Ancient Survivors in a
biology
+
medicine
Hostile Environment Evolve
Two Radically Different
Metabolic Processes
O
ctopus Spring in Yellowstone
National Park is one of the
most inhospitable places on the
planet. Yet, life flourishes there at
temperatures that reach nearly 90°C (194° F),
close to the temperature of the boiling water
that emanates from the source of the spring.
Living in effluent channels of the hot springs at
temperatures ranging from under 50°C to just
above 70°C are Synechococcus. These single-
celled cyanobacteria are part of a complex and
diverse microbial ecosystem forming mat-like
structures on the surface of the springs. shows the complex structure of the ecosystem.
This past summer, Stanford Scientists
Devaki Bhaya, Arthur Grossman, and Anne
Soisig Steunou from the Carnegie Institution’s
Department of Plant Biology collaborated with
22 stanford scientific
by Benjamin
Tran
genomicists, population biologists,
evolutionary biologists and physiologists
from Maryland, Montana, Connecticut
Arthur Grossman and Anne Soisig Steunou
and Denmark to investigate the question:
What are the interactions of different
species that are required for survival in
microbial communities?
Residents of the Mat
The microbial mats of Octopus
Spring are highly organized ecosystems
Cross section of gelatinous microbial mat where different organisms perform
different functions in the community.
At the top centimeter lives Synechococcus Synechococcus live in the top 1 mm layer
cyanobacteria, one of the many producers;
of the microbial mat and are the primary
below this level resides other heterotrophs,
including Acidobacterium and several green producers, using sunlight to convert
non-sulfur bacteria, such as Roseiflexus and carbon dioxide and water into oxygen
Chloroflexus (photoheterotrophs). and energy-rich sugar. Cyanobacteria are
believed to have evolved about 3 billion
years ago, making them the oldest known
microorganisms on the earth to perform
photosynthesis.
Other residents of the mat include
heterotrophs—organisms that cannot
produce their own food, and several
types of green non-sulfur bacteria
known as photoheterotrophs
– organisms that use light as an
energy source but can not convert
carbon dioxide into energy.
As a result of Synechococcus
photosynthesis, the other
organisms of the mat supplied
Octopus Springs in Yellowstone
National Park is seemingly
unwelcoming to life, even in
winter.
 biology
with carbon energy in the form of sugar, and oxygen which they This cartoon of the nitrogenase machine depicts the process for +
need for respiration. nitrogen fixation. To produce two molecules of NH3 and one H2 medicine
molecule, the machine takes in, among other things, 16 units of
ATP energy currency.
Nitrogen Fix
Nitrogen is one of the nutrients required by all organisms for
the building blocks of nucleic acids and proteins. Animals (and
carnivorous plants) can directly consume nitrogen by eating foods
that contain it, whereas plants and certain bacteria must reduce or
“fix” atmospheric N2 into larger carbon–containing compounds
in order to assimilate it into their systems. The microbial mat
receives some nitrogen and other required nutrients from organic
material that falls into and decomposes in the steamy waters.
However, it needs far more nitrogen than what is supplied by the
springs.
N2 fixation is a problem for photosynthetic cells, since oxygen
produced during photosynthesis inhibits the function of the
enzyme complex that fixes N2. Because Synechococcus performs The Nitrogenase Machine
photosynthesis, it was widely dismissed as a candidate for N2
fixation. T1 T2 T3 T4 T5 T6

When Night Falls

While investigating the extent of diversity within microbial nifK
mats and the interactions between the various members of the
mat environment, the group discovered a pronounced duality nifD
in the metabolic nature of the cyanobacteria that populate these Arthur Grossman and Anne Soisig Steunou
mats. During the day, Synechococcus proceeds normally with
photosynthesis, fixing inorganic carbon and releasing eight times 1200 T1
(μmol photon m-2 sec-1)

the amount of oxygen needed to fully saturate the mat. 1000

light intensity

However, as the intensity of light decreases from 1000 to 800

T2
around 50 –100 µmol photons per square meter per second, the 600

cyanobacteria stop photosynthesis and the organisms in the mat 400

T3 T4 T5 T6
200
0
Noon 2:00 4:00 6:00 8:00 10:00
A Question of Genetics time
John Heidelberg of The Institute for Genomic Research

{
{
(TIGR) sequenced genomes of two different Synechococcus Light Dark
strains, OS-A which can live up to 65°C and OS-B’ which can
live up to 60°C. While these two genomes had similar gene The plot of the light intensity as time progresses shows that as intensity falls
around 50 –100 µmol·m-2·s-1, the nif genes for nitrogen fixation “turn on.”

“It was as if at least one of the
genomes shattered, and the genes
were put back together in an almost
random order.”
content, the arrangement of the individual genes appeared
to be “scrambled.”
According to Steunou, “Analysis of orthologous genes
revealed that the cyanobacteria were very similar in terms of
gene content.” However, as coauthor Grossman states, “It
was as if at least one of the genomes shattered, and the genes
were put back together in an almost random order.” This
startling difference in genome architecture raises questions
about the evolution of Synechococcus ecotypes and the
relationships of these ecotypes to other microorganisms in
the mat community.
consume the oxygen more quickly than it can be produced. The
mat quickly becomes depleted of oxygen.
So what does Synechococcus do to survive? The group looked
at its gene expression to find out. Surprisingly, they discovered
that since Synechococcus cannot spatially separate photosynthesis
and N2 fixation, it solves the mat’s photosynthetic and nitrogen-
fixing needs instead by temporally separating these tasks. The
researchers found that the Synechococcus genome contains a nif
gene cluster - nifH, nifK, nifJ, nifF, and nifD – which encodes
for nitrogenase, the enzyme needed for N2 fixation. Previous work
suggested that Synechococcus growing at the high temperatures
of the hot spring mats were not able to fix N2. However, Steunou’s
group found that while there is no transcription of nif genes
during the day, the absence of oxygen at night allows the nif
transcripts to accumulate and nitrogenase activity can be readily
measured. Under these conditions, the cyanobacteria are able to
fix atmospheric nitrogen (N2) into ammonia (NH3), the form of
nitrogen they require for cell growth.

layout design:Loren Alegria volume iv 

biology
+
medicine
Fermentation – it’s not just for beer!
The energy cost of running what Steunou calls the Testing the Hypothesis
“Nitrogenase Machine” is high: to produce two molecules of The group’s method of testing for the presence and
ammonia and one molecule of hydrogen gas requires 16 ATP activity of the nitrogenase enzyme was based on a common
molecules (adenosine triphosphate, the cellular energy currency). procedure that has been used by many laboratories to
Where does all this energy come from? measure nitrogenase. Nitrogenase can be assayed for its
When photosynthesis shuts down at night, the mat becomes ability to reduce acetylene (C2H2) to ethylene (C2H4), which
oxygen starved, reducing the expression of the respiration is readily detectable by gas chromatography (GC).
genes coxA and cydA. Respiration is an efficient energy- “We took a cork bore sample of the mat, placed it in a
generating pathway that requires oxygen to release the energy test tube with acetylene, sealed it, and kept in it the effluent
stored in sugars. With respiration turned down, the cells must channel,” Steunou describes. “After a couple of hours we
rely on fermentation, a pathway that can proceed without stopped the reaction by the addition of formaldehyde
oxygen. However, fermentation produces little energy relative to and subsequently measured the amount of ethylene and
photosynthesis and respiration, and so nitrogenase activity during acetylene in the vial using GC.” This provided a quantitative
the evening is generally low. measure of nitrogenase activity in the mat.
During the first hour and a half of sunrise, the mats remain
anaerobic at the same time that photosynthesis just begins to
function. It is at this time that ATP availability increases, making
more energy available for N2 fixation. Thus, as Grossman states,
“Much of the N2 fixation seems to be driven by the generation
of energy by photosynthetic electron transport.” Even so,
once photosynthetically generated O2 accumulates in the mat,
nitrogenase activity is inhibited.
To sum it up, during the early morning, the rate of respiration

“…future research projects will

investigate the genetic relationships
between cyanobacteria and other
microbes in the mats and the
environmental parameters that trigger
metabolic switching”

in the mat is fast enough to prevent O2 accumulation and the

denaturation of the nitrogenase complex. As the morning wears

NPS Photo Bob Lindstrom

on, the light levels increase and O2 accumulates, which causes
destruction of nitrogenase activity and the switching off of the
nitrogenase genes.
The Future of Microbial Mat Research
These findings provide insight into the assimilation and
utilization of nitrogen in the hot spring environment and help to
define the metabolic processes and their interactions that shape
this community of organisms. Grossman and Steunou agree that
much work still needs to be done in order to fully understand these
complex ecosystems. Future research projects will investigate the
genetic relationships between cyanobacteria and other microbes
in the mats as well as the environmental parameters that trigger
metabolic switching. S
Benjamin Tran is currently a freshman who is looking to major in
mechanical engineering or chemical engineering. He is an avid sudoku
enthusiast and enjoys playing tennis and working on the Stanford
Solar Car Project in his spare time. Special thanks to Arthur Grossman
and Anne Soisig Steunou for their help with this article.
Testing the microbial mats
The Metabolic Switch
Gene expression of Synechococcus during the day and
night are summarized here:
Transcripts from photosynthesis (cpcF, cpcE, psaB,
and psbB) and respiration (coxA and cydA) genes decline
in the evening. In contrast, transcripts encoding enzymes
that may participate in fermentation fall into two categories.
Some (ldh, pdhB, ald, and ackA) decrease in the evening,
whereas others (pflB, pflA, adhE, and acs) increase at
the end of the day and remain high throughout the night.
Transcripts of nif genes are expressed only at the end of the
day when the mat becomes anoxic.

24 stanford scientific