100 U N I T 1 • T h e F o u n d at i o n s o f M i c r o b i o l o g y

to the ACP to form malonyl-ACP. As each malonyl residue is

donated, one molecule of CO2 is released (Figure 3.30). Table 3.5  Some nitrogen-fixing organismsa
The fatty acid composition of cells varies from species to spe- Free-living aerobes
cies and can also vary in a pure culture due to differences in Chemoorganotrophs Phototrophs Chemolithotrophs
growth temperature. Growth at low temperatures promotes the
Azotobacter, Azomonas, Cyanobacteria (e.g., Alcaligenes
biosynthesis of shorter-chain fatty acids whereas growth at higher Azospirillum, Klebsiella,b Anabaena, Nostoc, Acidithiobacillus
temperatures promotes longer-chain fatty acids ( Sections 5.12 Methylomonas Gloeothece,
and 5.13). The most common fatty acids in lipids of Bacteria are Aphanizomenon)
those with chain lengths of C12–C20. Free-living anaerobes
In addition to saturated, even-carbon-number fatty acids, fatty Chemoorganotrophs Phototrophs Chemolithotrophsc
acids can also be unsaturated, branched, or have an odd num- Clostridium Purple bacteria (e.g., Methanosarcina
ber of carbon atoms. Unsaturated fatty acids contain one or more Desulfotomaculum Chromatium, Methanococcus
double bonds in the long hydrophobic portion of the molecule. Rhodobacter) Methanocaldococcus
The number and position of these double bonds is often species- Green bacteria (e.g.,
Chlorobium)
specific or group-specific, and double bonds are typically formed Heliobacteria
by desaturation of a saturated fatty acid. Branched-chain fatty
Symbiotic
acids are biosynthesized using a branched-chain initiating mol-
ecule, and odd-carbon-number fatty acids (for example, C13, C15, With leguminous plants With nonleguminous plants
C17, etc.) are biosynthesized using an initiating molecule that con- Soybeans, peas, clover, etc. with Alder, bayberry, autumn olive, many
Rhizobium, Bradyrhizobium, other bushy plants, with the
tains a propionyl (C3) group.
Sinorhizobium actinomycete Frankia
a
Only some common genera are listed in each category; many other nitrogen-fixing
Lipid Biosynthesis genera are known.
In the assembly of lipids in cells of Bacteria and Eukarya, fatty b
Nitrogen fixation occurs only under anoxic conditions.
c
All are Archaea.
acids are first added to a molecule of glycerol. For simple triglyc-
erides (fats), all three glycerol carbons are esterified with fatty
acids. To form complex lipids, one of the carbon atoms in glyc- out the process completely independently. By contrast, others are
erol is embellished with a molecule of phosphate, ethanolamine, symbiotic and fix nitrogen only in association with certain plants
carbohydrate, or some other polar substance ( Figure 2.14a). ( Section 22.0). However, in symbiotic nitrogen fixation it is
Although in Archaea, membrane lipids are constructed from iso- the bacterium, not the plant, that fixes N2; no eukaryotic organ-
prene to form the phytanyl (C15) or biphytanyl (C30) side chains, isms are known to fix nitrogen.
the glycerol backbone of archaeal membrane lipids typically con-
tains a polar group (sugar, phosphate, sulfate, or polar organic Nitrogenase
compound) as well. Polar groups are important in lipids for form- Nitrogen fixation is catalyzed by an enzyme complex called
ing the standard membrane architecture: a hydrophobic interior nitrogenase. Nitrogenase consists of two proteins, dinitrogenase
with hydrophilic surfaces ( Figure 2.17).
Homocitrate
MiniQuiz
• Explain how fatty acids are constructed two carbon atoms at O–
a time while the immediate donor of these carbons is a three- O C
carbon compound. S
O–
• What differences exist in lipids from the three domains of life? Fe Fe
S S C
O
C H
3.17 Nitrogen Fixation Protein S Fe S Fe C Fe S Mo O C H
We conclude our coverage of biosyntheses by considering the
N
formation of ammonia (NH3) from gaseous dinitrogen (N2), a H
S C
process called nitrogen fixation. The ammonia produced is as- Fe Fe S
Protein H
similated into organic form in amino acids and nucleotides. The H
ability to fix nitrogen frees an organism from dependence on S H C
fixed nitrogen in its environment and confers a significant eco-
logical advantage when fixed nitrogen is limiting. The process of C
nitrogen fixation is also of enormous agricultural importance, as O–
O
it supports the nitrogen needs of key crops, such as soybeans.
Only certain species of Bacteria and Archaea can fix nitrogen, Figure 3.31  FeMo-co, the iron–molybdenum cofactor from nitrogenase. On
and a list of some important nitrogen-fixing organisms is given in the left side is the Fe7S8 cube that binds to Mo along with O atoms from homocitrate
Table 3.5. Some nitrogen-fixing bacteria are free-living and carry (right side, all O atoms shown in purple) and N and S atoms from dinitrogenase.
 Biological N fixaction is a Redox reaction
C H A P T E R 3 • M i c r o b i a l M e ta b o l i s m 101
and dinitrogenase reductase. Both proteins contain iron, and 4 Pyruvate CoA 4 Acetyl-CoA + 4 CO2
dinitrogenase contains molybdenum as well. The iron and
Pyruvate donates
molybdenum in dinitrogenase are part of the enzyme cofactor

UNIT 1
2 e– (×4) electrons to
Electrons
called the iron–molybdenum cofactor (FeMo-co), and reduc- for flavodoxin.
tion of N2 occurs at this site. The composition of FeMo-co is nitrogenase
MoFe7S8 # homocitrate (Figure 3.31). Two “alternative” nitroge-
4 Flavodoxin 4 Flavodoxin
(Ox) (Red)
nases are known that lack molybdenum. These contain either Flavodoxin reduces
vanadium (V) plus iron or iron-only in their cofactors and are dinitrogenase
made by certain nitrogen-fixing bacteria when molybdenum is reductase.
absent from their environment ( Section 14.12). 4 Dinitrogenase 4 Dinitrogenase
With one exception, nitrogen-fixing Archaea produce nitrogenas- reductase reductase
es with iron as the only metal in the enzyme. Nitrogen-fixing Archaea (Red) (Ox)
appear limited to a few methane-producing species (methanogens), 16 ATP
Electrons transferred to
at least one of which can grow and fix N2 at very high temperatures. dinitrogenase one at a
time. 2 ATP are
The species Methanosarcina barkeri, a metabolically versatile meth- Nitrogenase 16 ADP consumed per electron.
anogen ( Section 16.2), contains genes encoding molybdenum activity + 16 Pi
and vanadium nitrogenases as well as an iron-only nitrogenase, and
Dinitrogenase Dinitrogenase
so it likely contains the full suite of nitrogenase proteins. (Ox) (Red)
Nitrogen fixation is inhibited by oxygen (O2) because dinitro-
genase reductase is irreversibly inactivated by O2. Nevertheless,
many nitrogen-fixing bacteria are obligate aerobes. In these 2 NH3 N2
(a)

H2
4H 2H 2H
Sum: N N HN NH H2N NH2 2 NH3
(16 ATP 16 ADP + 16 Pi)
(b)

Figure 3.33  Biological nitrogen fixation by nitrogenase. The nitrogenase

complex is composed of dinitrogenase and dinitrogenase reductase.

organisms, nitrogenase is protected from oxygen inactivation by a

combination of the rapid removal of O2 by respiration and the pro-
duction of O2-retarding slime layers (Figure 3.32a, b). In heterocys-
Wael Sabra

Wael Sabra

tous cyanobacteria, nitrogenase is protected by its localization in a

differentiated cell called a heterocyst (Figure 3.32c; Section 14.3). cyanobacteria/
Anabaena

(a) (b) Inside the heterocyst, conditions are anoxic, while in neighboring
vegetative cells, conditions are just the opposite because oxygenic
photosynthesis is occurring. Oxygen production is shut down in
the heterocyst, thus protecting it as a dedicated site for N2 fixation.

Electron Flow in Nitrogen Fixation

Owing to the stability of the triple bond in N2, its activation and
Alicia M. Muro-Pastor

reduction is very energy demanding. Six electrons are needed

(c)
Figure 3.32  Protection of nitrogenase in Azotobacter vinelandii and in the
cyanobacterium Anabaena. (a) Transmission electron micrograph of nitrogen-fixing
cells of A. vinelandii grown with 2.5% O2; very little slime is evident. (b) Cells grown
in air (21% O2). Note the extensive darkly staining slime layer (arrow). The slime
retards diffusion of O2 into the filament, thus preventing nitrogenase inactivation by O2.
(c) Fluorescence photomicrograph of a cell of the filamentous cyanobacterium
Anabaena showing a single heterocyst (green). The heterocyst is a differentiated cell
that specializes in nitrogen fixation and protects nitrogenase from O2 inactivation.
to reduce N2 to NH3, and the successive reduction steps occur
directly on nitrogenase with no free intermediates accumulating
(Figure 3.33). Although only six electrons are necessary to reduce
N2 to two NH3, eight electrons are actually consumed in the pro-
cess, two electrons being lost as H2 for each mole of N2 reduced.
For unknown reasons, H2 evolution is an obligatory step in
nitrogen fixation and occurs in the first round of the nitrogenase
reduction cycle. Following this, N2 is reduced in successive steps
and ammonia is the released product (Figure 3.33).
The sequence of electron transfer in nitrogenase is as follows:
electron donor S dinitrogenase reductase S dinitrogenase S N2.
The electrons for N2 reduction are transferred to dinitrogenase
102 U N I T 1 • T h e F o u n d at i o n s o f M i c r o b i o l o g y
Atmosphere, 10% C2H2 in air (aerobes) or
10% C2H2 in N2 or argon (anaerobes)
( )
2H
HC CH H2C CH2
Acetylene Ethylene
Nitrogenase
Incubation Sample headspace
periodically and inject
into gas chromatograph.
Stoppered vial containing
cell suspension
Figure 3.34  The acetylene reduction assay of nitrogenase activity in nitrogen-fixing bacteria. The results show no
ethylene (C2H4) at time 0 but increasing production of C2H4 as the assay proceeds. As C2H4 is produced, a corresponding amount
of C2H2 is consumed.
reductase from the low-potential iron–sulfur proteins ferredoxin
or flavodoxin (Section 3.10); these proteins are reduced during the
oxidation of pyruvate (Figure 3.33). In addition to electrons, ATP
is required for nitrogen fixation. ATP binds to dinitrogenase reduc-
tase, and, following its hydrolysis to ADP, lowers the reduction
potential of the protein. This allows dinitrogenase reductase to
interact with and reduce dinitrogenase. Electrons are transferred
from dinitrogenase reductase to dinitrogenase one at a time, and
each cycle of reduction requires two ATP. Thus a total of 16 ATP
are required for the reduction of N2 to 2 NH3 (Figure 3.33).
Assaying Nitrogenase: Acetylene Reduction
Nitrogenases are not entirely specific for N2 and also reduce
other triply bonded compounds, such as acetylene (HC ‚ CH).
The reduction of acetylene by nitrogenase is only a two-electron
process, and ethylene (H2C “ CH2) is the final product. However,
the reduction of acetylene to ethylene provides a simple and rapid
method for measuring nitrogenase activity (Figure 3.34). This tech-
nique, known as the acetylene reduction assay, is widely used in
microbiology to detect and quantify nitrogen fixation.
Big Ideas
3.1 • Cells are primarily composed of the elements H, O,
C, N, P, and S. The compounds found in a cell are obtained
from or formed from nutrients present in the environment.
Nutrients required in large amounts are called macronutrients
while those required in very small amounts, such as trace
elements or growth factors, are micronutrients.
3.2 • Culture media supply the nutritional needs of
microorganisms and are either defined or complex. Other
media, such as selective, differential, and enriched media,
are used for specific purposes. Many microorganisms can
Chart recorder for
gas chromatograph
C2H2 C2H2
C2H2
C2H4
C2H4
Time 0 1h 2h
Although the reduction of acetylene is taken as strong proof
of N2 fixation, definitive proof requires an isotope of nitrogen,
15
N2, as a tracer. If a culture or natural sample is enriched with
15
N2 and incubated, the production of 15NH3 is firm evidence of
nitrogen fixation. Nevertheless, acetylene reduction is a more
rapid and sensitive method for measuring N2 fixation and can
easily be used in laboratory studies of pure cultures or ecologi-
cal studies of nitrogen-fixing bacteria directly in their habitat.
To do this, a sample, which may be soil, water, or a culture, is
incubated in a vessel with HC ‚ CH, and the gas phase is later
analyzed by gas chromatography for the presence of H2C “ CH2
(Figure 3.34).
MiniQuiz
• Write a balanced equation for the reaction catalyzed by
nitrogenase.
• What is FeMo-co and what does it do?
• How is acetylene useful in studies of nitrogen fixation?
be grown in liquid or solid culture media, and pure cultures
can be maintained if aseptic technique is practiced.
3.3 • All microorganisms conserve energy from either the
oxidation of chemicals or from light. Chemoorganotrophs
use organic chemicals as their electron donors, while
chemolithotrophs use inorganic chemicals. Phototrophic
organisms convert light energy into chemical energy
(ATP) and include both the oxygenic and anoxygenic
phototrophs.