Abstract
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.
Highlights
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]
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
Cyanobacteria possess sac-like membranes 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-oxygenaserich carboxysomes of cyanobacteria, nitrifying bacteria, thiobacilli, and the related polyhedral bodies or enterosomes of Salmonella enterica [1,2,3, 5]
Summary
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-oxygenaserich carboxysomes of cyanobacteria, nitrifying bacteria, thiobacilli, and the related polyhedral bodies or enterosomes of Salmonella enterica [1,2,3, 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 nature is attributable to the reaction of the polyphosphate (polyP) 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]. This paper is available on line at http://www.jbc.org
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