First published online June 16, 2005
Journal of Experimental Biology 208, 2569-2579 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01660
Life at acidic pH imposes an increased energetic cost for a eukaryotic acidophile
Mark A. Messerli1,2,*,
Linda A. Amaral-Zettler1,
Erik Zettler3,4,
Sung-Kwon Jung2,
Peter J. S. Smith2 and
Mitchell L. Sogin1
1 The Josephine Bay Paul Center for Comparative Molecular Biology and
Evolution, Marine Biological Laboratory, Woods Hole, MA 02543, USA
2 BioCurrents Research Center, Program in Molecular Physiology, Marine
Biological Laboratory, Woods Hole, MA 02543, USA
3 Sea Education Association, PO Box 6, Woods Hole, MA 02543, USA
4 Centro de Biología Molecular, Universidad Autónoma de
Madrid, Cantoblanco, Madrid 28049, Spain
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Fig. 2. (A) Transmitted light image of Chlamydomonas sp. isolated from the
Rio Tinto. (B) Pseudocolored images of BCECF fluorescence in
Chlamydomonas sp. The color bar to the right indicates relative pixel
intensity. Imaging parameters were adjusted to eliminate low-level
autofluorescence and to reduce exciting light intensity. The distribution of
dye in the 488 nm (H+-sensitive) is similar to the distribution of
dye in the 440 nm (H+-insensitive). Also the distribution of the
dye is relatively homogenous. The ratio pairs show homogenous intensity except
near the edges where the signal to noise ratio of the ratioed pixel intensity
will decrease due to lower signals in the raw images. Scale bar, 10 µm.
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Fig. 3. Mean cytosolic pH histogram of Chlamydomonas sp., together with
in vivo and in vitro calibration curves for BCECF, expressed
as ratio of fluorescence intensities (pH sensitive/insensitive wavelengths).
The calibration curves remain linear between pH 6 and 8. Weak acids and a weak
base were used to clamp the cytoplasmic pH to ensure that the dye retained its
H+ binding characteristics in the cells.
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Fig. 4. An example of an in vivo BCECF calibration record, obtained by
manipulation of cytosolic pH, expressed as ratio of fluorescence intensities
(pH sensitive/insensitive wavelengths). A weak acid and weak base were used to
clamp the cytosolic pH at 5 and 9 while a H+ ionophore was used to
collapse the pH gradient at pH 7. The cytosolic pH is reported from a single
Chlamydomonas sp. originally in pH 2 medium (arrow A) and then
transferred to pH 7 medium starting at arrow B. A H+ ionophore was
added (arrow C) to collapse the transmembrane pH gradient. A slight
acidification occurred just before the medium was exchanged with pH 5 medium
containing 10 mmol l-1 acetic acid. Once the cytoplasmic pH had
stabilized, the medium was exchanged with pH 9 medium containing 10 mmol
l-1 ammonia. This raised the cytoplasmic pH to 9 (arrow E), leading
to a full-range calibration for the pH indicator in vivo.
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Fig. 5. Current-voltage (I-V) relationships acquired from
acidophilic Chlamydomonas sp. at pH 7 and pH 2. (A,B) Current
recordings obtained at ±100 mV, acquired from the same acidophile at
external pH 7 (A) and pH 2 (B). (C) Summary I-V plot from
seven acidophiles normalized to cell surface area. The currents at pH 2 (open
circles) are shifted to more negative potentials to correct for the change in
glass charge during the change in extracellular pH. Inward currents are
linearly proportional to the change in membrane potential and are greater at
pH 2 (open circles) than at pH 7 (filled squares). Outward currents rise more
rapidly with voltage than inward currents, as displayed by the difference
between the plotted lines and the dotted lines representing a continuation of
the slope of the inward currents. Values are means ±
S.E.M.
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Fig. 6. (A) Relative amounts of cellular ATP over time. Control measurements
(filled squares) are displayed as the ratio of cellular ATP in cells placed at
pH 2 and pH 7. There is no significant difference in ATP between the cells for
at least 1 h. After 105 and 165 min, there is a 17% and 10% increase in the
ATP ratio, respectively, which is due to a decrease in ATP levels in the cells
at pH 7. Open squares, ratio of the relative amount of ATP in azide-treated
cells. Filled circles, normalization of the relative amount of cellular ATP in
azide-treated and control cells. There is a significant decrease in cellular
ATP in azide-treated cells in pH 2 medium at 5, 25, 45 and 65 min. At 105 and
165 min, ATP levels have recovered in the cells growing in pH 2 so that there
is no longer a significant difference between the cells growing at pH 2 and pH
7. (B) Rate of change of the relative amounts of ATP in azide-treated cells at
pH 2 and pH 7. The difference is plotted as the percent of the total cellular
ATP pool at the middle time point between the two measuring points in A
(filled circles). Relatively higher ATP consumption occurs for nearly 40 min,
after which time the cells in pH 2 medium produce relatively more ATP. At 135
min the rate of ATP change is nearly the same in cells growing at pH 2 and pH
7. The greatest rate of consumption was 0.027% of the total cellular ATP pool
per second. This was the average for the first 5 min of measurement in A
(filled circles), plotted here at 2.5 min.
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© The Company of Biologists Ltd 2005
7.5. Growth at pH 2
is not significantly different from growth at pH 3, 4, 5.5 or 6.0
(t-tests, P>0.2 for all). C. reinhardtii does
not grow at pH 2 and has lower growth at pH 3 and 4 but is growing faster than
Chlamydomonas sp. at pH