An obligately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent

1. An obligately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent J. Thomas Beatty, JörgOvermann, Michael T. Lince, Ann K. Manske, Andrew S. Lang, Robert E. Blankenship, Cindy L. Van Dover, Tracey A. Martinson, and F. Gerald Plumley Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada Catherine Cartino & Marianna Nava March 27 2011

2. About the Authors: • J. Thomas Beatty • Ph.D. Indiana University, Microbiology (1980); Postdoctoral, Stanford University School of Medicine (1983) His research interests: gene structure and expression in photosynthetic bacteria. • JörgOvermann • Professor Dr. JörgOvermann is director of the Leibniz-Institute German Collection of Microorganisms and Cell Cultures. His research interests are: bacterial speciation, adaptations to energy limitation, and bacterial interactions.

3. Important Definitions: • Anoxygenic Photosynthesis: light energy is captured and stored as ATP • Anaerobic: without oxygen • PCR: polymerase chain reaction- amplifies DNA to become DNA sequence • geothermal radiation - infrared and visible light from the hot magma at the vent site. The single-celled organisms, which are anaerobic, apparently use these photons to convert carbon dioxide in the presence of sulfur into organic carbon for use in the cell.

4. ALVIN • Discovered existence of black smokers around Galapagos islands in 1977 • Exploration of Titanic

5. Background • How are some species able to obtain light needed for photosynthesis when they are miles below the photic zone? • In these hydrothermal vents, microbial and invertebrate populations live on organic material that is made chemotrophic bacteria. They are able to oxidize inorganic compounds to make CO2, which is necessary for photosynthesis. • Hydrothermal vents may resemble primitive environments

6. Basis for experiment • To capture and describe anaerobic green sulfur bacterium from deep-sea black smoker • These bacteria appear to use CO2 reduction to make organic material • If volcanic or geothermal light can drive photosynthesis using these bacteria, it is possible that there is extra-terrestrial life?

7. Materials/methods • Water samples were added to tubes that contained medium 1 for cultivation of green and purple sulfur bacteria as well as with 0.02% yeast extract and 0.1% thiosulfate • Tubes were incubated and weekly illuminated with fluorescent light • GSB1 was grown anaerobically in saline SL10 medium. SL10 contains H2S as electron donor and CO2 for carbon source. SL10 had positive effect on growth of colonies when it was supplemented with 5mM acetate, 5 mM propionate, 0.05% peptone or elemental sulfur. • Colonies were then purified

8. PCR • DNA from pure cultures of GSB1 was obtained and used for PCR amplification • PCR products were sequenced and analyzed Pigment Analysis Absorption and fluorescence emission spectra of cells were obtained. Pigments were extracted from cells and spectra of individual HPLC peaks were taken. Electron Microscopy Negatively stained cells were examined in a TEM.

9. Fig. 1. Absorption (solid line) and fluorescence emission (broken line) spectra of GSB1 intact cells. Vertical axis gives absorbance/fluorescence (arbitrary units) and horizontal axis gives wavelengths in nanometers. Absorption Emission Wavelengths (nm)

10. Fig. 2. Morphology and ultrastructure of GSB1 cells. (a) Negatively stained cells viewed by transmission electron microscopy. (Bar, 500 nm.) (b) Cells deposited on a filter and viewed by scanning electron microscopy. (Bar, 800 nm.) (c) Thin section through cells viewed by transmission electron microscopy with electron-transparent structures characteristic of chlorosomes. (Bar, 300 nm.)

11. Fig. 3. Phylogenetic analyses of GSB1. (a) Tree of FMO protein amino acid sequences. (b) Tree of 16S rDNA sequences. Support values at nodes are given as percentages, and scale bars represent the expected number of changes per residue position.

12. Fig. 4. Survival of GSB1 during exposure to air in darkness and the absence of H2S. The vertical axis (log10 scale) gives percentages of viable cells based on most probable number (MPN) enumerations relative to microscopic counts (19, 20), and the horizontal axis gives the time of incubation. Points give average values, and vertical bars indicate 95% confidence limits.

14. Results: • the absorption spectrum had a high peak at 750 nm which indicated the presence of light-harvesting bacteriochlorophyll and absorption at the 450 nm region indicates light-harvesting carotenoid pigments • Thus the major chlorophylls of GBS1 are BChls on the basis of absorption/fluorescence spectra indicated that the major carotenoid is chlorobactene.

15. Electron microscopy showed that GSB1 is rod-shaped (0.3 x 1 micrometers) and revealed the presence of chlorosomes ( light harvesting structures found in the structures of green sulfur bacteria) • Also indicates that the bacteria divides by binary transverse fission • Lack of flagella indicates that the cells are not able to move in liquid media, which is consistent with other results as well

16. Green sulfur bacteria uniquely contain a light-harvesting protein called FMO. Oligonucleotides were designed by using specific sequences of FMO genes and GBS1 DNA was amplified by the use of PCR • The product of this PCR sequence was 71% to 91% identical in alignments with FMO sequences of 14 species of green sulfur bacteria. Figure 3a shows that the GBS1 FMO protein sequences are most closely related to Chlorobiumand Prosthecochloris species

17. Figure 3b shows that PCR was again used in order to amplify an approximately 1.5 kb segment of the GBS1 16S rRNA gene and a tree of 19 bacterial 16S rDNA sequences places GSB1 in a cluster that includes Chlorobiumand Prosthecochloris species. • From this, the scientists were able to conclude that the GSB1 cluster isolate is a previously unknown marine species of the green sulfur bacteria. This was unexpected because viable green sulfur bacteria were thought to be found only in environments where light from the sun is available

18. The growth of GSB1 requires anaerobiosis, light, H2S or elemental sulfur and CO2-. Of 102 substances tested, the only ones able to promote photosynthesis were: acetate, propionate, peptone, and elemental S. • Exposure of cultures to air in the presence of light and H2S reduced viability, but we found that GSB1 is resistant to exposure to air in the absence of light and H2S. There was no significant loss of viability after 2 weeks this is consistent with the thoughts that the bacteria would be able to survive in the fluctuating environments of deep-sea hydrothermal vents

19. Multiple samples were obtained from different depths and locations and a second portion of the water that contained the GSB1 bacteria did not yield growth under the same conditions. So, GSB1-like bacteria were not found around the vent • It is unlikely that green sulfur bacteria are direct descendents of other photosynthetic organisms, because there is evidence that these environments have been changing throughout the years therefore it is impossible for the bacteria to have continuously occupied these environments

20. Strengths/Weaknesses of Paper • Geothermal vents produce most of their radiation in the form of heat. "The rates at which photons are emitted from these vents are pretty low," Dr. Beatty said. "It's very dim." • So if the bacteria are photosynthesizing, it is happening very slowly. The researchers estimated that it would take a bacterium a couple of years to divide and grow into two cells.

21. Although the sample that contained GSB1 was collected directly from a black smoker plume, it can be argued that this kind of bacteria can be growing elsewhere, such as the surrounding bulk water, but this possibility was considered to be unlikely because the surrounding water is oxygenated and lacks a source of reduced sulfur and light.

22. There is no way of knowing that the samples were not compromised when they were transferred from the bottom of the ocean into the lab, since the conditions ( pressure, temperature, light exposure) are so different

23. Further Study • Scientists in the study cautioned that without further experiments there was no way of knowing for certain if the bacterium was actually photosynthesizing

24. The findings may have implications for life on other planets. Scientists have speculated that on planets far from a star, life would have to be chemotrophic, using chemical rather than solar energy to grow. By showing that an organism can use another form of light, Dr. Beatty said, "our results indicate that it's possible photosynthesis could form the foundation for an ecosystem" on such distant worlds.

25. Photo Credit/works cited • http://mahalie.com/notebook/2008/02/01/dr-gi-explains-origins-of-life-on-earth-and-elsewhere/ • Paper: www.pnas.org • www.wikipedia.org • http://www.microbiology.ubc.ca/beatty

26. PAPER: An obligately photosynthetic bacterial anaerobe from a deep-sea hydrotherman vent J. Thomas Beatty, JorgOvermann, Michael T. Lince, Ann K. Manske, Andrew S. Lang, Robert E. Blankenship, Cindy L. Van Dover, Tracey A. Martinson, and F. Gerald Plumley Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3; Department Biology I, University of Munich, 80638 Munchen, Germany; Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85069; Institute of Marine Science, University of Alaska Fairbanks, Fairbanks, AK 99775; Biology Department, College of William and Mary, Williamsburg, VA 23187; and **Bermuda Biological Station for Research, St. George’s GE 01, Bermuda Communicated by Bob B. Buchanan, University of California, Berkeley, CA, May 3, 2005 (received for review March 3, 2005)