, 907 11 of 18
concentration (2 mmol/L) is unable to disrupt the interaction between unmodified GlnK
and NifL. This suggests that GlnK is not sensitive to the concentration change of 2–OG
within the physiological range in A. vinelandii. Therefore, the regulatory signal of GlnK
uridylation is more crucial than 2–OG for GlnK– regulated NifL activity.
6. The Differences in the NifA–NifL System for the Different Nitrogen–Fixing Bacteria
The homologs of NifA can be identified in almost all nitrogen–fixing bacteria from pro-
teobacteria, and most of the NifA proteins encoded by these bacteria are sensitive to oxygen
or excess fixed nitrogen signals. In these microorganisms, NifA interacts with its sensor
NifL protein or PII family signal–transduction proteins when fixing nitrogen. However, the
regulation mechanisms are considerably varied among the different organisms.
NifA has been described to be regulated by the sensor NifL from the members in
γ
–
proteobacteria, such as the well–studied A. vinelandii and K. pneumoniae [
60
,
61
]. In spite of
the functions of NifL being similar to that in A. vinelandii and K. pneumoniae, the NifL–NifA
system may adopt a different mechanism to respond to the cellular nitrogen status. In
contrast to A. vinelandii, at least two homologs of PII–like proteins are involved in nitrogen
control in K. pneumoniae, designated glnB and glnK. The two PII proteins are structurally
similar, while the response of NifL to fixed nitrogen levels is only dependent on GlnK,
encoded by glnK. GlnK plays a key role in relieving the inhibitory effect of NifL on NifA,
but this regulation is associated with the concentration of GlnK other than the uridylation
state of GlnK. GlnB encoded by glnB can counteract this modulation of GlnK on the NifL–
NifA complex [
62
]. In addition, studies have shown that the NifL proteins from the two
organisms differentiate. The NifL protein from K. pneumoniae can only be synthesized in a
nitrogen–excess condition, and its C–terminal sequence has little sequence homology with
the GHKL domain and none of the characteristics of binding ATP/ADP. The N–terminal
PAS domain can bind and hydrolyze ATP, but only the C–terminal domain can form the
stabilized complex with NifA [
63
]. Besides
γ
–proteobacteria, NifL homologs have also
been characterized in
α
–,
β
–, and
ζ
–proteobacteria, but few studies have described the
regulation of NifL in these proteobacteria [64,65].
In
α
– and
β
–proteobacteria, few species can express NifL. This suggests that the
activity of NifA proteins does not require the synergistic regulation of NifL proteins in
most α– and β–proteobacteria. NifA is sensitive to oxygen and fixed nitrogen signals and
can directly interact with signal–transduction proteins from the PII family in response to
fixed nitrogen levels [
65
]. Studies have shown that NifA is diversely expressed in these
organisms. NifA in Rhizobias, such as Sinorhizobium meliloti and Bradyrhizobium japonicum
from
α
– or
β
–proteobacteria, contains a broadly conserved cysteine–rich motif located
at an inter–domain linker (IDL) region between the AAA domain and the HTH domain,
which confers its oxygen sensitivity. However, the IDL region does not exist in either A.
vinelandii or K. pneumoniae NifA [
66
]. These conserved cysteine residues are also present
in many non–rhizobia NifA homologs, such as Azospirillam brasilense (A. brasilense) and
Herbaspirillum seropedicae (H. seropedicae) [
67
]. Studies on A. brasilense and H. seropedicae
have shown that four conserved cysteine residues (C414, C426, C446, and C451) make NifA
sensitive to oxygen. The sensitivity of NifA to fixed nitrogen in A. brasilense is regulated by
its two PII proteins, designated GlnB (encoded by glnB) and GlnZ (encoded by glnZ). GlnB
and GlnZ are structurally similar but distinct in their regulation of nitrogen metabolism.
The uridylation activity and ATP–binding ability of GlnB require NifA activation [
68
].
GlnB can interact with the GAF domain to activate NifA in nitrogen–limiting conditions.
GlnZ may play a critical role in prohibiting the expression of the nitrogenase gene [
69
]. In
contrast, NifA can be inhibited via interacting with the PII protein to adapt to the nitrogen–
excess condition in Rhodobacter capsulatus [
70
]. These results suggest that NifA and NifL
in different nitrogen–fixing bacteria may play completely different roles in regulating the
expression of the nitrogenase gene. As a result, it is of great significance to further improve
the regulation network of nitrogen–fixing to facilitate agricultural applications in different
model strains.

Int. J. Mol. Sci. 2023,24, 907 12 of 18
7. The Potential Applications of the NifA–NifL System in Agricultural Development
The application of industrial nitrogen fertilizers, such as urea and nitrate, satisfies the
demand for high crop yields but also generates some global “nitrogen problems” [
71
]. Thus,
biological nitrogen fertilizers can substitute industrial nitrogen fertilizers in sustainable sys-
tems. Biological nitrogen fixation depends on functionally active nitrogenases. The complex
NifL–NifA regulatory system ensures that the synthesis of functionally active nitrogenases
only occurs under the proper physiological conditions. This regulation mode can ensure the
normal growth of nitrogen–fixing bacteria, other than facilitating the discharge of ammonia
into the environment. Thus, it is desirable for engineering methods to achieve stabilized
and efficient nitrogen fixation that provides an alternative to synthetic nitrogen. In recent
decades, various approaches have been proposed to enhance the efficiency of biological
nitrogen fixation by offering synthetic nitrogen alternatives (Figure 5). Some of them have
been successfully applied in agriculture to achieve high crop yields.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 12 of 19
C451) make NifA sensitive to oxygen. The sensitivity of NifA to fixed nitrogen in A. bra-
silense is regulated by its two PII proteins, designated GlnB (encoded by glnB) and GlnZ
(encoded by glnZ). GlnB and GlnZ are structurally similar but distinct in their regulation
of nitrogen metabolism. The uridylation activity and ATP–binding ability of GlnB re-
quire NifA activation [68]. GlnB can interact with the GAF domain to activate NifA in
nitrogen–limiting conditions. GlnZ may play a critical role in prohibiting the expression
of the nitrogenase gene [69]. In contrast, NifA can be inhibited via interacting with the
PII protein to adapt to the nitrogen–excess condition in Rhodobacter capsulatus [70]. These
results suggest that NifA and NifL in different nitrogen–fixing bacteria may play com-
pletely different roles in regulating the expression of the nitrogenase gene. As a result, it
is of great significance to further improve the regulation network of nitrogen–fixing to
facilitate agricultural applications in different model strains.
7. The Potential Applications of the NifA–NifL System in Agricultural Development
The application of industrial nitrogen fertilizers, such as urea and nitrate, satisfies
the demand for high crop yields but also generates some global “nitrogen problems”
[71]. Thus, biological nitrogen fertilizers can substitute industrial nitrogen fertilizers in
sustainable systems. Biological nitrogen fixation depends on functionally active nitro-
genases. The complex NifL–NifA regulatory system ensures that the synthesis of func-
tionally active nitrogenases only occurs under the proper physiological conditions. This
regulation mode can ensure the normal growth of nitrogen–fixing bacteria, other than
facilitating the discharge of ammonia into the environment. Thus, it is desirable for en-
gineering methods to achieve stabilized and efficient nitrogen fixation that provides an
alternative to synthetic nitrogen. In recent decades, various approaches have been pro-
posed to enhance the efficiency of biological nitrogen fixation by offering synthetic ni-
trogen alternatives (Figure 5). Some of them have been successfully applied in agricul-
ture to achieve high crop yields.
Figure 5. The strategy of engineering nitrogen fixation. (1) Adding nitrogenase into plant cells. Ni-
trogenase requires a lot of energy and a low–oxygen environment to execute the nitrogen–fixing
function. The mitochondrion provides a suitable place to accommodate the enzyme. (2) Creating a
novel partnership between azotobacter and plants. Under natural conditions, non–symbiotic azo-
Figure 5.
The strategy of engineering nitrogen fixation. (
1
) Adding nitrogenase into plant cells.
Nitrogenase requires a lot of energy and a low–oxygen environment to execute the nitrogen–fixing
function. The mitochondrion provides a suitable place to accommodate the enzyme. (
2
) Creating
a novel partnership between azotobacter and plants. Under natural conditions, non–symbiotic
azotobacter are not capable of infecting crops. Several key modifications, such as expressing rhizopine
in the crop cells and the receptor of rhizopine in azotobacter, can specifically allow crops to recognize
azotobacter. The elongated root hair can create a tunnel for azotobacter to infect the root hair and
form nodules. (
3
) Disrupting the transcriptional regulation of nitrogenase. As the transcriptional
regulation of nitrogenase is the crucial rate–limiting step in nitrogen fixation, several modifications can
disrupt the original regulatory pathways, allowing them to evolve in ways beneficial to agricultural
development. (4) Other strategies.
Studies have shown that the application of nitrogen–fixing bacteria can stimulate
the growth of crops, and the direct inoculation of azotobacter can improve the yield of
crops such as cereal, potatoes, corn, and vegetables. [
8
]. In nature, most azotobacter are
autotrophic heterotrophic bacteria, which can fix about 20 kg of nitrogen per hectare per
year. In a farmland ecosystem, most of the fixed nitrogen can be utilized in crop production
as a substitute for a portion of nitrogen fertilizer. Although nitrogen–fixing bacteria can be
directly introduced into agricultural production as biological nitrogen fertilizer to improve
crop yields, the nitrogen–fixation efficiency of nitrogen–fixing bacteria is still insufficient to

Int. J. Mol. Sci. 2023,24, 907 13 of 18
meet the demand of agricultural development for nitrogen fixation. Researchers believe that
these biotechnological approaches, which allow crops to make their own nitrogen fertilizers,
engineer legume symbiosis into cereals, and genetically manipulate azotrophic bacteria
for efficient nitrogen fixation and ammonia excretion, with the potential for even greater
returns. Nearly half a century ago, Ray Dixon and his colleagues managed to transfer
a complete nif gene cluster from K. pneumoniae to E. coli to confer the ability of nitrogen
fixation, which laid the groundwork for its application to crops [
72
]. This technique has
been successfully applied to tomatoes as well, which enabled the plant to fix nitrogen and
increase its yield. Moreover, the technology is now being applied to wheat, corn, and
other large–scale applied experiments [
1
]. Despite some amazing breakthroughs in the
ability of plants to fix nitrogen, several technical obstacles remain to be overcome. It is the
basic prerequisite for engineering nitrogen fixation that the nitrogenase system is expressed
stably and intactly in plant cells. In addition, protecting nitrogenase from oxygen is a key
barrier since nitrogenase is sensitive to oxygen, as described previously, and plant cells
generate large amounts of oxygen. Recent work on the transfer of nitrogenase into plant
cells has shown that mitochondria and chloroplasts can be used as suitable locations for
nitrogenase expression, as they can provide a large amount of reducing agents and ATP for
the nitrogen–fixation process [
73
–
78
]. In addition, the aerobic respiration of mitochondria
creates an oxygen–depleted environment for the expression of active nitrogenase, similar
to that of aerobic azide–trophic bacteria, such as A. vinelandii [
73
,
79
,
80
]. In fact, the direct
introduction of nitrogenase into plant cells can confer crops to make their own nitrogen
fertilizer, but the plant cells cannot express the components of the regulatory system,
leading to an out–of–control NifL–NifA regulatory system. The process of nitrogen fixation
requires a large amount of ATP and reducing agents, so extra–fast or excessive nitrogen
fixation may cause irreversible damage to plant cells and lower the yield of crops. TCA
in eukaryotic cells is carried out in the mitochondrial matrix, which provides multiple
regulators for the NifL–NifA regulatory system, including 2–OG, FAD/FADH
2
, ATP/ADP,
and Glu/Gln [
81
,
82
]. Studies on the subject suggest that mitochondria may be used as the
working site for the NifL–NifA regulatory system. Thus far, no successful application of
the NifL–NifA regulatory system in plant cells has been reported.
Even if such work was technically successful, it is difficult for such strategies to
gain public approval because they involve genetic modification. Therefore, focusing
on the nitrogen–fixing bacteria themselves, and adjusting the nitrogen–fixing patterns
of the nitrogen–fixing bacteria to generate more fertilizer, may achieve higher rewards.
Studies have shown that diazotrophic bacteria can be engineered for excess production
and excretion of NH
3
using several strategies [
12
,
83
,
84
]. Precise disruption of the nif L
component in the nif LA operon results in phenotypic dysregulation, the production of
ammonium far exceeding intracellular requirements, and the release of up to 30 mM of
ammonium into the growth medium [
39
,
60
,
85
,
86
]. However, in the absence of active NifL,
the activity of NifA becomes uncontrollable, which results in the generated bacterial strains
being disadvantaged in the environment due to the energetic burden [
85
,
86
]. This suggests
that it is unrealistic to engineer NifL to remove nitrogen regulation. Researchers believe
that both excessive ammonia emission and the minimization of the energy burden can
be achieved by controlling the activity or activation mode of NifA in a nitrogen–limiting
environment [
36
,
83
]. In a nitrogen–limiting environment, the NifA–E356K mutant in
engineering A. vinelandii is regulated by the 2–OG level instead of NifL [
36
]. After the key
residues of NifA in other proteobacteria are replaced by that of NifA in A. vinelandii, this
variant exhibits analogous properties to that of NifA in A. vinelandii. This may contribute to
the engineering of carbon–source–dependent ammonia excretion, which is distinct among
the different members of this family [
83
]. In addition, PII proteins, such as GlnK, modified
by UTase/UR, can directly or indirectly control NifA activity in response to fixed nitrogen
signals [
51
,
54
]. Based on this mechanism, the engineering of fixed–nitrogen bacteria for
insensitivity to the intracellular nitrogen state, to continuously fix nitrogen and exhaust
ammonia, has been accomplished by mutating the PII protein in Azorhizobium caulinodans

Int. J. Mol. Sci. 2023,24, 907 14 of 18
(A. caulinodans) and A. brasilense [
87
–
89
]. However, this engineering strategy does not seem
to be universally applicable to other bacteria and is even fatal, limited by differentiated
functions of PII in the regulation of NifA and nitrogenase activity [90–92].
From the perspective of agricultural development, engineering diazotrophic bacteria
for excessive nitrogen fixation and ammonia excretion may cause two major detriments.
First, uncontrolled NifA activity and active nitrogenase expression impose a severe ener-
getic burden and eliminate the competitiveness of root colonization. Second, ammonia
excretion is not directed, which results in low utilization of ammonia in the target crop.
This is expected to establish symbiotic engineering diazotrophic bacteria that can target
crops for ammonia excretion, similar to the symbiotic relationship between soybean and
rhizobia. Poole and colleagues developed a symbiotic model in which an engineered
diazotrophic bacterium, the endophyte A. caulinodans, sensed only the nitrogen–fixing
synthetic rhizopine signaling released by barley, which could induce ammonia excretion in
the barley direction [
93
–
95
]. Subsequently, this technology was successfully established on
the symbiotic model between A. caulinodans and cereals [
96
]. This highly complex control
circuit represents an important milestone in the development of “synthetic symbiosis” in
which N
2
fixation and NH
3
excretion can be activated in bacteria–specific colonizing target
rhizopine–producing cereals, targeting the delivery of nitrogen to the crops while avoiding
potential interactions with non–target plants.
8. Summary and Prospect
The “Nitrogen problem” restricts the sustainable development of agriculture. The
application of the biotechnology approach to the fabrication of biological nitrogen fer-
tilizer presents an opportunity to address this issue [
97
]. Currently available biological
nitrogen fertilizers, either wild–type or engineered, cannot match the ability of chemically
synthesized fertilizers to improve crop yields. However, it cannot be denied that biological
nitrogen fertilizers have great potential in agricultural applications.
A. vinelandii is widely used because of its high efficiency of nitrogen fixation and
simple genetic material that make it an easy subject for genetic manipulation, as well
as its agricultural value being gradually embodied. Several engineered bacteria have
been designed and developed with respect to A. vinelandii that promise a wide range of
applications in agriculture. However, a significant amount of work has to be completed on
agricultural applications of A. vinelandii. Improving the value of agricultural applications
depends on the development of new biotechnological data. To advance and develop
new biotechnological applications and products, a better understanding of their nitrogen–
fixation and –regulation mechanisms is required. Although the nitrogen–fixing regulatory
process of A. vinelandii is becoming clearer, an accurate explanation of the molecular
mechanism is still missing due to a lack of relevant structural biological evidence. For
example, how does 2–OG mediate the allosteric of GAF domain to activate NifA? When
NifA activates transcriptional activity of the RNAP–
σ
54–DNA holoenzyme, what is the
molecular mechanism by which an L1/L2 loop binds to
σ
54 and leads to partial uncoupling
of the DNA double strand? What is the molecular mechanism by which NifL inhibits
NifA activity under the conditions of excess nitrogen? In addition, structural information
on how unmodified GlnK forms ternary complexes with NifL and NifA is still unclear.
These questions need to be answered by reliable biochemical and structural biological
data theoretically supporting the artificial design and efficient utilization of nitrogen–
fixation modules.
Author Contributions:
W.Z.: conceptualization, validation, writing—original draft, writing—review
and editing, investigation, data curation, and visualization. W.Z., Y.C. and K.H.: producing all of the
figures. Z.M.: conceptualization, data curation, writing—review and editing. F.W.: conceptualization,
writing—review and editing, supervision, funding. All authors have read and agreed to the published
version of the manuscript.

Int. J. Mol. Sci. 2023,24, 907 15 of 18
Funding:
This work was funded by the National Natural Science Foundation of China grant (grant
number 31870791) to Z.M.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest:
The authors declare that they have no known competing financial interest or
personal relationship that could have appeared to influence the work reported in this paper.
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