Large amounts of apicoplast nucleoid DNA and its segregation in Toxoplasma gondii

Apicoplasts (apicomplexan plastids) are nonphotosynthetic, secondary endosymbiotic plastids that are found in most apicomplexans. Although these organelles are essential for parasite survival, their functions, activities, and structures are not well understood. We examined the apicoplast nucleoid of Toxoplasma gondii from a morphological aspect by high-resolution epifluorescence microscopy and electron microscopy. We found unexpectedly large amounts of DNA in the nucleoid and the presence of several division-related structures. Initially, we identified the organellar nucleoids by staining with the DNA-specific dye 4',6-diamidino-2-phenylindole. A single nucleoid was observed per apicoplast, and the fluorescent spot representing the nucleoid was bright and spherical in contrast to the weak and filamentous spot representing the mitochondrial nucleoid. We also measured the DNA content of each apicoplast nucleoid by a video-intensified microscope photon-counting system and determined that the genomic copy number was at least 25, a figure over four times greater than that reported previously. Moreover, several groups of apicoplasts had significantly higher genomic copy numbers. The DNA molecules were accurately divided into two daughter apicoplasts just before nuclear division. In addition, we examined nucleoid segregation and the division apparatus using electron microscopy. However, we failed to observe nucleoid structures, suggesting that the apicoplasts are predominantly composed of nucleoid material. In addition, we observed "cap" structures at the termini of dividing apicoplasts, a possible plastid-dividing ring, and a microbody-like granule around the constriction. These structures may be involved in apicoplast division.