Transcript An obligately photosynthetic bacterial anarobe from a deep

An obligately photosynthetic
bacterial anaerobe from a deep-sea
hydrothermal vent
J. Thomas Beatty, Jörg Overmann, 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
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.
• Jörg Overmann
– Professor Dr. Jörg Overmann is director of the LeibnizInstitute German Collection of Microorganisms and
Cell Cultures. His research interests are: bacterial
speciation, adaptations to energy limitation, and
bacterial interactions.
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 singlecelled 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.
ALVIN
• Discovered existence of
black smokers around
Galapagos islands in
1977
• Exploration of Titanic
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
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?
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
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.
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)
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 electrontransparent structures
characteristic of chlorosomes.
(Bar, 300 nm.)
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.
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.
Results:
• the absorption spectrum had a high peak at
750 nm which indicated the presence of lightharvesting 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.
• Electron microscopy showed that GSB1 is rodshaped (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
• Green sulfur bacteria uniquely contain a lightharvesting 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 Chlorobium and Prosthecochloris species
• 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
Chlorobium and 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
• 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
• 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
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.
• 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.
• 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
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
• 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.
Photo Credit/works cited
• http://mahalie.com/notebook/2008/02/01/dr
-gi-explains-origins-of-life-on-earth-andelsewhere/
• Paper: www.pnas.org
• www.wikipedia.org
• http://www.microbiology.ubc.ca/beatty
PAPER:
An obligately photosynthetic bacterial anaerobe from a deep-sea
hydrotherman vent
J. Thomas Beatty, Jorg Overmann, 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)