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Characterization
of a Hot-Spring Bacterium Resembling Heliothrix oregonensis An Honors
Thesis Written in 1989 for Bev Pierson, Reformatted for On-Line Presentation,
March 2005 by S. Boomer – Click HERE for pdf. Some
old graphs & images replaced as fading has occurred or current software
does not open them. INTRODUCTION Extreme temperature [45-60°C] microbial communities such as
those thriving in
Stromatolite, micrographs of
stromatolites (A-C), and living filamentous bacteria (C). Images provided to me as slides
for thesis presentation (Pierson). Such modern mat communities are often
composed of two types of organisms that form distinct laminations. The top layer of these mat systems is
generally composed of oxygenic, photoautotrophic cyanobacteria. Beneath this lies a layer of anoxygenic,
photoheterotrophic Chloroflexus [Castenholz, 1984]. Incident light striking the mat surface is
filtered through the mat such that the quality of light penetrating each
layer of the mat differs. At each
layer, the phototrophic organisms consume those wavelengths absorbed by their
pigment-protein complexes. In this
way, the light-absorbing properties of the bacteria within the layers of the
mat are believed to correlate with their positions in the mat [Castenholz,
1984]. Another extant species of
filamentous, phototrophic bacterium considered a possible analogue to
stromatolitic bacteria is Heliothrix oregonensis [Pierson, et al.,
1984, 1985]. This bacterium constructs
unusual mats found only in
Left: Heliothrix mats at Warm Springs
Indian Reservation, Right: Heliothrix mat turns red after low
light (image by Pierson). A second, unidentified, extant
bacterium was reported by D'Amelio et al.
[1987]. This organism is a
filamentous purple sulfur bacterium that possesses an extensive intracellular
structure of lamellae. The bacterium
was observed in mats found in hypersaline (65 - 128 ppt) ponds in Guerrero
Negro, Baja California Sur,
Left: Rabbit Creek spouter and mat at Right: Published Rabbit Creek core (Castenholz,
1984). Dr. Richard Castenholz first observed
an unusual filamentous organism in a thick hot spring mat sustained by a
six-inch spouting geyser near Rabbit Creek in MATERIALS AND METHODS Field
Observations Field measurements were made in late
June, 1988. Temperatures were measured
using a YSI Model 42SC Tele-Thermometer.
pH was measured using Colorphast indicator strips (6.5 - 10.0 pH range) and a battery-operated pH
meter. Spectral irradiance within the
mat was measured using a Li-Cor LI-1800 portable spectroradiometer connected
to a fiber optic probe that was inserted into the mat. Measurements were taken at 1 mm depth
intervals while the mat was exposed to natural solar radiation.
Li-Cor spectroradiometer and
schematic diagram of its set-up, click to enlarge (images by Boomer) Light
Microscopy Light microscopy was carried out
using a Zeiss 16 light microscope.
This microscope was equipped with a Zeiss Mercury arc lamp with BG 12
and BG 38 filters to induce fluorescence from Bchl a in living
filaments. In combination with a
Wratten 88A barrier filter, an MTI video camera permitted the visualization
of infrared fluorescence on a Lenco PMM-925 monitor. Oil immersion light
microscopy was utilized in conjunction with the Gram stain to characterize
the cell wall structure of the bacterium.
Pigment
Analysis Methanol extraction was carried out
on mat suspensions for thirty minutes over ice and in the dark. In vivo extracts were carried out
using suspensions of homogenized mat in Tris-Sodium-Magnesium buffer (pH
8.0). These were sonicated using a
Branson 200 sonifier cell disruptor [Pierson
et al., 1987]. Spectra from
both extracts were recorded using a Varian Cary 2300 spectrophotometer. Methanol extract spectra were run from 350
to 850 nm. In vivo extract
spectra were run from 350 to 1100 nm. Electron
Microscopy
Fixation of samples for transmission
electron microscopy was carried out according to procedures by D'Amelio
(personal correspondence) and adapted by Kaake (personal
correspondence). Primary fixation was
done in situ using 2.5% EM
grade glutaraldehyde and spring water.
Samples were refrigerated until further processing. Two fifteen-minute buffer rinses in 0.2 M
sodium cacodylate (pH 7.4) were then carried out. Secondary fixation was in 1% osmium
tetroxide buffered in 0.1 M sodium cacodylate (pH 7.4) for two hours. Samples were dehydrated via an ethanolic
dehydration series (50%, 70%, 90% 100% X3).
Each step lasted fifteen minutes and was carried out under a vacuum of
approximately 5 lb/square inch. Following
dehydration, samples were prepared for resin infiltration and encapsulation
by passing them through three propylene oxide transfers (once through 50:50
propylene oxide/ethanol, and twice through 100% propylene oxide, under a
vacuum of 5 lb/square inch). Samples
were embedded in Spurr's resin/propylene oxide, polymerized for twelve hours
at 60° C, sectioned on a Sorvall MT 5000 ultramicrotome, collected on mesh
copper grids. Following post-staining
in uranyl acetate and lead citrate, samples were viewed on a Zeiss EM 109
electron microscope.
Action spectrum uptake
experiment and schematic diagram of its set-up, click to enlarge (images by
Boomer). Photosynthetic
Activity Photosynthetic activity was
determined by measuring the uptake of [14C]-acetate. Uniformly labeled [14C]-acetate (specific activity, 56
mCi/mmol) was obtained from Amersham/Searle Corp. Experiments were carried out with freshly
collected filaments suspended in spring water. Suspended cells were incubated in 2-ml
screw-cap vials containing 10-4 M
3-(3,4-di-chlorophenyl)-1,1-dimethylurea (DCMU) and 0.01uCi/ml [14C]-acetate. DCMU was added to inhibit oxygenic
photosynthesis by the cyanobacteria.
All incubations were carried out for 60-90 minutes under in situ
temperatures (39-42°C) and were halted with the addition of 0.1 ml
formalin. Vials were refrigerated
until further processing in the laboratory.
To measure the effects of different conditions on acetate uptake,
vials were incubated on their sides in petri plates. Covers were blackened
except for a central window and fitted over the plates so that the only
radiation illuminating the samples was that which entered through the window. Over this window, filters were placed
according to the tested variables.
Left: Setting up uptake experiments (image by
Boomer). Right: Light intensity/IR uptake experiment (image
by Boomer). To measure the effect of wavelength
of acetate uptake (action spectrum), interference filters [ESCO products,
Inc.] were placed over the window above the incubation vials. Filters with
wavelength maxima of 640, 670, 710, 740, 790, 840, 910, 950, 1020, 1060 nm
were used. The filters were placed at the base of 15 cm chimneys that were
used to collimate the light from the sun.
To measure the effect of light intensity, vials were incubated under
varying thickness of neutral density filters of vinyl film (Universal
Plastics,
Oxic/anoxic uptake experiment
and schematic diagram of its set-up, click to enlarge (images by Boomer). Acetate uptake was also measured in
the presence or absence of oxygen. To
measure acetate uptake under oxic conditions, 4.0 ml vials were fitted with
rubber serum caps and a steady stream of compressed air was passed through
the cell suspension during the incubation period. The air was introduced through a syringe
needle extending to the bottom of the cell suspension. A second syringe needle permitted to escape
of gas from the headspace so as to prevent pressure from building up in the
vial. To create an anoxic environment,
a similar apparatus to above system was used;
In this case, argon was passed through the incurrent syringe during
the course of the incubation. All
incubation vials were placed in an opaque dish and covered with neutral
density nylon so that incubation was carried out with approximately 50% full
solar radiation. In the laboratory, all acetate uptake
experiment samples were processed by the following procedures: Settled filaments were resuspended and a
known volume was withdrawn and vacuum filtered (Gelman membrane filter, pore
size 0.22 um) so that cells were retained on the paper. Cells were immediately washed with
approximately 3 ml of 0.10 M unlabelled sodium acetate followed by 2 mls of
glass distilled water to ensure that the only radioactivity on the paper was
that taken up by the cells during the course of the experiment. Samples were dried overnight in uncapped
scintillation vials (Wheaton Wheaton 180 scintillation vials, 20 ml). 10 ml of scintillation fluid (Biofluor, NEN
Research Products, Dupont) were then added to the dried cells in each
vial. Samples were counted using a
scintillation counter (Beckman, LS 3133T) to obtain raw counts per
minute. Culture No previous attempts to culture this
organism have been reported. Agar plates were prepared using a standard
YED-medium containing sodium bicarbonate, glycylglycine buffer, yeast
extract, and DCMU. The first parameter
examined and varied was carbon source: acetate, glucose, glycolate, casamino
acids, bicarbonate and yeast extract. Filaments
grown on these media were observed for three successive days unless growth
proceeded. Each day, a sample of the
inoculated filaments was aseptically removed from the agar and examined using
fluorescence microscopy to evaluate the presence of Bchl a. This property, combined with observing the
color and quality of the filaments, was used to estimate the survival and
possible growth of the filaments on each type of medium used. Using the optimal carbon source, the pH
was then varied: 6.0, 7.0, 8.0, 8.5, 9.0, 9.5, 10.0. Growth was evaluated using the previously
described criteria. Using the optimal
carbon source and pH determined previously, the third parameter, temperature,
was examined. The following
temperatures were used: 14°, 25°, 35°,
45°, and 55° C. Growth was evaluated
using the previously described criteria.
Combining all optimal parameters, culturing was attempted using
different conditions of light and oxygen.
Conditions tested included full solar light measuring 450 watts/m2
(45-50% of the solar radiation measured in RESULTS Field
Observations The Rabbit Creek Spouter mat is
maintained by its hot-spring source through the constant splashing which
erupts from the geyser. The mat forms
a thick, gelatinous mass, approximately 1.5 m by 0.6 m, which curves a third
of the way around the geyser source.
The water splashing from the spouter is approximately 85° C. The
internal mat temperature is 40°C. The
spring water has a pH of 8.1. The red layer is located at a depth between 6.5
mm and 8.5 mm from the surface of the mat.
Left: Rabbit Creek mat, June 1988 (image by
Boomer). Right: Spectrum of full solar radiation available
on mat surface, click to enlarge (team data, 1988). Analysis of the light throughout the
Rabbit Creek mat demonstrated which layers attenuated which wavelengths of
light. The image above shows that
spectrum of solar radiation available upon the surface of the mat. The images below (team data, 1988) show the
spectrum of the light available just above the red layer (left) and directly
beneath the red layer (right). The
former (left) demonstrates that the green layer attenuates most wavelengths
between 400-700 nm. The latter (right)
demonstrates that the red layer attenuates specific wavelengths at 804 and
910 nm. Click on each to enlarge.
When cores were removed from the mat
and examined in full solar light, orange conglomerates of filaments puffed
from the red layer to the surface of the mat.
This phenomenon occurred at daytime temperatures of approximately 30°C
within fifteen minutes of exposure to full solar radiation. Samples placed immediately in cold, dark
environments were not observed to puff.
Left:
Rabbit Creek mat puffs orange after exposure to light (image by Pierson). Right: Rabbit Creek filaments, light microscopy
(image by Boomer). Light
Microscopy Phase contrast microscopy
demonstrated the red layer bacterium to be filamentous and septate. Granular spots (inferred to be PHB) were
observed inside the cells. The lengths
of filaments were in the millimeter range.
When exposed to intense blue radiation from the mercury ultraviolet
lamp, filamental Bchl a fluoresced in the infrared region. Gram staining demonstrated the organism to
be gram negative. Electron
Microscopy Filaments comprising the red layer
and orange puffs were identical in structure. Cytoplasmic PHB-granules and stacked
membrane systems existed throughout both cells. The dimensions of individual cells were 1
um by 8 um. A multilaminate wall
composed of three distinct layers was 0.03 um.
Left:
electron micrograph of Rabbit Creek red layer filament (image by Boomer). Right:
electron micrograph of Rabbit Creek orange puff filament (image by Boomer). Pigment
Analysis A single absorption maximum for Bchl a
from the methanol extract existed at 770 nm. In vivo absorption maxima
for the Bchl a-protein complex were
at 804 and 912 nm. The presence of
large amounts of carotenoids (responsible for the orange and red color) was
indicated by broad maxima at 475 nm in methanol and 475 nm in vivo. Photosynthetic
Activity Carbon uptake data from acetate
uptake experiments were plotted as disintegrations per minute (dpm). Dpm was calculated from counts per minute
by correcting for the efficiency of the scintillation counter. The action spectrum [left image below]
revealed that maximal carbon uptake occurred between 790 nm and 800 nm and at
910 nm. Variation of light intensity
[middle image below], revealed maximal uptake at fifty percent light
intensity. Infrared intensity
experiments [right image below] demonstrated high levels of uptake at both
high and low intensities. Finally,
oxic/anoxic uptake analysis demonstrated the organism's similar uptake of
carbon in the presence or absence of oxygen [data not shown].
Culture Optimal growth was obtained using
yeast extract as a carbon source, a temperature of 45°C temperature, and a pH
of 8.5 in 50% intensity solar light.
Cultures were observed to fluoresce maximally and maintain strong
orange color under these conditions.
The presence or absence of oxygen did not appear to affect
growth. The organism was shown to
persist on media containing sodium acetate and glycolate as carbon
sources. Bicarbonate, the autotrophic
carbon source, was the least successful.
The organism tolerated temperatures between 35° and 45° C. Temperatures below 25°C and above 50°C
proved immediately detrimental to the filaments. The organism tolerated pH's over the range
of 8.0 and 9.5. The organism did not
tolerate pH's below 7. Attempts to
culture the organism in the absence of light proved entirely
unsuccessful. The organism tolerated
near infrared radiation. In either the
presence or absence of light, culture growth was the same under both oxic and
anoxic conditions. DISCUSSION In this study, an unidentified
bacterium resembling the known bacterium Heliothrix oregonensis and an
unknown purple sulfur bacterium was characterized. Although several features of this unknown
bacterium are similar to those of the aforementioned bacteria, it is
currently impossible to determine any relatedness. The red layer organism was observed to form
mats at a site known as Rabbit Creek Spouter in Morphology All organisms were observed to be
septate filaments in which the individual cells measured approximately 1 x 7
um. Internal poly-ß-hydroxybutyrate
(PHB) granules were evident inside the cells of all organisms. All bacteria stained Gram negative. The red layer organism was strikingly
similar to the unidentified purple sulfur bacterium [D'Amelio et al., 1987] in their possessions of a
complex cytoplasmic lamellar system.
This feature contrasted with H. oregonensis that possesses no
internal structures. Pigmentation The major light-harvesting pigment of
the red layer organism is of Bchl a (methanol extract). The pigment/protein complex absorbed at 804
and 912 nm in vivo. Carotenoids
absorbed at 450 nm in both methanol and in vivo. H. oregonensis also possessed Bchl a as its light-harvesting pigment but its
pigment/protein complex exhibits absorbance maxima at 795 and 865 nm.
[Pierson, et al. 1984].
The dissimilarity between the two specific pigment/protein absorbance
maxima suggests that these two bacteria are evolutionarily distinct at the
species level. No such comparisons can
be drawn with respect to the unidentified purple bacterium as no pigment data
is currently available on this organism.
Habitat Positioned in a distinct layer 4-8 mm
beneath the surface of the mat, the habitat of the red Reflecting the different habitats of
the three organisms, the metabolisms of each organism were observed to
vary. The red layer organism was shown
to be photoheterotrophic (Hocson, 1987).
In this study, the photosynthetic metabolism, as a function of carbon
uptake, was maximal at wavelenths of 790 and 910 nm, reflecting both the
radiation absorbed by this layer in the mat and the cell's photosynthetic
pigment/protein absorbance maxima. Uptake was also maximal in low levels of
light intensity (60% full solar radiation or less). The organism was shown to be aerotolerant
by virtue of similar carbon uptake in both the presence and absence of
oxygen. H. oregonensis was
described as an aerotolerant photoheterotroph (Pierson et al., 1984,
1985). The purple sulfur bacterium was
observed to undergo marked changes in its metabolism in response to its
fluxuating environment. During the
morning, when sulfide was present in the photic zone of the mat, the
bacterium exhibited photoautotrophic metabolism with sulfide as the electron
donor. As the day progressed, the
photic zone was observed to become highly oxygenated at which point the
purple bacteria either used oxidized species of sulfur such as elemental
sulfur and thiosulate in the photoautotrophic mode or used organic carbon
excreted by cohabiting cyanobacteria in the photoheterotrophic mode. The organism, then, possesses a metabolic
mode similar, if not divergent, from that of the red layer organism and H.
oregonensis. Of the three compared organisms, only
H. oregonensis has been successfully co-cultured with an aerobic
chemoheterotroph in a medium containing glucose and casamino acids (Pierson,
1985). No attempts to culture the
purple sulfur bacterium have been reported to date. Attempts to culture the red layer organism,
though unsuccessful, have demonstrated persistence on a medium reflecting its
natural habitat: optimal conditions
include 45°C, pH 8.5, yeast extract carbon source, and low intensity
radiation. At this time, it is impossible to
conclude the relationship of the three organisms. Based on dissimilar absorbance maxima of
the pigment protein complexes between the red layer organism and H.
oregonensis, it is hypothesized that these two organisms are
distinct. It is difficult to make any
assertions concerning the purple sulfur bacterium based on currently
available data. Immediate future work should
concentrate specifically on the culturing of the red layer organism. Ultimately the technique of ribosomal RNA
sequencing should be employed to determine the evolutionary relatedness of
the three organisms. Additional
analysis of the low-light intensity red
H. oregonensis should be undertaken with an emphasis on examining
ultrastructure of the organism using electron microscopy and pigment/protein
absorbance maxima as a function of exposure to light; that is, does H. oregonensis develop
a lamellar complex and undergo a shift of pigment-protein absorbance in
response to low levels of light intensity.
Finally, infrared uptake analysis should be re-examined to clarify
ambiguities observed in this report. Additional
Data From 1988 Grant Report Given that I received funding over
the summer of 1988, I wrote up a separate fieldwork report for the Murdock
Foundation. Although most of these
data were included in my final thesis, one important dataset was not: the examination of other
Left:
Spray Geyser, prospective red layer community (image by Boomer). Right: Bev's Sink/Mammoth, prospective red layer community
(image by Boomer).
Left:
Imperial Geyser Pool (image by Boomer). Right: Pierson and Castenholz teams at Fairy
Geyser (image by Castenholz). ACKNOWLEDGEMENTS There are numerous individuals who supported,
contributed to, or assisted with this thesis project; I with to thank and recognize these
outstanding individuals for their contributions: First, I laud Dr. Beverly Pierson, my
thesis director, for her expertise, her inspiration, and her guidance. Her primary assistance with field research,
with experimental design, and with public and written presentations of my
work have been phenomenal. Second, I
thank Scott Sheffield, my advisor and departmental reader, for his primary
expertise using the electron microscope.
More importantly, though, I heartily praise him for encouraging me
very early to think about and develop research skills, introducing me to this
project in the process of teaching me the skills associated with electron
microscopy (i.e. stamina and patience).
Third, I wish to thank Dr. Jay Mueller, my extra-departmental reader
who intently, thoughtfully, and patiently assisted in the writing and
rewriting (and rewriting) of my thesis.
His attentive interest in and support of my work has been
extraordinary over the past year. Aside from thesis director and
readers, a substantial number of individuals partook in various activities in
support of my work. The most
outstanding group was involved in field activities - running samples through
timed experiments, hiking, measuring, recording, etc. Among them, I thank Judith Frederick
(research assistant to Bev Pierson) for her training skills in basic lab and
field procedures. I also wish to
praise Dr. Richard Castenholz and his crew of graduate students - visiting or
otherwise - whose dedicated presence and labor was exemplary. Of these students, I wish to thank Diane
Holmstrom whose assistance, constructive criticism, and friendship in and out
of the field has been extraordinary [RETROSPECTIVE COMMENT - Diane was my
best friend through college and remains one of my best friends today; after college, she began a doctoral degree
with Dick but left the program because of a serious family emergency]. In
addition to Diane, members of my family and several friends were engaged in
the re-working of my presentation. I
also was to single out Russ Kaake, whose primary work with electron
microscopy on this organism allowed me to continue a fascinating project. I commend the Honors Program for praising
my work in the form of the Best Honors Thesis Presentation Award; the wonderful experience of this
presentation and the honorary value of this award has been one of the most
rewarding events of all my work at the University. Finally, my
work has been funded by several sources.
First, I thank the Murdock Foundation and the University Murdock
Committee who approved this funding.
Second, I thank the Phi Sigma Biological Honor Society who honored my
work through a Slater Research Award.
Finally, I wish to recognize the National Science Foundation and NASA
whose support of Dr. Pierson has allowed her to continue such gratifying work
with students in the field. The
support of all these organizations has made my work all the more fulfilling
and outstanding.
Best Friends to this day -
Diane in Left: |
