Bacterial Origin of Acidocalcisomes - UNIVERSITY OF ILLINOIS

Goals / Objectives
Our specific aim is to demonstrate that acidocalcisomes are present in the bacterium Rhodospirillum rubrum and in pathogenic bacteria. This demonstration will provide further support to our recent report on the presence of these organelles in bacteria [1], that has had considerable recent impact (see comments in Nature, Science, and New Scientist, [2-4]). Eukaryotic organelles are said to have appeared during evolution only as a result of endosymbiosis of prokaryotes with early eukaryotes. The presence of acidocalcisomes (defined as membrane-bound "granules" containing polyphosphate (polyP), and different elements, enzymes and transporters) in many microorganisms such as bacteria, fungi, algae, and protozoa and its apparent absence in mammalian cells would make them promising targets for chemotherapy.

Project Methods
The characterization of acidocalcisomes in R. rubrum will involve the following work: (1) We will improve the isolation procedure to obtain pure acidocalcisomes. This will be accomplished by changes in the protocol used, such as changes in the material used (Percoll, Iodixanol, or Sucrose), gradient used, use of enzymes to degrade the cell wall or eliminate DNA or RNA that could trap the organelles because of their viscosity; (2) Investigation of the chemical composition of the acidocalcisomes as compared with intact cells, by using biochemical techniques or 31P-NMR spectroscopy to detect polyP and PPi (in collaboration with Dr. Eric Oldfield's group at the Department of Chemistry); (3) Ultrastructural, biochemical and molecular characterization of pumps and transporters present in their membranes, such a V-H+-ATPase, H+-PPase, Ca2+-ATPase, Na+/H+ exchanger, Ca2+/H+ exchanger, polyP kinases and phosphatases; and (4) Investigation of the presence of acidocalcisomes in other bacteria of medical importance such as those of the genera Corynebacterium, Mycobacterium, Brucella, and Helicobacter. All the methods that will be used have been used before in the study of acidocalcisomes from A. tumefaciens [1], from different trypanosomatid and apicomplexan parasites [10-32], the green algae Chlamydomonas reinhardtii [33], and the slime mold Dictyostelium discoideum [34], and we do not see any problem with their application to prokaryotes.

Progress 07/01/03 to 06/30/04

Outputs
Acidocalcisomes are acidic, calcium storage compartments with a H+ pump located in their membrane that have been described in several unicellular eukaryotes, including trypanosomatid and apicomplexan parasites, algae, and slime molds, which have also recently been found in the bacterium Agrobacterium tumefaciens. We report that the H+-pyrophosphatase (H+-PPase) of Rhodospirillum rubrum, the first enzyme of this type that was identified, is located in its acidocalcisomes, in addition to the plasma membrane. The identification of the acidocalcisomes of R. rubrum was carried out by using transmission electron microscopy, X-ray microanalysis, and immunofluorescence microscopy. Purification of acidocalcisomes using iodixanol gradients indicated co-localization of the H+-PPase with pyrophosphate (PPi) and short and long chain polyphosphates (polyPs), but lack of markers of the plasma membrane. PolyP was also localized to the acidocalcisomes by using 4',6'-diamino-2-phenylindole staining and identified by using 31P-NMR and biochemical methods. Calcium in the acidocalcisomes increased when the bacteria were incubated at high extracellular calcium concentrations. The number of acidocalcisomes and chromatophore membranes as well as the amounts of PPi and polyP, increased when bacteria were grown in the light. Taken together, these results suggest that the H+-PPase of R. rubrum has two distinct roles depending on its location acting as an intracellular proton pump in acidocalcisomes but in PPi synthesis in the plasma membrane.

Impacts
Our specific aim was to demonstrate that acidocalcisomes are present in the bacterium Rhodospirillum rubrum and in pathogenic bacteria. The presence of acidocalcisomes (defined as membrane-bound granules containing polyphosphate (polyP), and different elements, enzymes and transporters) in many microorganisms such as bacteria, fungi, algae, and protozoa and its apparent absence in mammalian cells would make them promising targets for chemotherapy.

Publications

Seufferheld, M., Lea, C., Vieira, M., Oldfield, E., and Docampo, R. (2004) The H+-pyrophosphatase of Rhodospirillum rubrum is predominantly located in polyphosphate-rich acidocalcisomes. J. Biol. Chem. 279, 51193-51202.