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Applied and Environmental Microbiology, December 2001, p. 5410-5419, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5410-5419.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Motility of Marichromatium gracile
in Response to Light, Oxygen, and Sulfide
Roland
Thar* and
Michael
Kühl
Marine Biological Laboratory, University of
Copenhagen, DK-3000 Helsingør, Denmark
Received 9 July 2001/Accepted 7 September 2001
 |
ABSTRACT |
The motility of the purple sulfur bacterium Marichromatium
gracile was investigated under different light regimes in a
gradient capillary setup with opposing oxygen and sulfide gradients.
The gradients were quantified with microsensors, while the behavior of
swimming cells was studied by video microscopy in combination with a
computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen
and sulfide. The observed migration patterns could be explained solely
by the various phobic responses. In the dark, M. gracile formed an ~500-µm-thick band at the oxic-anoxic interface, with a
sharp border toward the oxic zone always positioned at ~10 µM O2. Flux calculations yielded a molar conversion ratio
Stot/O2 of 2.03:1 (Stot = [H2S] + [HS
] + [S2]) for
the sulfide oxidation within the band, indicating that in darkness the
bacteria oxidized sulfide incompletely to sulfur stored in
intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 µM
O2. The cells also preferred low sulfide concentrations if
the oxygen was replaced by nitrogen. A light-dark transition experiment
demonstrated a dynamic interaction between the chemical gradients and
the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at
oxygen concentrations of >100 µM with no sulfide present the cells
remained viable and motile for ca. 3 days both in light and darkness.
Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as
effective protection strategies against high oxygen concentrations and
might represent first stages of biofilm formation.
| |
INTRODUCTION |
Purple sulfur bacteria perform
anoxygenic photosynthesis utilizing reduced sulfur compounds as
electron donors. Phototrophy is regarded as the ecologically most
important mode of their metabolism (28). However, several
species can grow chemotrophically under microaerobic conditions by
respiring reduced sulfur compounds with molecular oxygen
(21). Mass developments of purple sulfur bacteria are
often observed in anaerobic water columns and in benthic habitats,
where reduced sulfur compounds, as well as sufficient light intensity,
are abundant. Such habitats can, for example, be found in the
hypolimnion of stratified lakes (40) or in the upper
millimeters of sulfidic sediment and microbial mats (28). Opposing sulfide and oxygen gradients characterize these ecological systems (19, 42).
The various light conditions during a diurnal cycle are accompanied by
changes of the oxygen and sulfide distribution (19). Purple sulfur bacteria have adapted to such conditions by different strategies: the physiology of species such as Thiocapsa
roseopersicina is adapted to withstand a wide range of
environmental conditions (6, 33), whereas motile species
follow their preferred environment by migration (13). The
latter was reported for the gas-vacuolated species Thiopedia
rosea, which is able to migrate in the water column of lakes by
changing its buoyancy (23). Diurnal migration has also
been reported for flagellated Chromatium spp. (the former genus Chromatium was recently reclassified into several new
genera [17]). Sorokin (40) observed
migration of these bacteria in Lake Belovod, whereas Jørgensen
(19) described their motile behavior in sulfidic microbial
mats dominated by Oscillatoria and Beggiatoa spp.
During daytime Chromatium stayed below the oxic zone at a
depth of ca. 2 mm. After sunset, the sulfidic zone expanded toward the
mat surface, finally resulting in the release of sulfide into the
overlying water. This process was accompanied by the migration of the
Chromatium population into the sulfidic surface water. Soon
after sunrise with the onset of oxygenic photosynthesis, the swarming
bacteria retreated rapidly into the sediment.
The migration patterns found in nature should on principle be
explainable by the chemotactic and phototactic behavior of the cells.
Extensive studies exist about the characteristic photophobic step-down
response of Chromatium spp. (termed by other authors "photophobotaxis"). If swimming cells experience diminishing light intensities, they stop and reverse their swimming direction, whereby Chromatium cells effectively accumulate at optimal
irradiance (15, 16, 38). A combined effect of chemotaxis
and photoresponses can be observed under the microscope.
Chromatium cells accumulate around air bubbles in the
absence of light but move away if illuminated (1) (an
observation that has been erroneously cited to be already observed by
Engelmann [9] in 1883). Beside the photophobic response,
Chromatium exhibits also photokinesis (29),
i.e., swimming velocities are correlated to the illumination intensity.
In recent years much progress has been made in understanding the
molecular mechanisms behind the motile behavior of purple bacteria. The
bacteria sense the rate of electron transfer in their electron
transport chains (1). Phototactic and chemotactic signals
are integrated, because photosynthesis and respiration share parts of
their electron transport chains (37). Both processes compete for electrons, but photosynthesis is preferred under electron donor-limiting conditions, and respiration rates decrease with increasing light intensities (5).
Our goal was to study and quantify the motility behavior of
Marichromatium gracile in defined gradients and light
regimes in order to explain how the behavior of single cells leads to the migration patterns observed in natural systems. We used a gradient
capillary setup, which allowed the preparation of defined opposing
sulfide and oxygen gradients inside a flat microslide capillary that
could be illuminated with various light intensities. Chemical gradients
and light fields inside the capillary were measured with microsensors,
while tracks of individual bacteria in relation to light and chemical
gradients could be analyzed via a computerized cell tracking system
based on video microscopy. Similar gradient capillaries in combination
with oxygen microsensor measurements and microscopy have been used
previously for studies of motile colorless sulfur bacteria
(11), ciliates (3), and sulfate-reducing
bacteria (10).
| |
MATERIALS AND METHODS |
Sulfide nomenclature.
The total sulfide concentration
(Stot) is calculated as follows:
![<UP>S<SUB>tot</SUB> = </UP>[<UP>H<SUB>2</SUB>S</UP>]<UP> + </UP>[<UP>HS<SUP>–</SUP></UP>]<UP> + </UP>[<UP>S<SUP>2–</SUP></UP>]](/content/vol67/issue12/fulltext/5410/img001.gif)
|
(1)
|
The H2S microsensor (see below) detects
only [H2S] (18, 26).
[S2-] can be neglected at pH values of <9.
Thus, Stot can be calculated from
[H2S] and the pH value as follows
(18):
![<UP>S<SUB>tot</SUB> = </UP>[<UP>H<SUB>2</SUB>S</UP>] (<UP>1 + </UP><IT>K</IT><SUB><UP>1</UP></SUB><UP>/</UP>[<UP>H<SUP>+</SUP></UP>])](/content/vol67/issue12/fulltext/5410/img002.gif)
|
(2)
|
where K1 is the first
dissociation constants of the sulfide equilibrium system, assuming a
pK1 of 7.05 (24).
Bacterial strain and culture condition.
The purple sulfur
bacterium M. gracile strain CE2205 (previously named
Chromatium gracile and reclassified by Imhoff et al. [17]) was received from the culture collection of the
Laboratory of Biological Oceanography, University Bordeaux I, Arcachon,
France. The motile cells are ovoid to rod shaped, 1.0 to 1.4 µm by
2.2 to 4.5 µm, with polar flagellation. They grow phototrophically by
utilizing sulfide, intracellular sulfur, or thiosulfate as the electron
donor. Under microaerobic conditions, this species can also grow
chemolithotrophically with oxygen as the electron acceptor
(22).
Cultures were grown photoautotrophically under anaerobic condition with
sulfide as electron donor at 25
salinity and 20°C
in the medium
described by Eichler and Pfennig (
8). Illumination
was
provided by an incandescent lamp with a regime of 12 h of
light
and 12 h of dark. The applied scalar irradiance was 70 µmol
of
photons m
2 s
1 for
visible light (400 to 700 nm) and 190 µmol of photons
m
2 s
1 for near-infrared
light (700 to 950 nm). When intracellular sulfur
globules disappeared,
cultures were refed with neutralized sulfide
stock solution to a final
S
tot of ca. 1 mM. All experiments were
performed
with cultures in their exponential growth phase and
with all cells
showing intracellular sulfur globules. The samples
for the experiments
were taken from the upper water column in
the culture bottle in order
to exclude nonmotile cells which aggregated
at the
bottom.
Gradient capillary setup.
About one-half of a flat
microslide capillary (internal dimensions 8 by 0.8 by 40 mm3; VitroCom, Inc.) was filled with a
sulfidic agar plug (Fig. 1). The 1% agar
plug was prepared from filtered anoxic seawater (25 salinity) mixed
with neutralized sulfide stock solution to a final concentration of 1 to 10 mM Stot. The central part of the capillary was filled with liquid M. gracile culture by using a syringe
connected to a hypodermic needle. Air bubbles were carefully avoided.
The liquid culture extended over a distance of 7 to 10 mm between the
agar plug and the meniscus. The inner walls of the remaining gas-filled
part of the capillary were covered by a thin film of petroleum jelly
(Vaseline; Chesebrough-Pond's, Inc.), ensuring that the meniscus of
the liquid culture formed a straight line. Both openings of the
capillary were closed with Vaseline in order to avoid evaporation. The
composition of the gas in the gas-filled part could be controlled by
flushing the surrounding of the opening with defined gas mixtures. The
gas supply during the experiments was not limited by the Vaseline due
to the high diffusivity of gases within Vaseline. The gradient
capillary was mounted horizontally on the stage of a light microscope.
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FIG. 1.
Top view of gradient capillary setup with flat
microslide capillary (8 by 0.8 by 40 mm3) (a),
sulfidic agar plug (b), motile bacteria in liquid medium (c),
gas-filled space (d), Vaseline (e), microsensors (f), pH reference
electrode (g), border of illuminated region (dashed line) (h), and the
end of the tubing for flushing the capillary opening with defined gas
mixtures (i).
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Capillaries with homogeneous sulfide or oxygen
concentrations.
Liquid cell culture was added to the central 10 mm
of a flat microslide capillary (same dimensions as the gradient
capillary). For anoxic sulfidic conditions both ends were closed
with 1 mM Stot agar plugs (as described above)
and covered with Vaseline. For oxic conditions, the capillary was only
closed with Vaseline, allowing for diffusion of oxygen into the liquid
culture. The prepared capillaries were stored for ca. 1 h,
whereafter the oxygen concentration in the capillary had increased to
>100 µM and no free sulfide was detectable.
Illumination and irradiance measurements.
The bright-field
illumination of the microscope was used for illumination of the liquid
culture within the gradient capillary. In order to avoid an
inhomogeneous light field due to scattering or refraction at the
borders, only the part indicated by the broken lines in Fig. 1 was
illuminated by placing a quadratic field stop beneath the capillary.
Scalar irradiance within the gradient capillary was measured with a
microprobe consisting of a light-integrating sphere (70 µm in
diameter) fixed to the light collecting end of a tapered optical fiber
(27), which was connected to a light meter only sensitive
to visible light (400 to 700 nm) with a flat spectral responsivity
(25). The ratio between integral visible light (400 to 700 nm) and near-infrared light (700 to 950 nm) was 1:8.3, as measured in
micromoles of photons per square meter per second with a fiber-optic
spectrometer (Hamamatsu, Inc.). This ratio was kept constant throughout
the experiments, and all irradiance values presented below are given as
the scalar irradiance of the visible region.
Irradiances of >20 µmol of photons m
2
s
1 were not applied in the experiments in order
to avoid an increase in temperature inside
the gradient capillary. The
irradiance was varied by placing neutral-density
filters (Oriel, Inc.)
into the illumination path. The filters
were spectrally neutral to
visible light and near-infrared light.
Video microscopy for cell
tracking during experiments in the dark
(see below) was performed under
weak red light (650 to 700 nm)
by placing corresponding short-pass and
long-pass interference
filters (CVI, Inc.) into the illumination path.
The red light
apparently did not affect the swimming behavior of the
bacteria,
since shading did not cause a photophobic
response.
Microsensor measurements.
Dissolved oxygen measurements were
done with Clark-type O2 microsensors with a guard
cathode (35) connected to a picoammeter (Unisense A/S).
The microsensors had a tip diameter of 10 to 20 µm and <1 to
2% stirring sensitivity. A linear two-point calibration was
performed from microsensor readings in seawater (25 salinity, 20°C) flushed with air and nitrogen, respectively.
Dissolved hydrogen sulfide was measured with amperometric
H
2S microsensors (
18,
26) connected
to a picoammeter (Unisense
A/S). The electrodes had a tip diameter of
10 to 20 µm and <1
to 2% stirring sensitivity. Calibration
was performed in a closed
glass vessel filled with anoxic phosphate
buffer (50 mM, pH 7.0,
20°C). Sulfide standard stock solution (10 mM
S
tot) was added
to the buffer in several
consecutive steps up to a final S
tot concentration of 0.5 mM (corresponding to [H
2S] = 0.25 mM). The
obtained calibration points were fitted by linear
regression.
The linear equation obtained by the fitting procedure was
used
for converting the microsensor signal to H
2S
concentrations. The
exact molarity of the standard stock solution was
determined by
the titrimetric method described by Fonselius
(
12). S
tot profiles
were calculated
by equation
2 from measured [H
2S] and pH (see
below).
pH was measured with a potentiometric pH glass microsensor
(
36) connected to a high-impedance millivoltmeter (World
Precision
Instruments, Inc.). The pH-sensitive electrode tip had a
length
of ca. 100 µm and a diameter of ca. 20 µm. The reference
electrode
consisted of a chlorinated silver wire immersed into
saturated
KCl solution inside of a small capillary (tip diameter, ca.
0.5
mm). The pH microsensor was calibrated directly in the gradient
capillary after the actual measurements were finished (see below)
by
replacing the liquid culture with standard buffer solutions
of pH 7.0 and 11.0 for a linear two-point
calibration.
Both the O
2 and the H
2S
microsensor were mounted horizontally on a computer-controlled
motorized micromanipulator (Oriel,
Inc.; Märzhäuser GmbH)
with the sensor tips 3 mm apart from
each other. The microsensor tips
were moved into the gradient
capillary at its gas-filled side before
the opening was covered
with Vaseline (Fig.
1). The measured signals
were connected to
a computerized data acquisition system, which also
controlled
the micromanipulator (Unisense A/S). Horizontal profiles of
oxygen
and sulfide were measured with a step size of 100 or 200 µm.
pH
profiles were measured correspondingly by replacing the
O
2 and
the H
2S microsensors
by the pH microsensor. The tip of the reference
electrode was placed
within the agar plug (Fig.
1).
Molar conversion ratio of sulfide oxidation within bacterial
band.
Molecular diffusion fluxes along the gradient capillary were
determined according to Fick's first law for one-dimensional diffusion:
|
(3)
|
where
D0 is the free solution
diffusion coefficient and
C(
x) is the
concentration of the solute at position
x. Fluxes on
both
sides of the bacterial band were determined by linear regression
of the
measured concentration data within an interval of 500 µm
next to the
band. The change in flux across the band gave the
uptake rate,
R, of the bacterial band. The molar conversion ratio
of
sulfide oxidation (i.e.,
S
tot/O
2) was then given as:
|
(4)
|
The ratio of the diffusion coefficients of
S
tot and oxygen was needed for this calculation.
We used a value of
D0(S
tot)/
D0(O
2)
= 0.757 (
4,
34).
Cell tracking, swimming velocity, and relative cell density.
Swimming paths of motile cells were recorded on a digital video
recorder (Sony, Inc.) with a charge-coupled device camera (EHD GmbH)
attached to the microscope (Olympus BX50 WI). The focal plane was
adjusted to the middle of the gradient capillary in order to exclude
boundary effects of cells swimming near to the glass walls. The tracks
and swimming velocities of individual cells were subsequently analyzed
frame by frame (time steps, 0.04 s) by using the software program
LabTrack (DiMedia, Kvistgård, Denmark). Tracks and swimming
velocities reported in this study always represent their
two-dimensional projections into the focal plane. An average swimming
velocity was obtained by averaging the swimming velocities of all cell
tracks (n > 50) within 1 s of a video recording.
Relative cell density distributions of motile cells were obtained by
tracking over a time period of 2 s. The number of tracks
within
50- or 200-µm intervals along the gradient capillary were
taken as
the relative cell density. Thus, only actively swimming
cells were
taken into account, if not otherwise stated. Relative
cell densities of
nonmotile cells could not be quantitatively
analyzed with the existing
system.
Photokinesis and photophobic response measurements.
Capillaries with homogeneous oxic or sulfidic conditions were placed on
the microscope stage and illuminated with 4.8 µmol of photons
m2 s1 for 1 h
before the photokinesis measurements at different irradiances were
begun. Each irradiance was applied for 60 s before swimming velocities were measured by cell tracking over a period of 5 s.
For measurements of the photophobic response, a stepped neutral density
filter was placed into the illumination path of the
microscope so that
the straight border between two different optically
dense regions
(optical density [OD] = 0 and OD = ~1) was projected
into the
microslide
capillary.
| |
RESULTS |
General motility behavior.
The polar flagellated cells of
M. gracile always swam along their long axis. Straight
swimming paths were interrupted by reversals in swimming direction. The
cells stopped for ca. 0.04 s (corresponding to one frame of the
video recording) before they resumed swimming in the opposite
direction. The tracks before and after the reversal did not exactly
coincide with each other. The two-dimensional projection of the angle
between the tracks was on average 12.1° (standard deviation = 7.3°, n = 24). The motility patterns were apparently
symmetrical with respect to both swimming directions.
Photokinesis was analyzed under oxic conditions, as well as under
anoxic conditions, with 1 mM S
tot (Fig.
2A). In the first
case average swimming
velocities were not significantly influenced
by irradiance and showed
values of 33.4 ± 1.0 µm s
1. The same
was valid under anoxic conditions for >2 µmol photons
m
2 s
1, with swimming
velocities of 34.0 ± 2.3 µm s
1.
Swimming velocities decreased to 19.5 µm s
1
at 0.05 µmol of photons m
2
s
1. The dynamics of the swimming velocity in
response to sudden
changes in irradiance showed a biphasic behavior.
The average
velocity jumped within <1 s to a certain new value but
kept changing
within the next 60 to 120 s until a new steady-state
value was
achieved (see, for example, Fig.
2B).
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FIG. 2.
(A) Photokinesis of M. gracile under oxic
(>100 µM O2) and anoxic (1 mM Stot)
conditions. Datum points represent the mean ± the standard
deviation (n = 5) of the average swimming velocity.
(B) Dynamics of photokinesis under anoxic (1 mM Stot)
conditions. The illumination intensity of 0.1 µmol of photons
m2 s1 (shaded areas) was increased to 11.5 µmol of photons m2 s1 (bright area) for
30 s. The thick lines represent linear regressions.
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|
M. gracile exhibited phobic responses toward different
stimuli by reversing their swimming direction. Cells showed a
photophobic
step-down response when they crossed the border toward a
less
illuminated region, whereas cells crossing the border from the
other direction did not show any response (Fig.
3A). Thus, cells
were effectively trapped
within the more intensive illuminated
region. The photophobic response
was not influenced by the oxygen
or sulfide concentration. A phobic
response was also observed
toward certain lower and upper limits in
oxygen or sulfide concentration.
Figure
3B shows the response of
swimming cells toward oxygen gradients
in the dark. Cells swimming
toward higher oxygen concentrations
reversed their swimming direction
at 6 to 10 µM O
2. Cells approaching
from higher
oxygen concentrations did not show the phobic response.
Thus, cells
accumulated within the microoxic region.
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FIG. 3.
Swimming tracks of single M. gracile
cells. Points represent the positions of the cells with 0.04-s time
steps. Various point symbols were used in order to distinguish the
tracks of different bacteria. Arrows indicate the start of tracks. The
scale bar in panel A is valid for both panels. (A) Photophobic
step-down responses. Illumination intensities: 5 µmol of photons
m2 s1 (bright area) and ca. 0.5 µmol
photons m2 s1 (shaded area). (B) Phobic
response toward exceeding oxygen concentrations. The dashed lines
indicate oxygen isopleths.
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The reversals due to the photophobic response took place within a band
of 20-µm thickness around the light-dark boundary (Fig.
3A). In the
case of the phobic response toward oxygen, this band
was ca. 100 µm
thick (Fig.
3B). Otherwise the swimming tracks
revealed no apparent
difference between the photophobic response
and the phobic responses
toward oxygen or sulfide. All distribution
patterns of
M. gracile that are described below were caused by
the phobic
responses at the borders of the observed cell
distribution.
Observations in gradient capillaries.
The cells accumulated in
a band of 500 µm thickness within opposing oxygen-sulfide gradients
in the dark. The band was positioned between the oxic and the anoxic
parts of the capillary (Fig. 4A). The
boundary of the band toward the oxic part was found at ca. 10 µM.
More than 98% of the oxygen entering the band by diffusion was removed
within the band (Fig. 4B). In contrast, Stot was
only partially removed within the band. The molar conversion ratio Stot/O2 for sulfide
oxidation within the band was calculated to be 2.03:1. Preparations
with different oxygen and sulfide gradients revealed that the position
of the band was always at the oxic-anoxic interface independent of the
sulfide concentration at this position (data not shown). The vertical
band structure within the capillary could be revealed by moving the
focal plane of the microscope through the inner space (0.8 mm in
height) of the capillary. The cell density at the bottom was higher
than at the top of the capillary, and the band position at the bottom
was shifted 500 µm toward the oxic region relative to the band
position at the top. There was no apparent difference in motility
behavior between the cells in the top region and the ones in the bottom
region. If the capillary was turned upside down, the vertical band
structure readjusted within a few seconds. Again, the bottom part of
the band showed the highest cell density and was shifted toward the
oxic zone.
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FIG. 4.
(A) Relative cell distribution (bars, arbitrary linear
units) of swimming M. gracile in the dark, together with
oxygen ( ), sulfide ( ), and pH ( ) profiles. The
x axis represents the distance along the long axis of
the capillary. (B) Fine scale measurement of oxygen () and
Stot () profiles in the interval 4 to 7 mm of panel A. Thick lines represent linear regressions. The numbers next to the lines
give their slope in micromolar units/millimeter.
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Only few nonmotile cells were observed in fresh gradient capillary
preparations. If a preparation was kept for several hours,
cells
attached to the inner walls of the capillary. In the oxic
part (>10
µM O
2) this was more pronounced at the bottom
wall,
where the bacteria formed small colonies consisting of 10 to 50
cells. No cells in their dividing stage could be observed by
microscopic
examination. In contrast, cells in the anoxic, sulfidic
part attached
preferably on the top wall but did not aggregate in
colonies.
Many of these cells were in their dividing
stage.
If a bacterial band was illuminated with 18 µmol of photons
m
2 s
1, the cells
retreated within 1 min toward the anoxic region. The
dynamics of the
migration are shown in Fig.
5A. Within the initial
5 s, the band broadened symmetrically toward both sides. After
10 s, the cells which were initially swimming into the oxic
region,
reversed their swimming direction. Thus, most cells had
migrated
toward the anoxic region after 50 s. In order to
investigate the
photophobic behavior in response to the dark-light
transition,
the illumination path was shaded every 1 s for ca.
0.25 s. The
bacteria did not react to the shading immediately
after the illumination
was switched on. After 10 ± 2 s
(
n = 5), the photophobic response
could be observed
first, i.e., the cells reversed their swimming
direction upon
shading.
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FIG. 5.
Relative cell distribution (bars, arbitrary linear
units) of swimming M. gracile at the oxic-anoxic
interface. The x axis represents the distance along the
long axis of the capillary. (A) Dynamics of the cell distribution in response to dark-light transition. The
uppermost panel shows the steady-state distribution in the dark, where
the band of bacteria was positioned between the oxic and the anoxic
parts of the capillary. At time t = 0 s, the
band was illuminated with 18 µmol of photons m2
s1. The subsequent panels show the dynamics within the
next 50 s. (B) Steady-state distributions at different irradiances
(I).
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|
The retreat of the band was also examined in a different way by a
stepwise increase of the irradiance (Fig.
5B). A weak irradiance
of 0.2 µmol of photons m
2 s
1
did not significantly influence the bacterial band at the oxic-anoxic
interface. At an irradiance of greater than ~10 µmol of photons
m
2 s
1, the band
broadened and moved toward the anoxic region. The relative
cell density
still showed a maximum at its shoulder toward the
oxic-anoxic
interface, but there was no longer a sharp border
toward the anoxic
region. Eventually, all of the cells migrated
toward the anoxic region
when the irradiance was further
increased.
The resulting steady-state situation for an illuminated (18 µmol of
photons m
2 s
1) gradient
capillary with opposing oxygen and sulfide gradients
is shown in Fig.
6A. Most of the swimming cells (>90%)
were found
in the region with <10 µM O
2.
Apparently, the cells also avoided
high sulfide concentrations, which
resulted in a maximum of the
cell distribution at an
H
2S concentration of ~1.8 mM. If the oxygen
was
removed by flushing the surroundings of the gradient capillary
with
nitrogen, the cells migrated toward regions with less sulfide
concentration. In the case of high sulfide concentrations (Fig.
6A),
this migration finally resulted in an accumulation of all
cells at the
meniscus toward the gas-filled part of the capillary
(data not shown).
This migration stopped in the middle of the
liquid culture medium if
agar plugs with lower sulfide concentrations
were prepared for the
gradient capillary (Fig.
6B). A total of
67% of the cells accumulated
in regions with <10 µM H
2S, where
they formed
a band positioned at the end of the sulfide gradient.
Most of the
sulfide diffusing through the gradient was removed
within the bacterial
band.
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FIG. 6.
Relative cell distribution (bars, arbitrary linear
units) of swimming M. gracile in the light (18 µmol of
photons m2 s1), together with oxygen (),
sulfide (), and pH () profiles. The x axis
represents the distance along the long axis of the capillary. (A) Oxic
preparation with opposing oxygen and sulfide gradients (ca. 7.4 pH).
(B) Anoxic preparation (oxygen replaced by nitrogen).
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Dynamic changes of the oxygen and sulfide distribution could be
observed in response to changing irradiance. Figure
7A shows
the initial steady-state
situation in the light (18 µmol of photons
m
2
s
1). The cells accumulated in the micro-oxic
region (<10 µM O
2)
close to the sulfidic agar
plug. The oxygen penetration depth
in the capillary was ca. 6 mm. The
sulfidic and oxic zones overlapped
within 2 mm. Upon darkening, the
cells migrated toward the oxic
region and formed a band at the
oxic-anoxic interface (Fig.
7B).
After 1 h the position of the
oxygen and sulfide gradients were
shifted ca. 1.5 mm to the left, and
the overlapping zone decreased
to 1.5 mm. The steepness of the oxygen
gradient increased from
41 to 50 µM mm
1.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 7.
Influence of changing illumination intensity on the
relative cell distribution (bars, arbitrary linear units) of swimming
M. gracile together with oxygen (), sulfide (),
and pH () profiles. The x axis represents the
distance along the long axis of the capillary. The thick lines
represent linear regressions. The numbers next to the lines give their
slope in micromolar units/millimeter. (A) Steady state in light
with 18 µmol of photons m2 s1. (B)
Situation after 1 h of darkness.
|
|
Observations under homogeneous conditions.
In all previous
experiments the cells were exposed to oxygen and sulfide gradients,
wherein they could migrate to their "preferred" position. The
sulfidic agar plug could sustain the sulfide gradient for ca. 24 h. Within this period the majority of cells did not lose their
motility. In order to investigate the motility behavior under less
favorable conditions (i.e., conditions which were avoided by the cells
within oxygen or sulfide gradients), the cells were also exposed to
homogeneous oxygen or sulfide concentrations. In the beginning of the
following experiments all cells showed sulfur inclusions.
If the bacteria were exposed to 1 mM S
tot in the
dark, they lost motility within 1 h and attached to the walls of
the capillary.
In contrast, the bacteria remained motile for >3 days
if they
were exposed to oxic conditions (>100 µM
O
2) in the dark without
any sulfide present, and
many cells could be observed in their
dividing state. After 5 days,
only a few bacteria remained motile,
while most had attached to the
capillary walls. At this time the
cells did not show any sulfur
inclusions. When the cell density
in such an oxic preparation in the
dark was high enough, two bands
of swimming bacteria separated by an
anoxic region could be observed
in the capillary (Fig.
8A). When the illumination (18 µmol of
photons m
2 s
1) was
switched on, the bands approached each other and eventually
merged.
Within 30 min, the swimming cells concentrated to a dense
spot ~300
µm in diameter (Fig.
8B and C). The oxygen concentration
showed a
minimum of 9.5 µM at the center of the spot. During the
following 60 min, the spot broadened and finally measured 2 by
6 mm at steady state
(Fig.
8D to G). The broadening was accompanied
by an increase of the
oxygen concentration, which reached ~40
µM at the center of the
spot at steady state. Similar preparations
with lower cell densities
showed oxygen concentrations >100 µM
at steady state in the light.
Cells remained motile for >2.5 days.
After 3 days, most of the cells
were nonmotile and their sulfur
inclusions had disappeared.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 8.
Redistribution of swimming M. gracile
(shaded areas) in response to a dark-light transition in the absence of
sulfide. Note the different scales for panel A versus panels B to G. (A) Top view of flat microslide capillary with the steady-state
distribution in the dark showing two bands of bacteria. (B to G) The
bands merged to a single spot after the dark-light transition. Plots
show oxygen profiles through the center of the spot within 90 min after
illumination with 18 µmol of photons m2
s1 (x axis, millimeters; y
axis, micromolar). Spot dimensions: given by the x axis
in panels B to E; 2 by 3 mm2 in panel F; and 2 by 6 mm2 in panel G. The steady state in light was reached after
90 min.
|
|
| |
DISCUSSION |
Photokinesis and phobic responses.
Our photokinesis data point
to a correlation of swimming velocity and the amount of energy supply
in M. gracile (Fig. 2). Under oxic conditions and low
irradiances the cells can compensate the diminished phototrophic energy
supply by respiring their sulfur inclusions with molecular oxygen
(31). The flagellar motor of bacteria is driven by the
proton motive force across the cell membrane (
p), which is built up
as a result of photosynthetic and respiratory electron transport
(1). ATP can also build up p via reverse activity of
membrane-bound ATPase, and it was shown that externally supplied ATP
increases the swimming velocity of Rhodospirillum rubrum
(30). This might explain the biphasic velocity dynamics
under anoxic conditions exhibited by M. gracile (Fig. 2),
where the fast component of the dynamics was due to the direct coupling
between the photosynthetic electron transport and p, whereas some
intracellular energy storage, such as ATP, caused the slow component.
The motile behavior of
M. gracile can generally be described
as a random walk (
2,
29). Intervals of straight swimming
paths are interrupted by stops, followed by a reversal in swimming
direction. Reversal probability is increased if the cell experiences
a
decrease in electron transport rate (
1). In case of steep
gradients, this behavior results in the observed phobic responses.
A
bacterium crossing the light-dark border produced by a stepped
neutral
density filter (Fig.
3A) experienced the full decrease
in light
intensity within 10 µm. Consequently, the reversal positions
of the
bacteria lie within a band of 20-µm width. Oxygen or sulfide
gradients inside the gradient capillary always extended over several
millimeters due to diffusional processes. This explains the less-sharp
borders of the bacterial bands toward certain limits in oxygen
or
sulfide concentration (Fig.
3B).
The random walk pattern of
M. gracile is different compared
to the one exhibited by the enteric bacterium
Escherichia
coli,
which interrupts its straight swimming paths by tumbling,
resulting
in a random direction change (
2).
M. gracile does not tumble
but reverses its swimming direction. Thus,
M. gracile is trapped
within a region where it experiences
local maxima of transfer
rates in its electron transport chains. In
contrast, the random
direction changes of
E. coli result in
the possibility to move
from one local maximum of chemoattractants to
other local maxima.
We speculate that this difference in motile
behavior reflects
the different habitats of the species. The habitats
of
M. gracile are characterized by light, oxygen, and
sulfide gradients, where
the simple reversal of swimming direction is
sufficient to find
maxima, which provide good growth conditions (e.g.,
the oxic-anoxic
interface in darkness [Fig.
4]). In contrast, random
direction
changes are advantageous for
E. coli,
since enteric bacteria live
in complex three-dimensional environments
with many local chemoattractant
maxima. Furthermore, Duffy and Ford
(
7) speculated that reversing
swimming direction would
lead to fewer bacterium-obstacle collisions
in porous media, which
might be advantageous for motility in benthic
environments.
Engelmann (
9) is generally regarded to be the first to
have investigated the photoresponses of
Chromatium spp. in
the year
1883, when he described "
Bacterium
photometricum." The motility
pattern of this bacterium was not
symmetrical with respect to
both swimming directions. Rather, straight
swimming paths were
interrupted by stops, followed by a short period
with backward
swimming. Thereafter, cells reoriented randomly, and
resumed forward
swimming. We observed a different motile behavior for
M. gracile,
a finding consistent with the observation made
for
Allochromatium vinosum by Mitchell et al.
(
29).
The dynamic behavior of
M. gracile bacterial bands (Fig.
5A)
indicates that the phobic response toward oxygen adapts under
changing
irradiance conditions. It took ca. 10 s before the band
started to
retreat toward the anoxic region, indicating that the
phobic response
to oxygen became active. The comparable adaptation
times of ~10 s
observed for the phobic responses toward light
as well as toward oxygen
point to a tight coupling. In accordance
with the similar swimming
patterns observed for phobic responses
toward light and oxygen (Fig.
3), this underlines the theory that
phototactic and chemotactic
signaling are integrated by shared
components in the electron transport
chain of photosynthetic bacteria
(
1).
Motility behavior and metabolism in gradient chamber.
In
darkness M. gracile can grow chemotrophically if oxygen and
reduced sulfur compounds are both present (21, 22). This explains the accumulation of the bacteria at oxygen levels between 0 and 10 µM in the zone where the oxygen and sulfide gradients overlap
(Fig. 4). Kämpf and Pfennig (22) reported that the best chemotrophic growth conditions for M. gracile were 15 to 30 µM O2, whereas higher concentrations
caused cell lysis. Furthermore, oxygen concentrations of <10 µM
still allow bacteriochlorophyll synthesis, which is suppressed at
higher concentrations (22).
The two steps of sulfide oxidation are given by:
|
(5)
|
|
(6)
|
|
(7)
|
The incomplete oxidation with S
0 as an
endproduct requires 2 mol of sulfide for 1 mol of oxygen (equation
5),
whereas for
complete oxidation (equation
7) the
S
tot/O
2 ratio is 1:2. The
flux calculation for the bacterial band in the dark yielded a
S
tot/O
2 ratio of 2.03:1
(Fig.
4B). Thus, in the dark
M. gracile oxidized sulfide,
incompletely storing S
0 in its sulfur inclusions
(
32). Overmann and Pfennig reported
molar conversion
ratios of between 2.5:1 and 3.3:1 for other species
of purple sulfur
bacteria (
31).
In the light
M. gracile performs anoxygenic photosynthesis,
which does not require the presence of oxygen. Thus, the bacteria
spread into the anoxic part of the gradient capillary (Fig.
5B
and
6A).
The upper limit of the oxygen concentration was still
ca. 10 µM, but
the bacteria also preferred regions with low sulfide
concentrations
(Fig.
6B). Purple sulfur bacteria are well adapted
to such low
concentrations. Overmann reported
Km
values for the
oxygen and sulfide affinity of purple sulfur bacteria of
0.3 to
0.9 µM O
2 and 0.47 µM
S
tot, respectively (
31). Therefore,
the
bacteria can avoid possible harmful effects of high oxygen or
high
sulfide concentrations without limiting their substrate uptake
rates.
All observed distribution patterns of
M. gracile can be
explained by its phobic responses toward light, oxygen, and sulfide.
The photophobic response apparently overrules the other phobic
responses, since shading caused a reversal in swimming direction
of all
bacteria independent of their position in the oxygen or
sulfide
gradient. Furthermore, the phobic response toward oxygen
apparently
overruled the one toward sulfide (Fig.
6).
The influence of
M. gracile's metabolism on its chemical
microenvironment was seen under changing irradiance conditions. The
oxygen respiration of the bacteria increased after a light-dark
transition, and a steeper oxygen gradient indicated a higher oxygen
flux (Fig.
7). Thus, the bacteria and the chemical gradients interact
with each other. The distribution of the swimming cells is determined
by the gradients, but the gradients are simultaneously influenced
by
the metabolism of the
cells.
Motility behavior under adverse conditions.
The interaction
between the oxygen gradient and the cell's metabolism also caused the
dynamic distribution pattern in the experiment shown in Fig. 8. At
steady state in the dark, two bacterial bands removed all oxygen
diffusing into the capillary and kept the space that was in between
anoxic (Fig. 8A). As the respiration decreased in light, the oxygen
concentration increased, forcing the motile bacteria to retreat and
accumulate in the center (Fig. 8B to G). The bacteria were trapped
within their self-generated oxygen minimum due to their phobic response
toward oxygen. The highest bacterial density was observed when the
oxygen distribution showed a minimum of ~10 µM
O2 (Fig. 8D), corresponding to the upper oxygen
limit for the bacterial distributions in the gradient capillary. At
this low oxygen level, the trapping mechanism works most effectively,
but the number of accumulated bacteria was apparently not high enough
to respire all oxygen diffusing toward the center. Consequently, the
oxygen level increased until a steady state between respiration and
oxygen influx was reached. The minimum of the steady-state oxygen
profile (Fig. 8G) demonstrates clearly the ability of M. gracile to respire oxygen also in the light.
The latter observation might explain the formation of nonmotile
colonies in the oxic part of the gradient capillary. Within
colonies
cells experience lower oxygen concentrations, reducing
oxygen-related
damage (
22). The small colonies of 10 to 50 cells
observed
in our experiments are presumably not able to decrease
the oxygen
concentration significantly by their respiration. However,
the
experiment was conducted only over a period of several hours.
Over
longer periods the colonies may increase in size until oxygen
is
effectively removed by the respiring cells. This was, for example,
observed by Seitz et al. (
39) in a salt marsh where purple
sulfur
bacteria formed macroscopic aggregates on the sediment surface.
Tidal currents exposed the aggregates periodically to oxic water,
and
microsensor measurements showed that the inner parts of the
aggregates remained anoxic. The observed colony formation might
also
reflect the first stages of a biofilm
formation.
Cells remained motile under oxic conditions for >2.5 days until their
sulfur inclusions had disappeared. In darkness the bacteria
presumably
oxidize their sulfur inclusions to sulfate, whereas
in the light
anoxygenic photosynthesis with S
0 as an electron
donor is performed (
32). Although it was shown
that
M. gracile does not remain viable after long-term exposures
to >50 µM O
2 (
22), the
demonstrated short-term resistance might
be ecologically important. In
their natural environment
M. gracile can be exposed to high
oxygen concentrations and the motile bacteria
might be able to migrate,
within the next several days, back to
their favorite environment.
Further, bacteria which get trapped
in turbulent water above the
oxic-anoxic interface can remain
viable and may finally colonize other
sulfidic
habitats.
Under anoxic conditions in the dark it was shown that
Chromatium spp. can generate ATP by oxidizing
intracellularly stored
glycogen to poly-
-hydroxybutyric acid.
Elemental sulfur serves
as an electron acceptor, and sulfide is
released (
41). The lower
energy yield of this reaction
compared to phototrophic or chemotrophic
growth modes can explain our
observation that
M. gracile lost
its motility under
prolonged anoxia in the
darkness.
The vertical band structure observed in the gradient capillary under
dark conditions can be explained by the specific density
of the cells.
The specific density of
A. vinosum cells with sulfur
inclusions was determined to be 16% higher than the density of
water,
resulting in a sinking rate of 0.12 µm s
1
(
14). Negative buoyancy increases the probability that
motile
cells are found in the bottom part of the gradient capillary.
Thus, the bottom part of the band exhibited a higher cell density
accomplished with higher volumetric oxygen respiration rates than
the
upper part. The enhanced respiration rate at the bottom part
leads to a
shift of the 10 µM oxygen isopleth toward the oxic
part of the
capillary. Consequently, the bacterial band positioned
itself along the
isopleth resulting in the observed
pattern.
Conclusions.
Our investigation of M. gracile
has identified a range of cellular motility responses that can explain
migration patterns of purple sulfur bacteria in natural benthic
habitats (19). The observed patterns result from phobic
responses of the swimming bacteria. During night purple sulfur bacteria
can move toward micro-oxic regions. If the oxic-anoxic interface is
found below the surface, they presumably form an ~500-µm thin band
at this position. In case of complete anoxia during night, sulfide is released into the overlaying water. In this case, no clearly defined oxic-anoxic interface can build up due to turbulent mixing in the
water. Thus, the bacteria distribute as pink clouds in the overlaying
water, where sulfide and oxygen are simultaneously present. During the
daytime the oxic-anoxic interface moves into deeper layers due to
oxygenic photosynthesis in the upper layers, and purple sulfur bacteria
accumulate in a band below the oxic-anoxic interface. The upper limit
of the band is given by 10 µM O2, while the lower limit
is determined by the attenuation of irradiance with depth
(20). Additionally, M. gracile can
withstand adverse conditions for several days, and the observed
aggregation behavior provides a protection against high oxygen
concentrations. Altogether, M. gracile is well adapted
to dynamic changes in its habitat.
In line with earlier studies of colorless sulfur bacteria
(
11) and sulfate-reducing bacteria (
10), the
combination of
gradient capillaries, microscopy, and microsensor
measurements
is a powerful approach for studying the motility behavior
of single
microorganisms, as well as the migration patterns of cell
populations,
under defined oxygen, sulfide, and light conditions. It
allows
the preparation of microenvironments, which mimic conditions
found
in sulfidic benthic habitats, and also allows the establishment
of various stress scenarios. Microenvironmental conditions and
metabolic rates can be quantified with microsensors at the same
resolution at which simultaneous microscopy can reveal swimming
patterns and band
formation.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the European Commission
(grants MAS3-CT98-5054 and EVK3-CT-1999-00010) and the Danish Natural
Science Research Council (contract 9700549).
We thank Olivier Pringault and Henk Jonkers for providing media and for
advice in handling the cultures. Remy Guyoneaud generously provided the
culture of M. gracile. We thank Tom Fenchel, Andrea Wieland, and Nicholas Blackburn for advice regarding the gradient capillary setup, microsensor measurements, and video cell tracking, respectively. Finally, we thank Anni Glud for manufacturing excellent microsensors.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Marine
Biological Laboratory, University of Copenhagen, Strandpromenaden 5, DK-3000 Helsingør, Denmark. Phone: 45-49-21-33-44. Fax:
45-49-26-11-65. E-mail: roland.thar{at}gmx.net.
| |
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Applied and Environmental Microbiology, December 2001, p. 5410-5419, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5410-5419.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
