Researchers in ORNL's Environmental Sciences
Division (ESD) are seeking to understand the genetic structure, functions,
regulatory networks, and mechanisms of bacteria that might help keep
toxic metals out of the environment. To do this, they are integrating
genomic, biochemical, and physiological approaches to explore whole
genome sequence information. One of the genomic technologies being used
by ESD researchers is DNA array, or DNA microchip, technology. Basically,
genes are laid down in rows and columns on a glass slide in the order
in which they once existed in, say, a microorganism.
"We are using this
exciting new technology to study gene function and gene regulation in
bacteria capable of reducing metals," says Jizhong Zhou, an ESD researcher.
"Such bacteria may be useful for bioremediation of the Department of
Energy's waste sites." DNA microarray technology is also being developed
and used by Ken Beattie, Mitch Doktycz, and other researchers in ORNL's
Life Sciences Division engaged in functional genomics research and development.
Zhou, Tony Palumbo, and other ESD researchers are studying Shewanella
oneidensis MR-1, a group of bacteria that are found virtually anywhere,
including in water and soil, and at sites of infection. Just as humans
breathe in oxygen, these bacteria respire oxygen, nitrates, cobalt,
chromium, and uranium (U) to obtain energy. They move electrons from
each food molecule (e.g., organic compounds), changing the charge state
and the state of the respired material (e.g., U ions) on which they
dump electrons.
For example, when a Shewanella bacterium respires a
soluble U6+ ion in contaminated streams, it dumps two electrons on it
to create an insoluble U4+ ion, which sinks to the sediments. DOE is
interested in bacteria with this capability because they could help
keep water-borne uranium from leaving DOE sites.
About 99% of this bacterium's
sequence is known, thanks to work by the Institute for Genomic Research
(TIGR) in Rockville, Maryland, funded by DOE's Office of Biological
and Environmental Research. Using ESD's new genomic microarray equipment,
the ORNL researchers will lay down some 6 million bases and 6000 genes
from the Shewanella oneidensis bacterium on the gene chip. In the meantime,
while sequencing is being completed, the ORNL researchers are also working
with smaller subsets of the genes.
The goal of ORNL's first DOE-funded
project on microbial functional genomics and ecology is to identify
the genes in the bacteria that are responsible for metal reduction,
especially reduction of uranium to convert soluble ions into insoluble
ones. This work is being done in collaboration with the California Institute
of Technology and Michigan State University.
In a pilot study, ESD researchers
optimized the hybridization conditions with a partial gene chip containing
about 200 genes. "We are among the first groups to show that microarray
technology can be used to analyze gene expression in bacteria for environmental
applications," Zhou says. "We are using it to help us define gene functions
and gene regulatory mechanisms in microorganisms."
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The red dots on the gene chip represent bacterial genes expressing themselves in the presence of oxygen. The green dots indicate bacterial genes expressing themselves when exposed to metal. The yellow dots denote genes expressed by bacteria under both sets of conditions.
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To determine which bacterial genes are important
in the respiration of metals, ESD researchers grew the bacteria in oxygen
and on metals. They exposed some Shewanella bacteria to oxygen
only and stained them with a red fluorescent dye. They exposed other
Shewanella bacteria to metals only and dyed them green. Some
genes turned on in the presence of oxygen and other genes turned on
when exposed to metal. These expressed genes produce messenger RNA,
which carries the dye.
"Working with 200 selected genes,
we compared microarrays to see which genes were turned on under different
conditions," Zhou says. "Once we found out which of those genes turn
on in response to the presence of metal, we deleted two of these metal-reduction
genes from this bacterium to generate mutants using the genetic vectors
we recently developed. We will expose the mutant to uranium to see if
it still reduces uranium. If not, that suggests those missing genes
are important to uranium reduction."
More research will be needed to determine the precise
function of each metal-reducing gene by examining the role its protein
product plays in the organism. To carry out this research, ESD researchers
are collaborating with a group at Argonne National Laboratory. There
various tools are being used to find differences in proteins produced
by normal and mutant Shewanella bacteria and to characterize
enzymes that regulate gene expression by turning some genes on and others
off through mutagenesis.
ESD researchers have also used
DNA microarray technology to analyze the community structure and activity
of bacteria of environmental importance. "We are the first group," says
Zhou, "to demonstrate that DNA microarray technology works well for
analyzing complicated environmental samples."
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