Abstract
The formation of cytoplasmic organelles within prokaryotes is a matter of controversy among cell biologists as it reveals layers of complexity in microorganisms that were not long ago believed to lack complex internal organization. This controversy was furthered by analyses showing that archaea are more genetically similar to eukaryotes than their prokaryotic companions bacteria, revealing a tree of life with one branch for bacteria and another for eukaryotes and archaea. In this study, we investigated the molecular origins of a previous observation of possible organelle-like structures- intracellular honeycomb patterns visible by staining nucleic acids in phosphate-deprived cells of the haloarchaea Haloferax mediterranei. By deleting the genes responsible for polyhydroxyalkanoate (PHA) synthesis, we discovered that these Zones of DNA Exclusion (ZDEs) were PHA granules, which “crowded out” DNA from certain areas of the cytoplasm due to their hydrophobicity. We then used a known PHA dye to label granules in living cells and track their localization, size, and number within living cells over time. In doing so, we found that PHA granule number and size both decreased by a factor of ~2.5 over a 20-hour period, while over that same period, PHA granule distance from the cell border remained relatively constant. We also recorded what we believe to be the first video of PHA formation within living haloarchaea. Altogether, our work establishes a new framework for future research into dynamics of PHA granules as a membraneless organelle and their associated proteins in living haloarchaea.