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J. Biol. Chem., Vol. 278, Issue 32, 29971-29978, August 8, 2003
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Identification of Organelles in Bacteria Similar to Acidocalcisomes of Unicellular Eukaryotes*
From the Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
Received for publication, April 30, 2003 , and in revised form, May 29, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Acidocalcisomes are acidic calcium storage compartments described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds. In this work, we report that the volutin granules of Agrobacterium tumefaciens possess properties similar to the acidocalcisomes. Transmission electron microscopy revealed that each intracellular granule was surrounded by a membrane. X-ray microanalysis of the volutin granules showed large amounts of phosphorus, magnesium, potassium, and calcium. Calcium in the volutin granules increased when the bacteria were incubated at high extracellular calcium concentration. Immunofluorescence and immunoelectron microscopy, using antisera raised against peptide sequences conserved in the A. tumefaciens proton pyrophosphatase, indicated localization in intracellular vacuoles. Purification of the volutin granules using iodixanol density gradients indicated a preferential localization of the pyrophosphatase activity in addition to high concentrations of phosphate, pyrophosphate, short- and long-chain polyphosphate, but lack of markers of the plasma membrane. The pyrophosphatase activity was potassium-insensitive and inhibited by the pyrophosphate analogs, amynomethylenediphosphonate and imidodiphosphate, by dicyclohexylcarbodiimide, and by the thiol reagent N-ethylmaleimide. Polyphosphate was also localized to the volutin granules by 4',6'-diamino-2-phenylindole staining. The organelles were acidic, as demonstrated by staining with LysoSensor blue DND-167, a dye especially used to detect very acidic compartments in cells, and cycloprodigiosin, a compound isolated from a marine bacterium that has been shown to uncouple proton pyrophosphatase activity acting as a chloride/proton symport. The results suggest that acidocalcisomes arose before the prokaryotic and eukaryotic lineages diverged.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Intracellular membranous structures have been reported in several bacterial species (1–5). One group is formed by the abundant membrane-bound vesicles of photosynthetically grown cells of several purple bacteria, such as Rodospirillum rubrum and Rhodopseudomonas spheroides (4). These vesicles are usually designated as chromatophore membranes because of their similarity to particulate fractions known as chromatophores that could perform photophosphorylation in vitro, and that are obtained after sonication of these bacteria (4). Cyanobacteria possess sac-like membranes (thylakoids) with the same function (5). Another group of intracellular membranous structures is formed by a number of inclusions, such as the poly-
-hydroxybutyrate-containing granules of several bacteria,
the sulfur globules of the Thiorhodaceae, the gas vacuoles of aquatic
bacteria, the chlorophyll-containing Chlorobium vesicles, the
ribulose-1,5-bisphosphate carboxylase-oxygenase-rich carboxysomes of
cyanobacteria, nitrifying bacteria, thiobacilli, and the related polyhedral
bodies or enterosomes of Salmonella enterica
(1–3,
5). These are not considered to
be equivalent to the organelles found in the eukaryotes
(2). They usually consist, with
the exception of carboxysomes and perhaps enterosomes, of a material lacking
enzymatic function. They are surrounded by a non-unit membrane apparently
composed entirely of protein
(1,
2). Other, more recently
described intracytoplasmic membranes found in Planctomycetales are
the pirellulosome, which surrounds the cell DNA in Pirellula species,
and the anammoxosome of Candidatus species
(6). The anammoxosome is a
dedicated intracytoplasmic compartment where the anammox process (anaerobic
ammonium oxidation) takes place
(6,
7). All of these membranous
structures are different from the mesosomes described initially in
Gram-positive bacteria and that were shown to arise as a fixation artifact
(8). In addition to all of
these membrane-bound compartments, a number of apparently non-membrane bound
inclusions are also present in several bacterial species, such as the
multi-L-arginyl-poly(L-aspartic acid)-containing
cyanophycin granules of cyanobacteria, the polyglucoside granules containing
polymers of glucose, and the volutin granules
(1,
2,
5). Volutin or metachromatic granules were the first subcellular entities to be recognized in bacteria (9). Their metachromatic nature is attributable to the reaction of the polyphosphate (polyP)1 present in the granules with certain basic dyes, such as toluidine blue, which induces a characteristic shift in their absorption spectrum to a higher wavelength (10). They have been described as devoid of internal structure or limiting membrane (11) and have also been found in unicellular eukaryotes (12–16). In recent years, volutin granules of unicellular eukaryotes such as Chlamydomonas reinhardtii (17), Dictyostelium discoideum (18), and a number of human pathogens, including malaria parasites, Toxoplasma gondii, and trypanosomatids (19), were found to be surrounded by a membrane with a number of pumps and exchangers and were named acidocalcisomes. Acidocalcisomes are characterized by their acidic nature, their high electron density, and their high concentration of calcium, magnesium, and other elements in addition to pyrophosphate (PPi) and polyP. It has been postulated that acidocalcisomes may have important roles as energy stores and in intracellular pH, calcium, and osmotic regulation (19).
Because volutin granules in bacteria and unicellular eukaryotes are
morphologically similar, we investigated whether the bacterial volutin
granules are also surrounded by a membrane and have similar characteristics to
the acidocalcisomes. We chose for our initial studies the
-protobacterium Agrobacterium tumefaciens, which is known to
possess a H+-translocating pyrophosphatase (H+-PPase)
(20), a marker for
acidocalcisomes in unicelullar eukaryotes
(19). In this report, we
describe the isolation and biochemical properties of volutin granules of
A. tumefaciens and demonstrate that, as the acidocalcisomes, they are
surrounded by a membrane, acidic because of the presence of the
H+-PPase in their membrane, rich in PPi and polyP, and
able to accumulate calcium and other elements. Acidocalcisomes are therefore
the first organelles described in prokaryotes that are also present in
eukaryotes.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Cultures—A. tumefaciens (strain C58) was provided by Stephen K. Farrand (University of Illinois at Urbana-Champaign). Cells were grown in liquid Luria Bertani (LB) medium at 28 °C with agitation. Cells were cultured overnight and harvested at the stationary phase.
Isolation of Volutin Granules—Bacteria were collected by
centrifugation and incubated with a solution containing 0.5 M NaCl,
20 mM EDTA, 50 mM Tris-HCl, pH 8.0, and 1%
N-lauroyl-sarcosine, to disrupt the bacterial capsule, for 20 min at
room temperature. Bacteria were then centrifuged at 3,900 x g,
and the pellet was resuspended in lysis buffer (125 mM sucrose, 50
mM KCl, 4 mM MgCl2, 10 mM EDTA, 20
mM K-Hepes, 5 mM dithiothreitol, 0.1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 10 µM pepstatin, 10
µM leupeptin, 10 µM
trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane,
and 10 µM N-tosyl-L-lysine
chloromethyl ketone, pH 7.2) containing 2 mg/ml lysozyme and incubated on ice
for 30 min. Benzonase® (Novagen) (1 µl/ml) was added, and bacteria were
passed through a French press (SLM-Amico, Spectrometric Instruments) two times
at 1,000 psi. The lysate was incubated on ice under agitation for 1 h with an
equal volume of 0.5x silica (silicon dioxide) and 0.5x silicon
carbide solution to remove fragments of DNA and RNA. The lysate was then
centrifuged at 1,000 x g for 5 min and washed two times under
the same conditions. The supernatant fractions were combined and centrifuged
for 10 min at 14,500 x g. The pellet was resuspended in 2 ml of
lysis buffer with the aid of a 22-gauge needle. The suspension was diluted 1:1
in OptiPrep (60% iodixanol) and applied as the 30% step of a discontinuous
gradient of OptiPrep, with 4-ml steps of 24, 28, 30, 35, and 40% iodixanol,
diluted in lysis buffer. The gradient was centrifuged at 50,000 x
g in a Beckman SW 28 rotor for 60 min. The volutin granule fraction
pelleted at the bottom of the tube and was resuspended in lysis buffer.
Gradient fractions and markers were assayed as described previously
(21).
Analytical Methods and Immunoblotting—Bacteria were washed once with Dulbecco's phosphate-buffered saline (PBS) and PPi, and long-chain and short-chain polyPs were extracted as described before (22). Pyrophosphatase activity was assayed by measuring released phosphate using the EnzChek phosphate assay as described before (21, 23). The apparent Km for PPi was calculated by using a computerized nonlinear regression program (Sigma Plot 1.0, Jandel Scientific) using the Hill equation. Protein determination was done with the Coomassie Blue protein assay reagent from Bio-Rad. Proteins were separated by SDS-PAGE, using 10% gels, and blotted onto nitrocellulose using a Bio-Rad Transblot apparatus by standard techniques. Subsequent processing steps were done in Dulbecco's PBS containing 0.1% Tween 20. Blots were blocked for 1 h in 5% nonfat dry milk, washed three times, and incubated with polyclonal antibody against Trypanosoma cruzi H+-PPase (24) (1:5,000) for 1 h at room temperature. Blots were then washed three times, incubated for 1 h with horseradish peroxidase-labeled anti-mouse IgG (1:20,000), washed three times, and processed for chemiluminescence detection as per the manufacturer's (Amersham Biosciences) instructions. Molecular weights were calculated using prestained molecular weight markers.
Immunofluorescence Microscopy—For subcellular localization of H+-PPase, bacteria were washed with Dulbecco's PBS and fixed in 4% freshly prepared formaldehyde for 10 min at room temperature and 50 min at 4 °C, attached to poly L-lysine-treated glass slides, and permeabilized with 0.2% Nonidet P-40 in PBS for 5 min. Samples were blocked for 1 h with PBS containing 3% bovine serum albumin, 1% cold fish gelatin, and 50 mM NH4Cl and first incubated for 1 h at room temperature with the polyclonal antibody against the T. cruzi H+-PPase (24) diluted 1: 20 in 1% cold fish gelatin. Bacteria were subsequently incubated for 60 min at room temperature with rhodamine-conjugated secondary antibody diluted 1:100 in PBS plus 1% cold fish gelatin. Coverslips were mounted in glass slides with Vectashield® media and sealed. Images were collected with an Olympus laser scanning confocal microscope or an Olympus BX-60 fluorescence microscope.
For polyP localization, bacteria were washed twice with Dulbecco's PBS and resuspended in the same buffer and fixed for 30 min with 4% formaldehyde. 45 µl of this suspension was incubated at room temperature with 10 µg/ml 4',6-diamino-2-phenylindole (DAPI). After 10 min, the samples were mounted on a slide and observed with the fluorescence microscope.
For localization of LysoSensor blue DND-167 (9,10-bis(N-morpholinomethyl)anthracene), bacteria were centrifuged and resuspended in prewarmed (28 °C) LB medium containing 1 µM LysoSensor. Bacteria were incubated for 1 h at 28 °C, centrifuged, and resuspended in fresh prewarmed LB medium. Bacteria were mounted on a slide and observed with the fluorescence microscope using UV excitation. For cycloprodigiosin detection, bacteria were centrifuged, resuspended in Dulbecco's PBS containing 100 nM cycloprodigiosin, and incubated for 30 min. Bacteria were mounted on a slide and observed with the fluorescence microscope using a red emission filter. Bacteria resuspended in Dulbecco's PBS or LB medium, but without cycloprodigiosin or LysoSensor, respectively, were used as controls.
Electron Microscopy and X-ray Microanalysis—For routine electron microscopy, bacteria were washed with Dulbecco's PBS and fixed for 1 h with 2.5% grade II glutaraldehyde, 4% freshly prepared formaldehyde, 0.03% CaCl2, and 0.03% picric acid in 0.1 M cacodylate buffer, pH 7.2. Bacteria were post-fixed with OsO4 for 45 min and then for 15 min with potassium ferricyanide, washed, and treated with 2% uranyl acetate for 30 min. Subsequently, samples were dehydrated by successive incubations of 6 min with increasing concentrations of ethanol (10, 25, 50, 75, 95, and 100%) at room temperature. Epoxy embedding was carried out by resuspending the sample once in 1:1 ethanol/acetonitrile, twice in 100% acetonitrile, and then 30 min in 1:1 epoxy/acetonitrile, 1.5 h in 3:1 epoxy/acetonitrile, and 4 h in 100% epoxy. Embedded samples were polymerized for 20 h at 85 °C. Epoxy blocks were ultrathin-sectioned, sections were deposited on 300-mesh copper grids, and grids were stained with uranyl acetate for 30 min and triple lead stain (lead citrate, lead nitrate, and lead acetate) for 1 min.
For immunocytochemistry, bacteria were washed with Dulbecco's PBS, fixed for 1 h at 4 °C in a solution containing 0.5% grade I glutaraldehyde, 4% freshly prepared formaldehyde, and 1% picric acid in 0.1 M cacodylate buffer, pH 7.2. Fixed bacteria were washed with Dulbecco's PBS and dehydrated by successive incubations of 6 min with increasing concentrations of ethanol (10, 25, 50, 75, 95, 100, and 100%) at –20 °C. Samples were embedded in Unicryl at 4 °C by incubation with 1:1 ethanol/Unicryl for 1 h and 100% Unicryl for 1, 16, and 8 h. Embedded samples were polymerized under UV irradiation at –20 °C for 48 h. Thin sections were collected on 300-mesh nickel grids and blocked for 30 min with PBS containing 0.1% Tween 20 and 0.5% cold fish gelatin (PBS-TW-FG). Grids were incubated for 3 h with a mouse monoclonal antibody against T. cruzi H+-PPase (24) diluted 1:10 in PBS-TW-FG. After washing in PBS-TW-FG, grids were incubated for 1 h with a 5-nm gold-conjugate goat anti-mouse antibody. Subsequently, grids were washed with PBS, then in distilled water, and stained with uranyl acetate and lead citrate. Routine and immunocytochemistry samples were observed in a Hitachi H 600 electron microscope.
For imaging whole bacteria and electron-dense vacuole fractions, the preparations were washed in 0.25 M sucrose, and a 5-µl sample was placed on a Formvar-coated, 200-mesh copper grid, allowed to adhere for 10 min at room temperature, blotted dry, and observed directly with a Hitachi H 600 transmission electron microscope operating at 100 kV (18). Energy-dispersive x-ray microanalysis was done at the Electron Microscopy Center (Southern Illinois University, Carbondale, IL). Specimen grids were examined in a Hitachi H-7100FA transmission electron microscope at an accelerating voltage of 50 kV. Fine probe sizes were adjusted to cover the electron-dense vacuoles (or a similar area of the background), and x-rays were collected for 100 s by using a thin-window (Norvar) detector. Analysis was performed by using a Noran Voyager III analyzer with a standardless analysis identification program.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Volutin granules are recognized by their high electron density when the cells are observed by transmission electron microscopy without fixation and staining (5). A. tumefaciens typically shows a large granule toward one of the cellular poles (Fig. 1A, arrows) and additional smaller granules in different regions of the cells (Fig. 1A, arrowheads). The large granules have a diameter of about 210 ± 18 nm. X-ray microanalysis was performed on these granules (Fig. 1B). The spectrum shown was the one that yielded the most counts in 100 s (of 10 spectra obtained from granules of different bacteria), but all other spectra taken from volutin granules were qualitatively similar; counts for phosphorus were about 4-fold greater than counts for magnesium, which were about the same as counts for potassium. Counts for calcium were much lower. Counts for oxygen and phosphorus were about the same. Peaks for phosphorus, magnesium, potassium, and calcium were not present in spectra taken from the background (Fig. 1C). Peaks for copper, and in part for carbon and oxygen, arise from the grid.
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Examination of cells in thin sections showed a round vacuole of about 210 nm in one of the poles of the cell, corresponding to the location of the large granules in Fig. 1A. An electron-dense inclusion partially (Fig. 1, D and E, arrows) or completely (Fig. 1E, arrowhead) occupied its interior. Each intracellular granule appeared to be surrounded by a membrane (Fig. 1, E and F, arrow and arrowhead, respectively). The determination of the membrane was made difficult because of the so-called "phase" or "out of focus" effect often accompanying electron-dense particles of bacteria visualized by transmission electron microscopy (25). A better identification of the surrounding membrane was achieved with isolated volutin granules. As has been observed in acidocalcisomes of unicellular eukaryotes (19), a ring of electron-dense material was observed below the membrane of the isolated volutin granules (Fig. 1, G and H).
To purify the volutin granules and investigate their chemical and enzymatic content, we adapted the purification procedure used for the isolation of acidocalcisomes from T. cruzi (21). The utility of the method was assessed by assaying marker enzymes (Fig. 2). The yield of pyrophosphatase activity (a marker of acidocalcisomes) in the volutin granule fraction (fraction 12) was 80% (Fig. 2A), whereas the yield of protein in the same fraction was only 3% (Fig. 2H), a 27-fold purification. Plasma membrane (marked by succinate cytochrome c reductase) was not enriched in this fraction (Fig. 2B). The volutin granule fraction (fraction 12) contained around 20% of the total amount of PPi (Fig. 2D) and short chain polyP (Fig. 2E), and >35% of the total amount of Pi (Fig. 2C) and long chain polyP (Fig. 2F). Levels of PPi of 274 ± 77 nmol/mg protein and of polyP of 315 ± 79 and 217 ± 19 nmol/mg protein (in terms of Pi residues) in chains of <50 residues long and in chains of about 700–800 residues long, respectively, were found in total extracts of A. tumefaciens.
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Electron microscopy of the volutin granule fraction (fraction 12), by observation of air dried samples (Fig. 3A), showed the presence of granules with the same appearance as the granules observed in preparations of whole cells (Fig. 1A). When submitted to the electron beam, changes in their internal structure led to the appearance of a sponge-like structure, which has been described before in acidocalcisomes of T. cruzi (26), T. brucei (27), and C. reinhardtii (17). The results of x-ray microanalysis of the isolated granules were similar to those of whole cells, except that proportionally less potassium and calcium were detected (Fig. 3B), probably because of ionic changes occurring during the fractionation procedure. This phenomenon has also been described after isolation of acidocalcisomes of unicellular eukaryotes (17–19).
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To investigate whether A. tumefaciens volutin granules are similar to acidocalcisomes of unicellular eukaryotes (19), we used fluorescence microscopy and x-ray microanalysis methods to detect their ability to accumulate H+ and calcium and their polyP content. Fig. 3D shows that incubation of whole bacteria with LysoSensor Blue DND-167, a dye especially used to detect very acidic compartments in cells (28), stained granules located at one pole of the cells (arrowheads), at the same position where the large volutin granules were located by direct transmission electron microscopy (Fig. 1A). Cycloprodigiosin is a compound isolated from a marine bacterium that has been shown to uncouple H+-PPase activity acting as a chloride/proton symport (29) and that has been shown to stain acidocalcisomes of T. cruzi (21). Fig. 3E (arrowheads) shows that cycloprodigiosin-stained granules located at the same position as those stained by LysoSensor (Fig. 3D, arrowheads). DAPI has been shown to shift its maximum emission fluorescence from 426 to 525 nm in the presence of polyP, this change being specific for polyP and not produced by PPi or other anions (22, 30). We observed localization of DAPI-reactive material in the granules located at one pole of the cell (Fig. 3F, arrowheads). The medium used to grow A. tumefaciens for the previous experiments is calcium-deficient. This might explain the lower counts for calcium in the x-ray microanalysis of the volutin granules (Fig. 1B) as compared with acidocalcisomes (19). We therefore cultivated the bacteria in the presence of 100 mM CaCl2 for 16 h before preparing them for x-ray microanalysis. Fig. 3G shows a dramatic increase in the counts for calcium and no counts for magnesium and potassium in the volutin granules of these cells. The presence of these elements was not detected in spectra taken from the background (Fig. 3H) and demonstrates the ability of volutin granules to accumulate calcium.
Acidocalcisomes are characterized by the presence of a membrane-bound H+-PPase that maintains their acidity (19). The pyrophosphatase activity detected in the volutin granule fraction of A. tumefaciens (Fig. 2A), as measured by inorganic phosphate detection (23), was 0.69 ± 0.03 µmol of pyrophosphate consumed/min x mg protein (means ± S.E. of results from three separate experiments) and was totally inhibited by 20 µM aminomethylenediphosphonate (AMDP), a specific inhibitor of H+-PPases. As expected for a type 2 H+-PPase (31), the enzyme was potassium-insensitive (data not shown). The dependence of the initial rate of hydrolysis on pyrophosphate concentration in A. tumefaciens volutin granule fraction is shown in Fig. 4A. Activity was maximal at about 20 µM pyrophosphate with an apparent Km of 5.5 µM. Fig. 4B shows the effect of pH on the initial rate of pyrophosphate hydrolysis in A. tumefaciens volutin granule fraction. Activity was optimal at pH 7.5–8.0. Pyrophosphate hydrolysis of the volutin granule fraction was inhibited, in a dose-dependent manner, by pyrophosphate analogs AMDP (Fig. 4C) and imidodiphosphate (Fig. 4D). Fig. 4 also shows that potassium fluoride (Fig. 4E), dicyclohexylcarbodiimide (Fig. 4F) and the thiol reagent N-ethylmaleimide (Fig. 4G), agents known to inhibit the H+-PPases from plants (32), trypanosomatids (27, 33), and apicomplexan parasites (24, 34) were also effective in inhibiting the A. tumefaciens pyrophosphatase activity in a dose-dependent manner.
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To confirm that the pyrophosphatase in the volutin granules was a H+-PPase, we investigated its localization by immunocytochemistry using an antibody (24) against a peptide of T. cruzi H+-PPase (amino acids 735–761) (33) that is conserved (Fig. 5A) in the C-terminal region of the A. tumefaciens sequence available in GenBank (accession number AAL42186 [GenBank] ; amino acids 641–669). This antibody showed cross-reactivity with a band of 72 kDa present in the A. tumefaciens volutin granule fraction (Fig. 5B, left lane). No background staining was observed when pre-immune serum was used as a control (Fig. 5B, right lane). Immunofluorescence microscopy using these antibodies resulted in staining of granules located at one pole of the cells (Fig. 5C, arrowheads, and inset), at the same position where the large volutin granules were located by direct transmission electron microscopy (Fig. 1A) or by LysoSensor (Fig. 3D), cycloprodigiosin A (Fig. 3E), and DAPI (Fig. 3F) staining. These results are in agreement with the colocalization of polyP and the pyrophosphatase in the volutin granules as assayed biochemically (Fig. 2). To analyze in more detail the structures labeled with the anti-H+-PPase antibody, immunoelectron microscopy was performed on thin sections of bacteria embedded in Unicryl hydrophilic resin. The results obtained confirmed labeling in vacuoles with an "empty" appearance located at one pole of the cell (Fig. 5D, arrows).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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We report that volutin granules of A. tumefaciens are discrete intracellular acidic compartments rich in Pi, PPi, polyP, magnesium, potassium, and calcium that possess an enclosing membrane with a calcium-accumulating activity and a pyrophosphatase to maintain their acidity. A. tumefaciens volutin granules provide evidence for the existence of an organelle common to both prokaryotes and eukaryotes. The structural and biochemical resemblance of volutin granules of A. tumefaciens with eukaryotic acidocalcisomes suggests potential functional similarities.
H+-PPases (31, 35), as volutin granules (11, 19), are widely distributed phylogenetically. Heterologous expression studies have documented the attribution of K+ requirement to a particular sequence. Enzymes from the archeon Pyrobaculum aerophilum (36) the plant Arabidopsis thaliana (AVP2) (37) and the bacterium R. rubrum (38) are capable of PPi hydrolysis in the absence of K+, whereas those from the plants A. thaliana (AVP1) (39) and Vigna radiata (40), the bacteria Thermatoga maritima (41) and Carboxydothermus hydrogenoformans (31), and the trypanosomatid T. cruzi (33) require millimolar concentrations of K+ for activity (31). On the basis of phylogenetic analyses of a large set of H+-PPase sequences, it was suggested that K+-dependent and K+-independent enzymes form two independently evolving groups (35, 41). A comparison of the amino acid conservation patterns in K+-dependent and K+-independent H+-PPases revealed only two sequence positions where a residue conserved in one type is absent from the equivalent position in all the sequences from the other type (31). K+-independent H+-PPases (or type 2 H+-PPases) possess conserved Lys and Thr in two sequence positions that are absent in K+-dependent H+-PPases (or type 1 H+-PPases) and substitution of Lys for Ala in C. hydrogenofomans H+-PPase is sufficient to confer K+ independence to both PPi hydrolysis and PPi-energized H+-translocation (31). It was suggested that this is attributable to the substitution of K+ with the NH3+ group of Lys (31). In agreement with these conclusions, we found that the A. tumefaciens H+-PPase, the sequence of which has Lys and Thr in the positions investigated (31), is K+-insensitive. Only another proteobacterial H+-PPase has been biochemically characterized, the enzyme from R. rubrum (38, 42, 43). As this enzyme, the H+-PPase from A. tumefaciens, was sensitive to inhibition by aminomethylenediphosphonate, dicyclohexylcarbodiimide, and N-ethylmaleimide and has low sensitivity to the soluble pyrophosphatase inhibitor fluoride (Fig. 4).
Having a homolog enzyme in a similar organelle is a strong argument of the common origin of the two organelles. Many of the organisms described to possess this enzyme (31, 35) are also known to possess volutin granules or acidocalcisomes, such as bacteria of the genera Rhodospirillum, Rhodopseudomonas, Chlorobium, and Chromatium (4, 11), the apicomplexan parasites Plasmodium falciparum (34) and T. gondii (24), the trypanosomatids T. cruzi, T. brucei, and Leishmania spp. (19), the ciliate Tetrahymena pyriformis (14), the slime mold D. discoideum (18), and the green alga C. reinhardtii (17). Evidence for the presence of the H+-PPase in acidocalcisomes of several of these organisms has been published (17–19, 24, 34), but definitive evidence is still lacking for others. Further work is also needed to investigate the evolutionary relationship of acidocalcisomes to other organelles with some similarities in morphology and composition, such as the plant and yeast vacuoles, and the secretory granules of higher eukaryotes, such as the dense granules of platelets, the chromaffin granules of the adrenal gland, the atrial natriuretic factor granules of the heart, and many others, which are also acidic and contain calcium (44), and are often referred to as lysosome-related organelles or secretory lysosomes (45).
In conclusion, volutin granules or acidocalcisomes are widely distributed in organisms from different phylogenetic groups. The elucidation of the function of acidocalcisomes in eukaryotic organisms and in their bacterial counterparts may have significant and widespread implications on important issues, such as pathogenesis and adaptive mechanisms to changing environments as well as in the dynamics of the evolutionary process of prokaryotic and eukaryotic organisms. The origin of eukaryotic organelles is a matter of extensive debate, although the principle of endosymbiosis has been advanced as one of its possible mechanisms. Whether this organelle originated prior to the basal bifurcation of the universal tree of life (46) and was maintained in both prokaryotes and eukaryotes deserves further investigation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant AI23259 (to R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of Molecular
Parasitology, Dept. of Pathobiology and Center for Zoonoses Research,
University of Illinois at Urbana-Champaign, 2001 S. Lincoln Ave., Urbana, IL
61802. Tel.: 217-333-3845; Fax: 217-244-7421; E-mail:
rodoc{at}uiuc.edu.
1 The abbreviations used are: polyP, polyphosphate; PPi, pyrophosphate,
H+-PPase, proton pyrophosphatase; LB, Luria Bertani; PBS,
phosphate-buffered saline; DAPI, 4',6-diamino-2-phenylindole; AMDP,
aminomethylenediphosphonate.
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ACKNOWLEDGMENTS |
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We thank Arthur Kornberg for Escherichia coli CA38 pTrcPPX1, Hajime Hirata for cycloprodigiosin, Michael Martin for AMDP, John Bozzola and Steve Schmitt for help with the x-ray microanalysis, Stephen K. Farrand for the C58 A. tumefaciens strain, Yinping Qin and Shuhong Luo for technical advice, and David A. Scott for critically reading the manuscript.
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