Identification of Organelles in Bacteria Similar to Acidocalcisomes of Unicellular Eukaryotes*
Manfredo Seufferheld, Mauricio C. F. Vieira, Felix A. Ruiz, Claudia O. Rodrigues, Silvia N. J. Moreno and Roberto Docampo‡
Author Affiliations
Laboratory of Molecular Parasitology, Department of Pathobiology and Center for Zoonoses Research, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
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@uiuc.edu.
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 ofAgrobacterium 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 reagentN-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.
Footnotes
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.
* 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.
Received April 30, 2003.
Revision received May 29, 2003.
The American Society for Biochemistry and Molecular Biology, Inc.
