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DR. STEPHEN GIOVANNONI - Oregon State University, Corvallis
The Research: Culturing the Ocean's Microbial Diversity
Like a number of researchers described in this marine biotech website, one of
Stephen Giovannoni's key research goals is to discover new bacteria. But rather
than pharmaceutical, industrial, or agricultural potential, his interest in the
bacteria is in identifying and better understanding those species that are most
important in oceanic geochemical processes. More specifically, he focuses on
oceanic bacteria, mostly from the open ocean, that are involved in the carbon
cycle. This is the process by which carbon dioxide from the atmosphere is taken
up by plants and bacteria during photosynthesis and cycled and recycled by other
organisms in a complex web until it is either permanently buried or re-released
as carbon dioxide to the atmosphere. Oceanic bacteria are in fact responsible for
a significant percentage of the photosynthesis in the ocean, and most of the
carbon recycling.
Despite his carbon cycle focus, Giovannoni's work has made important contributions
to marine biotechnology because the techniques he has developed to study genetic
material from bacteria and to improve culturing techniques have been vital to the
field and are being used by a number of other groups, including Diversa,
which has licensed some of the technologies
Giovannoni's laboratory has developed.
 - VIDEO CLIP 1: "Marine Microbiology...With A Twist"
 Probing the Seas
An important first step in identifying a new bacterial species, especially if you want to
quantify its concentration, is coming up with a way to recognize it. "If it's just a cell
bobbing around in the water, they all look alike and you'll have no idea it's there," says
Giovannoni. One widely used means of recognition is DNA probes. Developing probes begins
with the collection and sequencing of DNA from bacteria found in samples. If unique genes
are identified in this mix, they typically indicate a novel species. Probes are gene
sequence snippets that match one of these unique genes. This DNA snippet is typically
denatured to make single strands and a fluorescent molecule is added as or label that
will fluoresce under the proper conditions to allow the probe to be spotted under
a microscope. The probe can then be added to a sample fixed in formaldehyde, where
it diffuses into the bacterial cells and binds only with its target gene. When the
sample is viewed under a microscope, the bound probes are visible due to the fluorescent
label, allowing the number of a particular species of bacteria to be counted.
- VIDEO CLIP 2: "Hunting Water Column Micobes With DNA Probes"
Rulers of the Sea
A good example of the Giovannoni lab's cloning work, and its benefits, is the story of
a bacterial clade known as SAR-11. It was discovered back in 1990 through cloning and
sequencing but its abundance and importance in the oceans had remained controversial.
Giovannoni's team developed a SAR-11 probe and found that during the summer, fully 50%,
and on average throughout the year about 25%, of the bacterial cells in the Atlantic
is SAR-11. Multiplying the number of SAR-11 cells by the mass of a cell suggests that
the bacteria's biomass is about equal to the total weight of all the fish in the seas.
"That's a lot of carbon," says Giovannoni, putting it mildly. A second important factor
in considering SAR-11's role in the carbon cycle is that small bacterial cells are in
a constant state of flux due to attack by viruses and grazing by small flagellates.
That coupled with the cells' own growth, makes for a massive turnover of carbon in
the oceans and, hence, a critical component in the overall oceanic carbon cycle. By
extension, that means that SAR-11 has a major role to play in the global carbon cycle,
which determines, among other things, the rate at which carbon dioxide builds up in
the atmosphere contributing to global climate change.
Other related research has led to the Giovannoni group's identification of the bacteria
chiefly responsible for the critical process of consuming the dissolved organic carbon
that is injected into the deep sea each year as winter storms churn the Atlantic. This
turnover sends blooms of phytoplankton into the depths that are ultimately broken down
to contribute large quantities of dissolved organic carbon to the food web.
Why Microbiologists Needed More Culture
For many years, researchers have struggled with the challenges of culturing bacteria from ocean
and other environments in the laboratory as conventional techniques only allowed a tiny fraction
of them to be grown. "If you look back fifteen or twenty years ago, microbiologists were mainly
working with things that were easy to grow, and they were largely unaware of most of the organisms
in nature," says Giovannoni. As more genetic research was performed on microbes, there was a major
change in thinking among microbiologists, he says, because they became aware that most microbial
diversity was organisms never grown in the laboratory. By extension, this meant that organisms
being studied in the laboratory were often poor models for environmental processes. Species
growing in the laboratory typically had some features that enabled them to adapt very well
to laboratory conditions, but that didn't necessarily make them very successful in the natural
world.
Armed with databases of gene sequences, researchers began to deliberately hunt for the
organisms they could prove were abundant in nature, and many of them turned out to be very
difficult to culture, highlighting the need for innovative new techniques. Overall, Giovannoni
believes one of the most important changes in microbiology since the 1990s has been a collective
change in attitude toward recognizing the need to culture the organisms most prevalent in nature.
As the importance of these organisms became apparent, researchers in many cases first turned to
study techniques that did not require cultivation, namely widespread sequencing of DNA out of
nature in attempts to determine what the microorganisms did. However, attempts to get whole
genomes in order to reconstruct the metabolism of an organism from gene sequences alone largely
failed. "So people are seeing how valuable it is to have a culture," says Giovannoni, because
then you can get a complete genome sequence. "To really make good scientific progress you need
all the parts, all the research approaches working together," he says, " You need genomics, you
need to have cells in culture in laboratories where experiments can be performed on them. And
you need to have natural observations that can tell you what sort of experiments are going to
be important and what you need to know in order to interpret organisms' impacts on natural
processes."
How Microbiologists Can Get More Culture
Giovannoni's group has worked extensively to extend culturing capabilities to more microbial
species. One approach they have taken is to closely analyze the environment in which marine
bacteria normally grow. For instance, Giovannoni, among other researchers, began to consider
that concentrations of phosphorus, dissolved organic carbon, and other critical components
are typically very low in ocean waters. In contrast, the media typically used to culture
microorganisms often had as much as 1,000 times higher concentrations of these components.
Recognizing this problem, groups began working to culture collected bacteria in seawater
instead of standard media. "We call it catch-and-release," says Giovannoni, "you catch it
in nature and let it go in seawater, and many cells will start to replicate.
A key contribution of the Giovannoni group has been to extend such work a step further to
develop techniques for applying this catch-and-release idea to culturing bacteria in tiny
wells on microtiter dishes. This was a challenging endeavor, because a typical well was
about 1 milliliter, which is enough volume to hold tens or hundreds of thousands of bacterial
cells, which the group needed to count. They were able to accomplish this, though, which has
led to the culturing and discovery of many new organisms. Doing that depended mainly on the
use of a blotter that can take cells and put them on a membrane that is then mounted on
a microscope for cell counting. This vastly accelerated the speed at which the team could
count the cells. Some, but not all, of the work they have been able to automate. A pipetting
robot is used to perform the blotting onto membranes that the team refers to as cell arrays,
and a Laser Scanning Cytometer performs actual cell counting in the various sectors of the
array, though it still needs some human direction regarding which spots should be counted.
The team is also experimenting with using a device called a flow cytometer to sort cells.
Diversa has licensed and is now using parts of the high-throughput technologies the Giovannoni
group has developed and is now using them to isolate new bacteria and screen them for the
pharmaceutical and other applications. "We haven't been involved in drug screening ourselves,"
says Giovannoni, "but we're aware that the technology we developed is good for getting new
organisms and probably lots of people are going to use it to search for new drugs."
Another aid to increasing culturing success has been careful analysis of the DNA of target
species. For instance, Giovannoni and others worked in collaboration with Diversa to sequence
the entire SAR-11 genome. This allowed them to, among other advances, determine that SAR-11's
genome includes the biosynthetic pathway for the production of the essential amino acid
histidine, meaning that it does not need to get histidine from growth media. "With that
sort of metabolic reconstruction it's been possible for us to get some idea of what sort
of organic compounds these organisms use out of seawater.
- VIDEO CLIP 3: "Hardware Roundup: Tools of the Trade"
Why Did the Oceanographer Climb the Volcano
Most of the Giovannoni group's fieldwork is conducted in Bermuda, because it's location about
700 miles east of North Carolina puts it on the western boundary of the Sargasso Sea. This
region of the ocean is known as a mid-ocean gyre, where water is very clean and overall
productivity low. Waters near Bermuda are therefore representative of the open ocean and
suited to the processes the team is working to understand. Open water work allows the team
to correlate the properties of cells they study in the laboratory with large-scale processes
they can observe at sea.
The group also worked for several years on Crater Lake, which fills the crater of an extinct
volcano now known as Mount Mazama at Oregon's Crater Lake National Park. The lake was chosen
as a study site because its exceptional clarity and depths up to 1,932 feet lead to the
mimicking of many processes that occur in the ocean.
Crater Lake's elevation is about 6,000 feet and to get there, you have to drive up to the
top of the mountain then hike down to the lake. "That's a pretty steep set of switchback
trails, so most of the gear was lugged down by hand," says Giovannoni. The park service
keeps several boatsÑinitially brought in by Chinook helicopterÑon the lake, which are available
for research.
"It was pretty exciting," says Giovannoni, "we discovered a lot of interesting new organisms
there."
- VIDEO CLIP 4: "Field Work in Bermuda"
- VIDEO CLIP 5: "Away From the Sea: Research at Crater Lake"
Education: Seemingly Random Steps to the Goal
Heading to college, Stephen Giovannoni wanted to be a marine biologist and was interested in
Scripps Institution of Oceanography, but since they did not have an undergraduate program,
he settled for the nearby University of California, San Diego. There he got what he describes
as a "hardcore biology" degree. "I never saw anything that was alive until I got my first job,"
he says, which, oddly enough, was working for a physicist, who was studying bacterial
photosynthesis. Giovannoni worked to isolate proteins from the bacteria for physical
measurements, but says, "that turned me into a microbiologist."
He then went to Boston University where he got a master's degree with Lynn Margulis,
whom he describes as "a wonderfully stimulating advisor." From there he went to Woods Hole
Oceanographic Institution to work as a teaching assistant for a marine microbiology class.
He remembers going fishing with the course professor and explaining that he wanted to be
a marine microbiologist, and the professor told him to go study thermophiles, because it
was the late seventies and hydrothermal vents that harbor thermophiles had just been
discovered and they were all the research rage in oceanography, meaning hiring in the
area was likely. So, Giovannoni headed to the University of Oregon, where he received
his Ph.D. and later his current faculty position, to work on bacteria from Yellowstone
hot springs. He couldn't figure out what some of the species he isolated were, so he
contacted Carl Woese
and then Norm Pace, pioneers in
the molecular
microbial field. This led him to become involved in the development of the emerging field of
microbial ecology, which brought him back full circle to marine systems. "So, sort of miraculously,
in a series of seemingly random steps," he says, "I ended up at my original goal of being
a marine biologist."
- VIDEO CLIP 6: "Tapping Marine Microbial Diversity"
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