Identification of Organelles in Bacteria Similar to Acidocalcisomes of Unicellular Eukaryotes

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.

Magnetosome vesicles are present before magnetite formation, and MamA is required for their activation

“…Recently, other specialized membranous organelles have also been described in prokaryotes (29,30). The presence of organelles in bacteria has significant evolutionary implications.…”

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

“…V-PPase may contribute to parasitic cytosolic pHstasis by pumping H + into the acidocalcisomes [17,23]. However this organelle accumulates also Ca and other divalent cations such as Zn and Fe which co-precipitates with large amounts of polyphosphates.…”

“…These were first described in Trypanosoma brucei [8] and Trypanosoma cruzi [9], then in Leishmania amazonensis [10], Leishmania donovani [11], Plasmodium berghei [12], Plasmodium falciparum [13], Toxoplasma gondii [14] and in organisms such as Chlamydomonas reinhardtii [15], Dictyostelium discoideum [16] and more recently in the bacterium Agrobacterium tumefaciens [17]. These acidic organelles are electron dense, posses a surrounding membrane, have variable size, ranging from 200 ± S.E.…”

Dynamics of polymorphism of acidocalcisomes in Leishmania parasites

“…These were first described in Trypanosoma brucei (Vercesi et al 1994) and Trypanosoma cruzi (Docampo et al 1995) and then shown to be present in different members of the Trypanosomatidae family, in apicomplexan parasites (reviewed in Moreno 1999, 2001;De Souza et al 2000), in other unicellular eukaryotes such as the slime mold Dictyostelium discoideum (Marchesini et al 2002) and the green algae Chlamydomonas rheinardtii (Ruiz et al 2001a), and more recently in the bacterium Agrobacterium tumefaciens (Seufferheld et al 2003). Acidocalcisomes of some of these cells are characterized by possessing a vacuolartype H + -ATPase and/or a vacuolar H + -pyrophosphatase for proton uptake, a Ca 2+ /H + countertransporting ATPase for Ca 2+ uptake, and a Ca 2+ /nH + antiporter for Ca 2+ release (Vercesi et al 1994;Docampo et al 1995;Lu et al 1997, Scott andRodrigues et al 1999a, b).…”