discovering dual cyanobacteria metabolism

8/14/2019 Discovering Dual Cyanobacteria Metabolism

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Octopus Spring in YellowstoneNational Park is one of themost inhospitable places on theplanet. Yet, life ourishes 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 efuent channels of the hot springs attemperatures ranging from under 50°C to justabove 70°C are Synechococcus. These single-celled cyanobacteria are part of a complex anddiverse microbial ecosystem forming mat-likestructures on the surface of the springs.

This past summer, Stanford ScientistsDevaki Bhaya, Arthur Grossman, and AnneSoisig Steunou from the Carnegie Institution’sDepartment of Plant Biology collaborated with

Ancient Survivors in a

Hostile Environment Evolve

Two Radically DifferentMetabolic Processes by Benjamin

Tran

genomicists, population biologists,evolutionary biologists and physiologistsfrom Maryland, Montana, Connecticutand Denmark to investigate the question: What are the interactions of differentspecies that are required for survival in

microbial communities?

Residents o the MatThe microbial mats of Octopus

Spring are highly organized ecosystems where different organisms performdifferent functions in the community. Synechococcus live in the top 1 mm layerof the microbial mat and are the primary producers, using sunlight to convertcarbon dioxide and water into oxygenand energy-rich sugar. Cyanobacteria are believed to have evolved about 3 billion

years ago, making them the oldest knownmicroorganisms on the earth to performphotosynthesis.

Other residents of the mat includeheterotrophs—organisms that cannot

produce their own food, and severaltypes of green non-sulfur bacteriaknown as photoheterotrophs

– organisms that use light as anenergy source but can not convertcarbon dioxide into energy. As a result of Synechococcus

photosynthesis, the other

organisms of the mat supplied

Cross section o gelatinous microbial mat

shows the complex structure o the ecosystem.

At the top centimeter lives Synechococcus

cyanobacteria, one o the many producers;

below this level resides other heterotrophs,

including Acidobacterium and several green

non-sulur bacteria, such as Roseifexus and

Chlorofexus (photoheterotrophs).

Octopus Springs in Yellowstone

National Park is seemingly

unwelcoming to lie, even in

winter.

A r t h u r G r o s s m a n a n d

A n n e S o i s i g S t e u n o u

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volume iv layout design:

This cartoon o the nitrogenase machine depicts the process or

nitrogen fxation. To produce two molecules o NH3

and one H2

molecule, the machine takes in, among other things, 16 units o

ATP energy currency.

with carbon energy in the form of sugar, and oxygen which they need for respiration.

Nitrogen FixNitrogen is one of the nutrients required by all organisms for

the building blocks of nucleic acids and proteins. Animals (andcarnivorous plants) can directly consume nitrogen by eating foodsthat contain it, whereas plants and certain bacteria must reduce or“x” atmospheric N

2into larger carbon–containing compounds

in order to assimilate it into their systems. The microbial matreceives some nitrogen and other required nutrients from organicmaterial that falls into and decomposes in the steamy waters.However, it needs far more nitrogen than what is supplied by thesprings.

N2

xation is a problem for photosynthetic cells, since oxygenproduced during photosynthesis inhibits the function of theenzyme complex that xes N

2. Because Synechococcus performs

photosynthesis, it was widely dismissed as a candidate for N 2

xation.

When Night Falls While investigating the extent of diversity within microbial

mats and the interactions between the various members of themat environment, the group discovered a pronounced duality in the metabolic nature of the cyanobacteria that populate thesemats. During the day, Synechococcus proceeds normally withphotosynthesis, xing inorganic carbon and releasing eight timesthe amount of oxygen needed to fully saturate the mat.

However, as the intensity of light decreases from 1000 toaround 50 –100 µmol photons per square meter per second, thecyanobacteria stop photosynthesis and the organisms in the mat

consume the oxygen more quickly than it can be produced. Themat quickly becomes depleted of oxygen.

So what does Synechococcus do to survive? The group lookedat its gene expression to nd out. Surprisingly, they discoveredthat since Synechococcuscannot spatially separate photosynthesisand N

2xation, it solves the mat’s photosynthetic and nitrogen-

xing needs instead by temporally separating these tasks. Theresearchers found that the Synechococcus genome contains a nif gene cluster - nif H, nif K, nif J, nif F, and nif D – which encodes

for nitrogenase, the enzyme needed for N2

xation. Previous work suggested that Synechococcus growing at the high temperaturesof the hot spring mats were not able to x N

2. However, Steunou’s

group found that while there is no transcription of nif genesduring 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 tox atmospheric nitrogen (N

2) into ammonia (NH

3), the form of

nitrogen they require for cell growth.

A Question o GeneticsJohn Heidelberg of The Institute for Genomic Research

(TIGR) sequenced genomes of two different Synechococcusstrains, OS-A which can live up to 65°C and OS-B’ which canlive up to 60°C. While these two genomes had similar gene

Loren Alegria

content, the arrangement of the individual genes appearedto be “scrambled.”

According to Steunou, “Analysis of orthologous genesrevealed 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.” Thisstartling difference in genome architecture raises questionsabout the evolution of Synechococcus ecotypes and therelationships of these ecotypes to other microorganisms inthe mat community.

“It was as if at least one of the

genomes shattered, and the genes

were put back together in an almost

random order.”

The plot o the light intensity as time progresses shows that as intensity alls

around 50 –100 µmol·m-2·s-1, the ni genes or nitrogen fxation “turn on.”

Arthur Grossman and Anne Soisig Steunou

nif K

nif D

T1 T2 T3 T4 T5 T6

Noon 2:00 4:00 6:00 8:00 10:00

time

1200

1000

800

600

400

200

0

l i g h t i n t e n s i t y

( μ m o l p h o t o n m - 2 s

e c - 1 )

{ {Light Dark

T1

T2

T3 T4 T5 T6

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The Nitrogenase Machine



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Testing the HypothesisThe group’s method of testing for the presence and

activity of the nitrogenase enzyme was based on a commonprocedure that has been used by many laboratories tomeasure nitrogenase. Nitrogenase can be assayed for itsability to reduce acetylene (C

2H

2) to ethylene (C

2H

4), which

is readily detectable by gas chromatography (GC).“We took a cork bore sample of the mat, placed it in a

test tube with acetylene, sealed it, and kept in it the efuentchannel,” Steunou describes. “After a couple of hours westopped the reaction by the addition of formaldehydeand subsequently measured the amount of ethylene andacetylene in the vial using GC.” This provided a quantitativemeasure of nitrogenase activity in the mat.

The Metabolic SwitchGene expression of Synechococcus during the day and

night are summarized here:Transcripts from photosynthesis (cpcF, cpcE, psaB,

and psbB) and respiration (cox A and cyd A) genes declinein the evening. In contrast, transcripts encoding enzymesthat may participate in fermentation fall into two categories.Some (ldh, pdhB,ald , andack A) decrease in the evening, whereas others ( pf B, pf A, adhE, and acs) increase atthe end of the day and remain high throughout the night.Transcripts of nif genes are expressed only at the end of theday when the mat becomes anoxic.

Fermentation – it’s not just or beer!The energy cost of running what Steunou calls the

“Nitrogenase Machine” is high: to produce two molecules of ammonia and one molecule of hydrogen gas requires 16 ATPmolecules (adenosine triphosphate, the cellular energy currency). Where does all this energy come from?

When photosynthesis shuts down at night, the mat becomes

oxygen starved, reducing the expression of the respirationgenes cox A and cyd A. Respiration is an efcient energy-generating pathway that requires oxygen to release the energy stored in sugars. With respiration turned down, the cells mustrely on fermentation, a pathway that can proceed withoutoxygen. However, fermentation produces little energy relative tophotosynthesis and respiration, and so nitrogenase activity duringthe evening is generally low.

During the rst hour and a half of sunrise, the mats remainanaerobic at the same time that photosynthesis just begins tofunction. It is at this time that ATP availability increases, makingmore energy available for N

2xation. Thus, as Grossman states,

“Much of the N2

xation seems to be driven by the generationof energy by photosynthetic electron transport.” Even so,once photosynthetically generated O

2accumulates in the mat,

nitrogenase activity is inhibited.To sum it up, during the early morning, the rate of respiration

in the mat is fast enough to prevent O2

accumulation and thedenaturation of the nitrogenase complex. As the morning wearson, the light levels increase and O

2accumulates, which causes

destruction of nitrogenase activity and the switching off of thenitrogenase genes.

The Future o Microbial Mat ResearchThese ndings provide insight into the assimilation and

utilization of nitrogen in the hot spring environment and help todene the metabolic processes and their interactions that shapethis community of organisms. Grossman and Steunou agree that

much work still needs to be done in order to fully understand thesecomplex ecosystems. Future research projects will investigate thegenetic relationships between cyanobacteria and other microbesin the mats as well as the environmental parameters that triggermetabolic 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.

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“…uture research projects will

investigate the genetic relationships

between cyanobacteria and other

microbes in the mats and the

environmental parameters that trigger

metabolic switching”

N P S P h o t o B o b L i n d s t r o m

Testing the microbial mats

discovering dual cyanobacteria metabolism

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