DNA Repair and Photoprotection in Halophilic Archaea
by Jason Rupp, Ashlee Allred, Bonnie K. Baxter
Halophilic Archaea are much more resistant to ultraviolet (UV) light damage than Escherichia coli and other species of Bacteria. This resistance is probably due, at least in part, to the large number of genes present in the genome that are involved in DNA repair. It has been shown that halophilic genomes often contain some repair genes homologous to those in prokaryotes and other genes homologous to repair genes found in eukaryotes (1). Extreme halophilic organisms use light-driven ion pumps to maintain an acceptable internal environment despite living in water that has salt concentrations up to 30% (2), and this need for light requires that the organisms experience more UV exposure than most other known microorganisms. For this reason, they have developed survival mechanisms, including efficient DNA repair processes (1). In addition, we propose that halophilic Archaea also use pigmentation as a photoprotective mechanism to protect from UV damage.
An unidentified strain isolated from the Cargill salterns near Great Salt Lake, Utah showed a much greater resistance to UV radiation than E. coli. The lethal UV dose for 50% of the population (LD50) was 187.34 J/cm2, which is nearly 13 times higher than the LD50 for E. coli. This is consistent with data from other Archaea. A mutant was then isolated based on pigmentation differences, and the LD50 for the mutant strain fell to 16.35 J/cm2, a value very close to that of E. coli. This preliminary data supports our hypothesis that pigmentation plays a role in UV protection of DNA.
DNA Repair and Photoprotection in Halophilic Archaea
Halophilic Archaea represent a diverse group of microorganisms that are able to sustain life despite hyper-saline surroundings. Archaea also represent a potential bridge between prokaryotic and eukaryotic organisms, as they share gene homology with both domains (3). Also, halophilic Archaea sport high levels of carotenoid production (1). Carotenoids, a class of very important antioxidants, may provide protection from UV damage and are usually bright orange, yellow or red in color.
Extreme halophilic organisms use light-driven ion pumps to maintain an acceptable internal environment despite living in water that has salt concentrations up to 30% (2). This need for light requires that the organisms experience more ultraviolet (UV) exposure than most other known microorganisms. However, halophilic Archaea are more resistant to UV light damage than Bacteria, such as Escherichia coli (1).
The experiments discussed below address the hypothesis that carotenoid compounds (found in halophilic Archaea, but not in E. coli), may work to protect the organisms' DNA from UV damage. This pigmentation, along with a GC-rich genome (4), may serve as mechanisms of survival for halophilic Archaea that dwell in such extrteme conditions.
Materials and Methods
Strains. The Cargill strain was isolated from the Cargill salterns near Great Salt Lake, Utah. E. coli DH5-alpha was purchased from Promega. Halobacterium NRC-1 was received as a gift from Dr. Shil DasSarma at the University of Maryland, Baltimore.
Growth curves. Growth curves for each organism were established by first taking optical density readings at 600 nm for a broth culture. The culture was then serially diluted and inoculated on an appropriate agar medium. The inoculum was spread on the plate and individual colonies were counted after a sufficient growth period (overnight for E. coli, 7-10 days for the Archaea).
UV survival. Once growth curves had been established, we were able to predict the number of colony forming units (CFUs) per milliliter of broth using optical density readings. Each sample was diluted accordingly and inoculated to the appropriate agar medium. Once spread, the plates were exposed to a measured amount of UV radiation, with one plate receiving no radiation and serving as the control. After a sufficient amount of time for growth, individual colonies were counted and relationships were established between UV exposure and the percentage of surviving colonies.
Mutant isolation. Cargill and NRC-1 mutants were selected by inoculating plates in order to get individual colonies. They were given time to grow, then each colony was inspected for pigment mutations. When a pigment mutation was observed, that colony was inoculated to a new plate and streaked for isolation. This process was repeated until the mutant grew pure.
Both the Cargill strain and E. coli were subjected to the procedures outlined in the UV survival section. The Cargill strain showed significantly more resistance to UV radiation than E. coli. The LD50 for each was 187.34 J/cm2 and 14.47 J/cm2, respectively (Figure 1).
After isolating a mutant from the Cargill strain based on pigmentation differences, the mutant was subjected to the same procedures to determine its survival rate. The results showed a significant loss of UV resistance in the mutant strain; its LD50 fell to 16.35 J/cm2, a value very near that of E. coli (Figure 2).
These preliminary results support our hypothesis that pigment production plays a role in DNA protection in halophilic Archaea. Judging by the color differences (the wild type is a light red color, but the mutant is pink), the Cargill mutant appears to produce less pigment than the wild type, or perhaps a different set of carotenoid intermediate compounds. In order to confirm this observation, we will use thin-layer chromatography (TLC) to look for the presence of carotenoids in both the wild type and the mutant. This procedure would allow us to detect variations in both the type of pigment present as well as variations in the amount of pigment.
In addition to the Cargill strain, data from Halobacterium NRC-1 support the hypothesis of the role of pigmentation in DNA protection. Though the data are preliminary and need further confirmation, it appears that the mutant is more resistant to UV radiation than the wild type. Unlike the Cargill strain, the NRC-1 mutant appears to have an abundance of pigment instead of a deficiency. Again, TLC will be used to look at differences between carotenoids in the wild type and mutant strains. In addition, NRC-1 has been studied extensively and much data concerning the carotenoid synthetic pathways is available. In fact, many of the genes involved in the pathway are located in a gene cluster (Figure 3), facilitating examination on a genetic level.
Our ultimate goal is to examine the significance of the carotenoid biosynthetic pathway, gene by gene, in halophilic archaea. Understanding the significance of these compounds in UV photoprotection may shed light on the function of carotenoids in human skin cells.
1. DasSarma, S. et al. 2001. Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototrophic, phototactic, and UV-tolerant haloarchaeon. Photosynthesis Research 70: 3-17.
2. Oesterhelt, D. and J. Tittor. 1989. Two pumps, one principle: Light-driven ion transport in Halobacteria. Trends Biochem. Sci. 14: 57-61.
3. Barinaga, M. 1994. Archaea and eukaryotes grow closer. Science 264: 1251.
4. Ng, W. et al. 2000. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. 97: 12176-12181.