Visualizing a problem is often the key to seeing its solution. An INEEL research team is using a gene cloned from a northern pacific jellyfish to provide useful illumination. The gene — which codes for green fluorescent protein (GFP) — is being used as a molecular marker. The marked bacteria brightly fluoresce, making them easy to see using epifluorescent or confocal microscopy.

Conventional techniques for visualizing bacteria require preparation steps that are often toxic to the cells and can change bacterial surface properties. “We are working to create new, non-invasive tools for the environmental microbiologist,” said INEEL microbiologist and team leader Joni Barnes. “GFP tagging has great potential. It can be used for anything from studying the dynamics and distributions of microbial populations to developing biosensors.” (See sidebar about biosensors.)

GFP is widely used in the research community as a reporter gene for tracking cells and assessing metabolic activity in plants, animals, and bacteria. The gene is an excellent marker because the cells make the protein and generate fluorescence without researchers having to introduce other chemicals.

To mark environmental microbes with the GFP gene, Barnes and her team use plasmid cloning vectors. Plasmids — small, genetic elements that reside in bacteria and replicate independently from the chromosome — can be manipulated and modified in the laboratory to carry genes of interest that can then be inserted into host bacteria. These genes can provide the organism with specific traits, such as resistance to antibiotics or heavy metals.

The team constructed a plasmid that carries both the GFP gene and a gene for tetracycline resistance and can be inserted into a broad range of bacteria using chemical or electrical techniques. Then they grew the cells in the presence of the antibiotic tetracycline to identify clones that carried the GFP plasmid.

INEEL Researchers Begin Developing an Environmental Arsenic Biosensor An INEEL research team, led by molecular biochemist Frank Roberto, has been working on a biosensor for arsenic. Biosensors are devices that use biological molecules to detect other biological molecules or chemical substances. The results can be measured either qualitatively or quantitatively. If a biosensor monitoring technique was available, it could potentially reduce the high costs currently associated with arsenic monitoring. The team began by identifying microbes tolerant to arsenic. These microbes produce various protein enzymes that allow them to survive. Since their biochemical survival mechanism is genetically determined — their toxic-response genes are organized as operons, which are controlled by genetic trigger mechanisms called promoters — marking the location with a reporter gene offered a potential biosensor system. The team isolated the toxic-response genes for the bioavailable compounds arsenate and arsenite. Then they co-located the GFP reporter gene behind the promoter for arsenic resistance. When the microbes were exposed to arsenic, the biosynthesis of proteins was triggered, as well as the production of GFP with its characteristic glow. The team was able to detect submicrogram quantities of arsenate and arsenite compounds at concentrations relevant to public health concerns and current regulations. According to Roberto, their research effectively demonstrated that inexpensive, effective whole cell biosensors can be produced. “Biosensors may not replace accepted methods of instrumental analysis,” said Roberto. “But they have significant potential and may eventually offer a low-cost alternative. That alone is reason to continue this line of research.” Results of the team’s research were recently published in Talanta1, the international journal of pure and applied analytical chemistry. Contact: Frank Roberto (208) 526-1096 ffr@inel.gov Reference: Roberto, F.; J. Barnes; D. Bruhn. Evaluation of a GFP reporter gene construct for environmental arsenic detection. 2002, Talanta, 58, p.181-188.

Barnes is also interested in learning more about the research potential offered by other fluorescent proteins, including GFP variants that produce blue, cyan, and yellow fluorescent proteins and a red fluorescent protein recently isolated from sea anemones. The variants have different chemical properties, maturation rates, and color intensities in addition to producing a variety of colors. “Because these proteins have different fluorescent excitation and emission spectra, researchers could use different colored tags to simultaneously monitor the interaction of up to three unique cell populations present in a mixed culture,” said Barnes.

The variants also give researchers the ability to customize their experiments. “Colors could be used that offer better visual contrast with the background,” said Barnes. “For example, in mineral samples that appear yellow under epifluorescent light, a blue glow would be more easily detected than a yellow glow.”

One of Barnes’ goals is to define the advantages and limitations of each protein under environmental conditions. She would also like to develop techniques for their use in laboratory and field studies as well as biosensor development.

So far, Barnes and her team have successfully tagged several different bacterial species with GFP, including iron, sulfate, and nitrate reducers. These groups of microbes are environmentally significant because they influence contaminant mobility in the subsurface. To visualize these labeled cells, the team developed effective epifluorescent and laser confocal microscopy methods that allow real-time monitoring of the GFP-tagged populations.

GFP plasmids clearly provide an effective means of tagging environmental bacteria for use in short-term studies. However, according to Barnes, “Bacterial strains should be chromosomally marked for long-term use or field studies. Chromosomal marking maximizes genetic stability and reduces the chance that the GFP marker will be transferred to other microbes.”

Transposon: a piece of DNA that has the ability to move from one site on the chromosome to another.

In anticipation of field studies, the team is currently using transposon mutagenesis to insert the GFP gene onto the chromosome of environmental bacteria. So far, the team has tagged the Pseudomonas putida F-1 chromosome, a microbe capable of degrading trichloroethene.

“New tools and techniques often lead to new discoveries,” said Barnes. “We expect that the use of GFP will increase our understanding of microorganisms in the environment and be very useful for environmental research.”

This research is funded by the INEEL’s Laboratory Directed Research and Development (LDRD) program. It is being conducted by INEEL researchers Joni M. Barnes; Frank Roberto, Ph.D; Debbie Bruhn; and Bill Bauer, Ph.D.