June 18, 2003 CHAMPAIGN, Ill. -- Researchers looking inside a pathogenic soil bacterium have found an organelle, a subcellular pouch, existing independently from the plasma membrane. The discovery within a prokaryotic organism challenges the theory on the origin of eukaryotic organelles and suggests a targeted approach to killing many disease-causing organisms.
"The organelle we found in the bacterium Agrobacterium tumefaciens is practically identical to the organelle called acidocalcisome in unicellular eukaryotes," said Roberto Docampo, a professor of veterinary pathobiology in the College of Veterinary Medicine at the University of Illinois at Urbana-Champaign.
Docampo began researching these organelles in 1994. He soon determined that a tiny granule in yeast, fungi and bacteria, thought to be for storage, was a fully operational organelle containing pyrophosphatase, a pump-like enzyme that allows proton transport. He named it an acidocalcisome for its acidic and calcium components. In 2000, he reported its existence in Plasmodium berghei, a malaria-causing eukaryotic parasite.
The newest discovery appeared in a paper published online this month by the Journal of Biological Chemistry. The paper, by Docampo and colleagues at the Center for Zoonoses Research and Laboratory of Molecular Parasitology at Illinois, will be published in a later print edition of the journal.
Agrobacterium tumefaciens is a prokaryote, a unicellular organism lacking membrane-bound nuclei. It causes crown gall disease in many broad-leaved plants but also is a favored tool for plant breeding because of its model system of DNA transfer into the hosts it invades. Samples were provided to Docampo's team by biotechnology researcher Stephen K. Farrand, a professor of microbiology and crop sciences at Illinois.
Bacteria and other prokaryotes generally lack an endomembrane system. Thus bacteria are presumed to lack compartments such as organelles not somehow linked to the plasma membrane ringing the organisms.
"What we describe is a discrete organelle independent of the plasma membrane," Docampo said. "It has a proton pump in its membrane, which is used to maintain its interior acidic content. This has never been described before in a bacterium."
The existence of discrete organelles is a defining component of unicellular eukaryotes, which have membrane-bound nuclei and specialized structures in their cell boundaries. The evolution of eukaryotic organelles "is a matter of extensive debate," Docampo said. The principle of endosymbiosis says that as microorganisms engulfed others, then new, membrane-surrounded organelles emerged in eukaryotes.
"It appears that this organelle has been conserved in evolution from prokaryotes to eukaryotes, since it is present in both. This argues against the belief that all eukaryotic organelles were formed when early eukaryotes swallowed prokaryotes," he said.
Using transmission electron and immunoelectron microscopy and X-ray microanalysis on the bacterium, researchers got a highly magnified and illuminated view.
They applied a fluorescent dye into the suspected organelle. They saw a membrane around it. The dye stained areas only within it, not in the cytosol. Serum containing antibodies to peptides related to pyrophosphatase unveiled this pump-like enzyme, and other staining techniques revealed high levels of polyphosphate only in the organelle.
Many parasites such as those that cause malaria, African sleeping sickness and toxoplasmosis and bacteria that contain these acidocalcisome organelles are pathogens.
Some pharmaceutical approaches have targeted pyrophosphate-related enzymes, Docampo said. "Our suggestion is that if drugs specifically targeted these organelles, you may be able to kill the entire organisms."
In addition to Docampo, other Illinois researchers were Manfredo Seufferheld, Mauricio C.F. Vieira, Felix A. Ruiz, Claudia O. Rodrigues and Silvia N.J. Moreno.
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