Ok, I know everyone has been waiting eagerly to hear all about a certain radiation-resistant bacterium. Don’t worry, there is no need to hold your breath any longer. Here is everything you wanted to know and more, plus some nice pin-ups:
Conan the Bacterium: The Ancient Microscopic Hero
(aka Deinococcus radiodurans)
By Karmen Lee Franklin
Was Mother Nature ready for the A-bomb? Nature is filled with built in checks and balances; all parts of the ecosystem are involved with one another with intricate detail. Typically, if something is available, something will eat it; but what could eat radioactive waste? In 1956, researchers in Corvallis, Oregon were sterilizing canned meat with gamma radiation, when something unusual happened: the food spoiled. A.W. Anderson, leading the study at the Oregon Agricultural Experiment Station, was surprised. What sort of organism could survive high doses of radiation and keep on snacking? Upon closer inspection, he found a cluster of odd, thick-celled bacteria, which he called Micrococcus radiodurans. The name was later changed to Deinococcus radiodurans, but the organism had already earned a nickname: Conan the Bacterium.
Since its discovery in the 1950s, D. radiodurans has been studied with great detail. In the 1960s, scientists isolated the mechanisms for repairing mutations in the bacterium’s DNA. It was also found to exhibit a unique cell wall structure, much like archaeabacteria, but remained classified in the kingdom of bacteria. Later, researchers charted the entire genome, enabling specific classification and close comparison with other bacteria. They discovered close cousins of D. radiodurans living in the oddest places, from hot springs to animal feces to the human stomach.
No one ever quite expected to find a bacteria that thrives on radioactive waste, especially since there hasn’t been much waste on the planet until very recently (within the last century.) On the other hand, life has been present on earth—and perhaps prepared for radioactive conditions—for at least the past 4 billion years. The latest studies suggest that all life shares a common ancestor, a thick-walled bacterium, able to withstand the harsh atmosphere and high temperatures of the early earth (Ciccarelli, et al., 1284.) D. radiodurans is not too far removed from this original ancestor, and so it is highly likely that it evolved billions of years ago, when extreme climates were the norm. Of course, the climate of Earth has settled considerably since. So, where has Conan the Bacterium been hiding out all this time?
Structure of the D. radiodurans Cell
First, it is necessary to understand the structure and abilities of D. radiodurans before identifying its ideal home. The name Deinococcus radiodurans means “strange berry that withstands radiation” (Lottman.) Indeed, the bacterium looks a bit like a berry, round, with a reddish hue. It grows in clusters of two to four cells, depending on its stage in the reproductive process. The non-pathogenic, non-spore-forming, D. radiodurans is considered to be an obligate aerobic organism, meaning that it lives in a specific niche where oxygen is present. While the bacterium expresses these rather run-of-the-mill features, it displays some rather unusual ones as well, notably, the unique structure of the cell envelope. As Kira Makarova and colleagues describe:
“the cell envelope of D. radiodurans is unusual in terms of its structure and composition. Although the cell envelope of D. radiodurans is reminiscent of the cell walls of gram-negative organisms, Deinococcus often stains gram positive; this may result from the inability of its thick peptidoglycan layer to decolorize” (Makarova, et al. 45.)
Most bacteria are easily classified as either Gram-positive, containing a thick cell wall of peptidoglycan which stains purple after treatment, or Gram-negative, lacking the cell wall and staining pink. Not only does the cell wall of D. radiodurans manage to stain either way, but it also exhibits a variety of unusual layers. The inner layer of the cellular envelope is the plasma membrane common to all cells. (See layers 1 and 2 below.) The lipids of the plasma membrane are atypical, containing unique fatty acids. Unlike phosphoglycolipids in other organisms, these lipids contain alkylamines.
Atop the plasma membrane is the typical peptidoglycan layer, (3) riddled with perforations, and so dubbed “the holey layer.” No significant purpose for these holes has been identified. The next layer is unique as well, containing a fine matrix of tiny compartments (4). Above the compartmentalized layer is another plasma membrane (5) (with a similar unusual composition as the inner plasma layer,) topped with an electrolucent zone. The sixth identified layer (6) is known as the S-layer, consisting of tightly-packed hexagonal proteins. Above this, some strains of D. radiodurans contain an extra layer, (7) consisting of densely packed carbohydrates (Makarova, et al. 45-46.)
D. radiodurans Metabolism
In most ways, D. radiodurans metabolizes in a similar manner to all bacteria. It is an obligatory heterotroph, taking sustenance from just about anything it can get. In this manner, D. radiodurans acts like well-known bacteria, such as Escherichia coli or Mycobacterium tuberculosis. On the other hand, D. radiodurans contains a particular vacuolar type of proton ATP synthase, which is more typical of archaea and eukaryotes than bacteria. Makarova explains:
“….Unlike most other free-living bacteria, it uses the vacuolar type of proton ATP synthase instead of the F1F0 type. Vacuolar (V)-type H1-ATPase is typical of eukaryotes and archaea; all archaea have a conserved operon that consists of eight genes encoding the ATPase subunits. This operon is partially conserved (with some of the subunits missing) in a minority of characterized bacteria, where it replaces the F1F0 ATPase, e.g., in Deinococcus, Thermus, spirochetes, chlamydiae, and Enterococcus. The scattered distribution of the VATPase operon among bacteria, in contrast to its conservation in archaea, suggests that this operon has been disseminated in the bacterial world by horizontal transfer.” (Makarova, et al. 48.)
D. radiodurans depends on a steady metabolism in order to survive chronic irradiation. As one study described, “Under nutrient-limiting conditions during chronic irradiation, DNA repair was found to be limited by the metabolic capabilities of the organism and not by any nutritionally induced defect in genetic repair.” According to the authors of the study, this supports the hypothesis that “there are several defects in its metabolic pathways.” (Venkateswaran, et al. 2621.)
Reproduction of D. radiodurans
When a D. radiodurans cell divides, only the cytoplasmic membrane and the peptidoglycan layer are used. The other layers form on the surface as the daughter cell grows. During the early stages of reproduction, D. radiodurans is found in pairs, two connected cells known as diplococci. Later, as the cells undergo mitosis, they remain in clusters of four cells, known as tetracocci. Within these clusters of cells, D. radiodurans keeps four to eight extra copies of its genome. Within one tetracocci, there are 16 to 32 copies of the genome (Daly 20.) This redundancy accounts for some of the survival abilities of the bacterium in the face of radiation, but not all. Not only does the organism maintain extra copies of its genome, it also contains other unusual repair mechanisms, in order to keep the genome consistent.
Typically, when a living cell is exposed to radiation, point mutations within the DNA are likely to occur. A small dose of radiation can cause an amino acid to be mistaken for another, so when the cell is copied, something turns out slightly different. This is typically bad for the organism, as most mistakes result in death. For instance, a small dose of radiation 5 Gy (a Gy, named for Harold Gray, is the standard unit of radiation that can be absorbed) is enough to kill a human being. 1,000 Gy will render an entire culture of Escherichia coli sterile in moments. D. radiodurans, on the other hand, manages to withstand doses of radiation up to 10,000 Gy! It can also stand constant doses of radiation, up to 60 Gy in an hour (Daly 20.)
How does it survive? Robyn Seipp of the University of British Columbia explored the question, “does this bug wear a lead vest, or what?” (Seipp, 57.) While the unusual layering of D. radiodurans may well constitute a lead vest, the endurance of the organism may be credited to proteins responsible for repairing errors in DNA. All bacteria contain a protein called RecA (eukaryotes carry a similar protein) responsible for nucleotide repair. This protein uses an undamaged copy of the DNA strand from another section or another chromosome to replace the damaged section. Since D. radiodurans contains extra copies of its DNA, it may be easier for RecA to find replacement copies. This protein alone does not explain the resistance of D. radiodurans; other bacteria given the D. radiodurans’ version of RecA do not display resistance (Seipp 57-59.)
It is likely that a combination of special proteins give the bacterium its hardiness. The genome of D. radiodurans was recently sequenced, and more than a dozen unique proteins have been identified (Makarova, et al. 67.) It has also been suggested that the bacteria is capable of preventing DNA degradation, and has an efficient habit of expelling damaged bases from the nucleoid region (Seipp 58.)
A variety of proteins have been shown to act in response to stress; D. radiodurans nearly has them all. (Click figure 4 for a more intensive description.) Many proteins deal with general stress, increasing or decreasing metabolic production as necessary, while others fit more specific needs. Several proteins specifically manage high temperatures, working as “molecular chaperones,” while others repair damage from extreme cold. Some proteins regulate the organism in the face of starvation, preventing growth if the food sources run low. Proteins help D. radiodurans keep shape, regulating osmosis and the uptake of potassium. One protein deals with alkaline shock, another with acids, while still others defend against oxidative stress. A few proteins (including one only previous found in the plant kingdom, in Craterostigma plantagineum) prevent desiccation. Other proteins protect D. radiodurans in the presence of antibiotic drugs, phages, and of course, radioactive toxins (Makarova, et al. 55-58.)
With such a diverse soup of protective proteins, redundancies in the genome, and a virtual “lead vest,” D. radiodurans is one of the hardiest organisms around. However it manages to do it, Conan the Bacterium has no trouble remaining consistent in extreme environments, such as in the presence of high doses of radiation. While there is no shortage of explanations for such toughness, discovering how D. radiodurans came to be presents many difficulties.
Finding the Origins of D. radiodurans
Since the first experiments with nuclear fission in the 1930s, quite a bit of nuclear waste has accumulated. As a result, there are now many environments where D. radiodurans may thrive. Before this time, however, radioactive materials were relatively rare. So where was Conan hanging out in the meantime? A few Russian scientists suggested in 2002 that it may have evolved on Mars, where it would have faced higher levels of cosmic radiation (Clark, 1.) Even further out in the Solar System, potential signs of D. radiodurans have been found.
Galileo’s Near Infrared Mapping Spectrometer captured a false-color image of Jupiter’s moon, Europa, (figure 6) revealing an unusual spectrum. Some have speculated that the discoloration is caused by something like Conan the Bacterium. “Though speculative, it is conceivable that explosions of icy slush or melt-throughs ferried extremophile organisms to Europa’s surface, where they stained the ice,” wrote Kristin Leutwyler in her book, The Moons of Jupiter (Leutwyler 126.)
NASA scientists ran experiments to test this hypothesis. “Brad Dalton of the NASA Ames Research Center imposed Europa-like conditions on acid-loving Sufolobus shibate and Deinococcus radiodurans, the most radiation-resistant organism known. Both species happen to be pink and brown in color, not unlike the hues on Europa.” Dalton discovered both similarities and differences. “[The organisms] produced spectra that shared certain features with the Europan plots. Some of the spectral bands that didn’t match corresponded to amine bonds, which would disintegrate were the bacteria subjected to radiation levels like those on the moon.” Since D. radiodurans is tough, but not that tough, it is most likely that it evolved locally, rather than in some extraterrestrial location.
Some suggest that the bacterium evolved its repair mechanisms in response to conditions other than radioactivity, including Kira S. Makarova and her colleagues. “Notwithstanding a few natural fission reactors like those that gave rise to the Oklo uranium deposits 2 billion years ago, the radiation levels in the Earth’s surface environments have provided only about 0.05 to 20 rads/year over the last 4 billion years,” she writes. “DNA damage is readily inflicted on organisms by a variety of other common physicochemical agents… or nonstatic environments… and it seems more likely that radiation resistance evolved in response to chronic exposure to nonradioactive forms of DNA damage” (Makarova, et al. 44.)
If the necessary conditions are so ubiquitous, then why should D. radiodurans exclusively have these abilities? Just because the overall levels of natural radiation on Earth are relatively low doesn’t mean there are not pockets of higher levels of radiation scattered about. Some hot springs dissolve uranium or radium within the earth, bringing the unstable isotopes to the surface. The hot springs of Ramsar, Iran, for instance, contains levels of radium which give off doses of radiation ranging up to 260 Gy (Mortazavi 1.) Could Conan have been hiding there?
Petrified forests and fossil beds can also contain high levels of radiation. If located by any natural outcropping of uranium (as are the famed dinosaur fossils of the Morrison formation) the remains will naturally “soak up” the radiation over time. The amount of radiation acquired is high enough that many museums use special storage for certain fossils and collections of petrified wood (Frame, 1.) While these forests and bones have been considered “dead” for centuries, perhaps they have been home to members of the Deinococcus family.
A Deinococcal Family Tree
Relatives of D. radiodurans have been found in strange places already. Some were found in animal feces, like Deinococcus proteolyticus, found in the feces of a Llama-like pack animal, or D. grandis in the feces of an elephant. A few, Deinococcus geothermalis and Deinococcus murrayi were discovered in Mediterranean hot springs. Still others have been found in the flesh of haddock (Deinococcus radiopugnans) and Bombay duck (Deinococcus radiophilus.) A strain of D. radiodurans was even found in the ventilation system of a hospital in Ontario. These organisms all fall under the genus Deinococcus, in the order of Deinococcales, in the Deinococcus-Thermus phylum of the kingdom of bacteria (Makarova, et al. 45.)
The Deinococcales are closely related to the thermophiles. Similarities between the two groups may represent the transition between gram-positive and gram-negative bacteria. Members of the two groups share a variety of proteins, specific to the Deinococcus-Thermus group, including the special S-layer protein. Other features are shared with members of the archaeal and eukaryotic kingdoms (Makarova, et al. 72-74.)
While many of the proteins found in the Deinococcus family are homologous to others, many are distinct. At least 24% of the D. radiodurans genome is particular to the species. In addition to producing the unique cellular envelope and specific types of DNA polymerase (used in repair jobs,) there are several repeating sections of unused code (small noncoding repeats, or SNRs.) These SNRs, which fall in “complex mosaic patterns,” help to make D. radiodurans unique (Makarova, et al. 71.)
The similarities in D. radiodurans to Archaea and Eukaryotes indicate that the bacterium may have played an important role in evolution, through horizontal gene transfer. It is theorized that some obligate bacteria pass genetic information to and from its host, sharing useful bits of DNA that allow either organism to adapt. This theory describes sort of a co-evolution of species, a phylogenic tree with many interwoven branches. Does D. radiodurans have a special place in this tree, sharing stress responses with various species over time? Maybe Conan has been hiding closer to home.
It was once thought that no living thing could withstand the harsh acidic environment of the human stomach. This myth was dispelled with the discovery of Helicobacter pylori, bacteria thought to cause ulcers. Realizing that the belly could be a previously unexplored ecosystem, they took a closer look and found a wealth of bacteria. A number of the organisms which were discovered living in the stomach were unusual to the researchers.
“Ten percent of the phylotypes found in this study were previously uncharacterized…. Because little is known about their closest relatives, the biological and clinical significance of these putative organisms is unclear.” Elisabeth M. Bik and her team were surprised to find Conan’s cousin, above all the other microbes, living among thermophiles and fusobacterium and the now-popularized H. pylori. “Of the previously uncharacterized phylotypes found in this study, the sequence belonging to the Deinococcus -Thermus phylum is particularly interesting…. To our knowledge, this Deinococcus-related sequence is the first identified from a human.” (Bik, et al. 736.) The organism, for the time being known as GMC-T94, most closely resembled Deinococcus indicus, which has been found in water naturally contaminated with arsenic.
Perhaps Conan the Bacterium (or a close relative) has been living inside of us from the beginning, helping us adapt to stressful conditions. It might seem especially appropriate then, that D. radiodurans is being engineered to help tackle some of humanity’s largest messes.
Usefulness of D. radiodurans
In 1999, Hassan Brim and others explored the potential of engineering D. radiodurans to clean toxic waste. By adding a gene isolated from E. coli known to provide resistance to high levels of mercury to the D. radiodurans genome, they were able to make Conan the Bacterium even beefier. By combining the radioactive resistance of D. radiodurans with the ability to process heavy metals, they created a powerful tool for the processing of toxic waste. Their goal was to create a strain of D. radiodurans that could both “confer resistance to the most common metallic waste constituents” and “transform those metals to less toxic and less soluble chemical forms.” (Brim, et al. 85.)
The experiment was quite successful, as the modified bacteria thrived in a radioactive, mercury-rich environment. Brim commented, “This remarkable genome plasticity shows that D. radiodurans is able to maintain, replicate, and express extremely large segments of foreign DNA, and that it will probably be able to accommodate the large number of gene cassettes required for bioremediation of complex waste mixtures.” In other words, if Conan the Bacterium is put to work, he does the job.
Where is Conan the Bacterium going from here? With nuclear power and weapons disposal, we are creating new habitats in which it can flourish. Is diversity in these habitats possible—or does the unique system of DNA repair indicate that it is unlikely to evolve? Perhaps, it was, until we came along to give it a job or two. We still have much to learn about the potentials and abilities of D. radiodurans. Whether it comes to the natural disposal of radioactive waste, or the simple adaptation to stress, Conan the Bacterium, is truly our ancient hero in microscopic form.
Cover Photo “The circular structure of the gene COM could cause the strength of the red pigmented bacterium Deinococcus radiodurans.”
S. Levin. Courtesy of Levin-Zaidmann. 2006.
Figure 1: D. radiodurans. Courtesy of Michael Daly, Uniformed Services University of the Health Sciences. 2005. (Website contains an animation as well.)
Figure 2: Cell wall model of D. radiodurans. Courtesy of Franck Karrenberg. ”Topographische Studien an den äußeren Zellwandschichten von Deinococcus Radiodurans,” Dissertation, Universität Düsseldorf, 1985.
Figure 3: Electron microscopy photos of bacteria in growth. Courtesy of Science. 2006.
Figure 4: Conventional Metabolic Pathways of D.radiodurans and Systems that Generate and Defend Against Free Radicals (See site for full size diagram and description) Courtesy of Michael Daly, Uniformed Services University of the Health Sciences. 2005.
Figure 5: “D. radiodurans… has been found in many extreme environments including contaminated soil at the U.S. Department of Energy’s Hanford Nuclear Site.” Courtesy of Planet Quest: Alien Safari: Most Extreme Organisms. 2006.
Figure 6: “Europa: Sea Salts or Battery Acid?” (Surface of Europa; false color spectrometry shows a similarity to D. radiodurans.) From Galileo Near Infrared Mapping Spectrometer. NASA Planetary Photo Journal. 2003.
Figure 7: “Deinococcus Family Tree.” By K. Franklin. 2006.
Figure 8: “Circular genome of D. radiodurans.” Courtesy of the Institute for Genomic Research. 2004.
Figure 9: “Barrel of radioactive waste on the steep continental slope at a depth below sea level of about 2,000 feet (610 meters) [in the Gulf of Farallon].” Courtesy of R. Dryer, U.S. Environmental Protection Agency. 2003.
Bik, Elisabeth M., et al. “Molecular analysis of the bacterial microbiota in the human stomach” The National Academy of Sciences. Vol. 103, no. 3. p. 732–737. January 17, 2006.
Brim, Hassan, et al. “Engineering Deinococcus radiodurans for metal remediation in radioactive mixed waste environments.” Nature Biotechnology. Vol 18, p. 85-90. 2000.
Ciccarelli, Francesca D. et al. “Toward Automatic Reconstruction of a Highly Resolved Tree of Life.” Science. Vol. 311, no, 5765, p. 1283-1287. March 2006.
Clark, Stuart. “Tough Earth bug may be from Mars.” New Scientist. September 2002
Daly, Michael J. “Deinococcus radiodurans” Uniformed Services University of the Health Sciences. December, 2005.
Frame, Paul. “Answer to Question #1952 Submitted to ‘Ask the Experts’” Health Physics Society, Specialists in Radiation Safety.
Lottman, Tim. “Deinococcus radiodurans” Microbe of the week. 2000.
Leutwyler, Kristin. The Moons of Jupiter. W. W. Norton & Company, New York, NY. 2003.
Makarova, Kira S. et al. “Genome of the Extremely Radiation-Resistant Bacterium Deinococcus radiodurans Viewed from the Perspective of Comparative Genomics.” Microbiol Mol Biol Rev. Vol. 65, no. 1, pg. 44–79. March, 2001.
Mortazavi, S. M. “High Background Radiation Areas of Ramsar, Iran” Javad Biology Division, Kyoto University of Education, Kyoto, Japan. 2005.
Seipp, Robyn “Deinococcus radiodurans: Does this Bug Wear a Lead Vest or what?” BioTeach Online Journal Vol. 1, pg. 57-62. Fall 2003.
Venkateswaran, Amudhan, et al. “Physiologic Determinants of Radiation Resistance in Deinococcus radiodurans” Applied and Environmental Microbiology. Vol. 66, no. 6, pg. 2620–2626. June 2000.