Studying bacterial genomes
In 1956 a new microbe was discovered in a can of spoiled ground beef thought to have
been sterilized by radiation. The bacterium was named Deinococcus radiodurans
because it can survive doses of radiation thousands of times higher than would kill most
organisms, including humans. The Department of Energy is interested in studying D.
radiodurans because of its efficiency in repairing its own radiation damage and because
it can reduce, or add electrons to, uranium, iron, chromium, and technetium. Thus, the
bacterium might be a strong candidate for remediating mixed wastes—combinations of
radioactive materials and toxic metals—found at DOE sites. For example, it could be
used to convert uranium in storage ponds from a soluble to an insoluble form so that it
sinks into the sediments instead of remaining in water that might flow off-site.
ORNL researchers are interested in knowing more about the
bacterium's genes that repair radiation-induced damage to its
DNA. Knowing how these DNA repair genes work together in
a network could help scientists better understand how living
organisms evolved and how microbes that may have lived on
Mars or other extraterrestrial sites tolerated extreme radiation
environments. This knowledge may lead to gene therapy in
which DNA repair genes are inserted into the body to prevent
radiation-induced cancer or to treat the disease. DNA repair
genes discovered in D. radiodurans could be transferred to
other bacteria that reduce toxic metals, to make them better
able to survive radiation as they treat mixed wastes.
Thanks to advanced technologies such as microarrays and mass
spectrometry and internal funding from the Laboratory
Directed Research and Development Program, six ORNL
researchers are improving the understanding of D.
radiodurans. One of their goals is to identify which genes in D.
radiodurans are expressed during exposure to high levels of
radiation, resulting in the production of DNA repair proteins.
Another goal is to discover the regulatory genes that control
other genes involved in radiation resistance. The researchers
are Jizhong Zhou, Bob Burlage, and postdoctoral scientists
Dorothea Thompson and Alex Beliaev, all in the Environmental
Sciences Division (ESD); Bob Hettich of the Chemical and Analytical Sciences Division,
and Randy Hobbs of the Research Reactors Division.
To identify the D. radiodurans genes involved in DNA repair, Zhou and his ESD
colleagues use microarrays, also known as gene chips. (See Complex Biological
Systems in Mice.) They will place the entire complement of genes from D. radiodurans
on the same gene chip and then use the chip to identify differences in global gene
expression between D. radiodurans cells exposed to high radiation and those not so
exposed. (The expressed genes produce messenger RNAs that can be detected.) Genes
expressed only in the irradiated bacteria are likely to play specific roles in DNA repair.
The ORNL researchers will then develop genetic vectors
for generating deletion mutants—bacteria from which
key regulatory genes involved in radiation resistance are
removed. These regulatory genes code for proteins that
may activate other genes involved in DNA repair. If the
mutant bacteria die when exposed to high radiation
doses, then the results of this experiment suggest that the
deleted genes play a key role in radiation resistance.
To understand the cellular response of the D.
radiodurans bacteria to radiation, it is necessary to
identify protein expression profiles and protein-DNA
interactions. Zhou and his colleagues will culture the
bacteria under both non-irradiated and irradiated
conditions. The bacteria cells then will be lysed and the
proteins will be extracted, purified, and finally
characterized by Hettich, using high-resolution
electrospray mass spectrometry.
"The capability of this technique for accurate
measurement of the protein masses, as well as
identification of sequence tags (short amino-acid sections
from the protein), provides unparalleled information for
unambiguous protein identification," Hettich says. Such
information will be compared to that obtained from more
conventional two-dimensional gel electrophoresis. The data should provide insights
about how the microbe's proteome—its total protein profile—is altered in response to
DNA damage, providing important details about gene activity.
"Knowing when and where a gene is expressed
often provides a strong clue to its biological
function," Zhou says. "Gene expression
patterns and protein profiles in a cell can
provide detailed information about its state."
The researchers' goal is to identify pathways of
interactions between DNA and proteins that
result in DNA repair of radiation damage.
Another group of researchers interested in D.
radiodurans is the Computational Biology
Section of ORNL's Life Sciences Division.
Using gene-recognition algorithms and other
tools, Frank Larimer and his colleagues analyze
completed DNA sequences of microbes and compare the identity and order of these
DNA bases with known sequences in databases. They identify the known genes
contained in these microbial genome sequences and predict the amino-acid sequences,
molecular sizes, and possible functions of the proteins these genes encode. They also
predict the makeup and functions of unknown genes and proteins.
"We have studied the completed sequence of D. radiodurans and can recognize many
of its DNA repair genes," Larimer says. "We inferred the function of their proteins
based on their degree of similarity with proteins in the database associated with repair
"The majority view is that there is a 'eureka' DNA repair gene to explain why D.
radiodurans is so resistant to high levels of radiation. I take the minority view that,
although the bacterium's DNA repair genes look like the repair genes in other organisms,
D. radiodurans does the repair job more efficiently. It is extremely adept at aligning and
recombining its broken chromosome parts in the proper order."
D. radiodurans is one of the millions of microbes that have been evolving on the earth
over the past 3.8 billion years. Many of the 1% of the microbes known to humans
survive and thrive in extremes of radiation, heat, cold, pressure, salinity, and acidity,
often where no other life forms could exist. DOE is interested in identifying and
harnessing the talents of some of these microbes for cleaning up hazardous wastes,
producing energy (e.g., methane), and sequestering carbon.
Since its establishment in 1994, DOE's Microbial Genome Program has focused on
determining the sequences of the genomes of selected bacteria and other microbes that
do not cause disease. The ORNL group provides computational and other bioinformatic
analysis of microbial sequences obtained by DOE's Joint Genome Institute (Lawrence
Berkeley, Lawrence Livermore, and Los Alamos national laboratories) at their
production facility in Walnut Creek, California. Larimer and his colleagues "annotate"
these microbial DNA sequences and others provided by academic groups. That is, they
add to the database "biological footnotes" about the genes, the coding and noncoding
regions of the genes, and the possible structure and function of the proteins encoded by
individual genes. For each microbe, the researchers translate roughly 2 billion DNA
bases into meaningful information.
Using the IBM RS/6000 SP supercomputer at DOE's Center for Computational
Sciences at ORNL, the group runs GrailEXP, the version of the ORNL-developed Gene
Recognition and Analysis Internet Link written for parallel supercomputers, along with
other gene modeling programs to determine the correct DNA sequences in the microbial
genes that code for proteins. "Ninety percent of bacterial DNA encodes protein
compared with less than 2% for humans," Larimer says. "Microbes are very efficient
this way. So we try to distinguish between what portions of the bacterial sequence are
genes and what is a statistical anomaly."
The typical bacterium has 2000 genes (each microbial gene has about 900 bases). The
ORNL group tries to find gene signatures in bacteria—unique combinations of genes
that indicate each bacterium's identity.
"What is startling is that we can assign functions for 50% of the genes in a microbial
gene, which might be 1000 genes. The other half of the genes have no known function.
Half of these genes are unique—we've never seen them before. There is a lot of genetic
biodiversity in microbes. Microbes acquire sets of genes from each other and throw
away large blocks of genes they don't need as they evolve."
To date, 50 microbial genes have been completely sequenced. Larimer's group has been
annotating them and working with 200 ongoing microbe sequencing projects. DOE's
goal is to sequence 250 microbes that could be useful for its energy and environmental
In addition to D. radiodurans, some of the bacteria being analyzed are particularly
fascinating to Larimer. One is Prochlorococcus marinus, a blue-green marine alga that
is the most abundant organism on earth in number and mass. These ocean algae, whose
individual cells are each less than 1 micron in diameter, together fix more carbon dioxide
than all of the terrestrial biosphere. Prochlorococcus marinus is found 100 meters deep
in the ocean yet it absorbs sufficient blue-green photons penetrating to that depth to get
enough energy to fix carbon.
Another interesting microbe that fixes carbon dioxide is
Nitrosomonas europa, which gets its energy not from sunlight
but from oxidizing ammonia. Using ammonia fertilizer, it is
responsible for putting nitrogen into the soil, making it fertile
Zhou is fascinated by gene-chip and mass spectrometry
studies of the metal-reducing bacterium Shewanella
oneidensis. "We compared gene expression patterns in
Escherichia coli bacteria and a mutant of Shewanella
oneidensis that we generated," Zhou says. "We found that
the functions of about 75% of their genes are similar. But
there are differences. In E. coli one gene activates nitrate
reduction, but the counterpart gene in mutant Shewanella
oneidensi represses nitrate reduction.
"Using bioinformatics, scientists often assign gene functions
based on the similarity of sequences of two different bacteria.
This approach may not correctly define all gene functions. To
get the complete picture, bioinformatics must be
supplemented by experimental approaches that analyze
expression in microbes, using microarrays and mass