608 UNIT 4 Metabolic Diversity and Microbial Ecology

evidence for the activity of nitrogenase. In an assay, the sam-

Atmosphere, 10% GH, in air (aerobes) or
a culture, or a cell extract, is
10% GH, in N, or Ar (anaerobes) ple, whichmay be soil, water,
incubated with acetylene, and the gas phase of the reaction
mixture is later analyzed by gas chromatograph for produc
tion of ethylene (Figure 20.39). This method is far simpler
can easily be adapted for
Stoppered vial containing and faster than other methods and
cell suspension use in ecological studies of Nz-fixing bacteria directly in their
habitats.

20.15 Genetics and Regulation

of N2 Fixation
2H Because the process of N2 highly energy demand
ixation is
HCECHHC=CH
nitrogenase ofand the many
Incubation Acetylene Ethylene ing, the synthesis and activity
other enzymes required for N hixation (Figure 20.36) is highly
Nitrogenase regulated.

Sample headspace Genetics of Nitrogen Fixation

periodically and inject into
Chart recorder for
gas chromatograph gas chromatograph The genes for dinitrogenase and dinitrogenase reductase in
Klebsiella pneumoniae, a well-studied nitrogen fixer, are part

of complex regulon
a (a large network of operons, c Sec-
CH2 tion 9.9) called the nif regulon. The K. pneumoniae nif
CHe CH
CpH2
kbp of DNA and contains 20 genes
regulon spans 24
CaH transcriptional units (Figure 20.40). In
JU arranged in several
addition to nitrogenase structural genes, the genes for
FeMo-co synthesis, genes controlling the electron transport
2h and number of regulatory genes are also present
Time 0 1h proteins, a

in the nif regulon.

Figure 20.39 The acetylene reduction assay for nitrogenase Dinitrogenase is composed of two subunits, a (the prod-
The results show no ethylene (C2Ha) when the experiment be-
activity uct of and B (the product of nifK), each of which is
nifD)
as the assay proceeds.
gins (time 0), but increasing production of CaHa copies in the nitrogenase enzyme complex.
Note how as CpHa produced,
is acetylene (CpH2) is consumed. lf the vial present in two

contained an enzyme extract, conditions would be anoxic, even if nitro-

genase was trom an aerobic bacterium

Nitrogenase

Dinitrogenase
reductase

FeMo-co
FeMo-co
synthesis
synthesis Dinitrogenase
Dinitro-
genase Homocitrate FeMo-co Electron
reductase synthesis synthesis transport
processing Pyruvate
Mo Regulators Metal center
FeMo-co
insertion into
flavodoxin
process Positive Negative Oxido-
biosynthesis dinitrogenase
ing Flavodoxin reductase

nif
NE YTK DH DNA
OB M ZWVSU

Transcripts
Figure 20.40 The nif regulon in Klebsiella pneumoniae, the best-studied nitrogen-fixing bacterium.
The function of the nifTgene product is unknown,. The mRNA transcripts are shown below the genes, arrows
indicate the direction of transcription. Proteins that catalyze FeMo-co synthesis are shown in yellow. Other
colors match those of Figure 20.36
 Chapter 20 I Metabolic Diversity. Phototrophy, Autotrophy, Chemolithotrophy, and Nitrogen Fixation
Dinitrogenase reductase is a protein dimer consisting of two
identical subunits, the product of nifH. FeMo-co is
sized by enzymes encoded by several
synthc
genes, including nifN, V
Z, W, E, and B, as well as nif Q, which encodes a
molybdenum
processing enzyme. The nifA gene encodes a positive regula-
tory protein that activates transcription of other nif genes
(Figure 20.40).
Nitrogenase is a highly conserved protein, and the
nifHDK genes that encode it have been used as molecular
probes to screen DNA from various prokaryotes for homolo-
gous genes, signaling their ability to fix
nitrogen. In all
nitrogen hxers examined (except for the highly unusual
Streptomyces thermoautotrophicus, Section 20.14), nifHDK-
like genes are present,
suggesting that the genetic requirements
for nitrogenase are rather
specific. Alternative nitrogenases
(see Section 20.14) are encoded
by their own sets of genes,
vnfHDK for the vanadium system and anfHDK for the iron-
only system, but these genes show signihcant
sequence
similarity to nifHDK.
Regulation of Nitrogenase Synthesis
Nitrogenase is subject to strict regulatory controls. Nitrogen
fhxation is repressed by O2 and by fixed forms of
nitrogen, in-
cluding NH3, NO3 , and certain amino acids. Amajor part of
this regulation is at the level of
transcription, as shown in
Figure 20.40. Transcription of nif structural genes is activated
by the NifA protein (positive regulation,
Section 9.4). By
contrast, NifL is a negative regulator of nif gene
expression
and contains a molecule of FAD (recall that FAD is a redox
coenzyme for flavoproteins, o Section 5.11) that is involved
in O-sensing. In the presence of sufficient O, NifL represses
synthesis of other nif genes, which blocks synthesis of the
Oxygen-labile nitrogenase.
Ammonia represses N2 fixation through a second pro-
tein, called NirC, whose activity is regulated by the nitrogen
status of the cell. When ammonia is limiting, NtrC is active
and promotes transcription of nifA. This produces NifA, the
nitrogen fixation activator protein, and nif transcription
begins.
The ammonia produced by nitrogenase does not block
enzyme synthesis because it is incorporated into amino
acids (co Section 5.16) and used in biosynthesis as soon as
it is made. But when ammonia is in excess (as in natural en-
vironments or culture media high in ammonia), nitrogenase
synthesis is repressed. In this way, ATP is not wasted in
making ammonia when it is already available in ample
amounts.
Regulation of Nitrogenase Activity
Besides repressing the synthesis of nitrogenase, nitrogenase
activity is also regulated by ammonia in many nitrogen-fixing
bacteria; that is, nitrogenase proteins that already exist in the
cell can be turned on o r off. Although there are likely different
mechanisms for shutting off the activity of nitrogenase, they
seem to function by shutting
down electron flow to dinitro-
all
609
genase in response to exCess ammonia, thus making nitroge-
nase inactive.
The mechanism of nitrogenase shut down by ammonia is
known in many phototrophic and some chemotrophic nitrogen-
fixing Proteobacteria and is called the ammonia switch-off
effect. In this mechanism, excess ammonia causes a covalent
modification of dinitrogenase reductase, which results in a
loss of enzyme activity. When ammonia becomes limiting, the
modified dinitrogenase reductase is converted to its active
form and
N, fixation can resume. Ammonia switch-off is thus
a
rapid and reversible method of controlling ATP consump-
tion by nitrogenase.
Ammonia switch-off has also been observed in nitrogen-
fixing Archaea. In Methanococcus species, for example,
ammonia quickly inhibits the activity of nitrogenase. Here,
however, covalent modification of dinitrogenase reductase is
not involved. Instead, it appears that an
ammonia-sensing
protein exists in the cell that can bind to nitrogenase or in
some other way inactivate it when ammonia is in excess in the
cell. Interestingly, the ammonia-sensing system appears to
function not by detecting ammonia directly, but by detecting
a shortage in the carbon compound a-ketoglutarate, a direct
precursor of the amino acid glutamate (Section 5.16). In this
model for nitrogenase regulation, a shortage of a-ketoglutarate
is sensed
as an excess of
glutamate, and thus nitrogenase
activity would be unnecessary because it would produce
ammonia and lead to the
more
synthesis of more glutamate
cSection 9.8 and Figure 9.16). This interesting regulatory
system also controls nitrogenase activity in some species of
Bacteria, in particular nitrogen-fixing species of the fermen-
tative anaerobe Clostridium ( Section 16.2).
20.14-20.15 MiniReview
Nitrogen fixation is the reduction of Na to NH3 and requires
the enzyme complex nitrogenase. Most nitrogenases contain
molybdenum or vanadium plus iron as metal cofactors, and
the process of nitrogen fixation is highly energy demanding.
Nitrogenase and most associated regulatory proteins are
encoded by the nif regulon. Certain substances that are
structurally similar to N2, such as acetylene or cyanide, are
also reduced by nitrogenase. Nitrogenase and nitrogen
fixation are both highly regulated, with Oz and NH being the
two main regulatory effectors
IWrite a balanced equation for the reaction carried out by
the enzyme nitrogenase.
IWhat is FeMo-co and what metals does it contain?
IHow is CaH2 useful for studies of nitrogen fixation?
What chemical and physical factors affect the synthesis or
activity of nitrogenase? How does the Streptomyces
thermoautotrophicus nitrogenase system differ in this
regard?
I How do alternative nitrogenases differ from classical
nitrogenase?