The synthesis of granules : elements for a theory

There is no consensus about the formation of the PHB granules. Two models have been proposed, and all the experiments realized until May 2009 (Jendrossek 2009) can’t confirm or refute one of them. But it’s admitted that three proteins play a major part in the biogenesis of the granule. The first one is the PHB synthase. PHB synthase (phaC) This protein catalyzes the polymerization of hydroxyacyl-coenzyme A (CoA) to PHA and free CoA, as we can see on the schema n°1. Four types of PHB synthase had been discovered, and distinguished by the number of subunits and the substrate specificity. Despite these differences, all the PHB synthase have a conserved cysteine, acting as a catalytic site in the association of two others amino acids (generally: histidine and aspartate). The growing polymer of PHB is covently attached to the cysteine. Many results show that PHB synthase is located on the surface of the granule. PhaPs and PhaR PhaPs, also called phasins, are proteins regulating the size and the morphology of granules and preventing the fusion of PHB granules by formation of a protein layer between the hydrophobic polymer and the hydrophilic cytoplasm. There are not constitutive, and their expression is regulated by the PHB accumulation, thanks to a repressor, PhaR. Studies show that PhaR can bind by growing PHB granules during PHB accumulation. The consequence is a low concentration of soluble PhaR allowing the expression of more PhaP and PhaR. Once all binding sites for PhaR at the PHB granules are occupied by PhaR and PhaP, excess soluble PhaR binds to DNA upstream regions of PhaP and PhaR, repressing the expression of the two proteins. 5% of the granule is composed by these proteins, called Granules Associated Proteins. Biogenesis of the granule Two models of PHA granule formation have been proposed. The fist one is called “micelle model”. It is based on the fact that soluble PHB synthase reacts with its substrate, hydroxyacyl CoA, in the cytoplasm. After the synthesis of the first PHB chain, the others will aggregate to the “primer” by hydrophobic interactions, to make small PHB granules. The enzyme remains in the surface of the granule, and the other proteins of the granule (phasins, PhaR) bind to the growing surface. Figure 2. "Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase",Stubbe and Tian, 2003 The second one is called “budding model”. In this one, the PHB synthase is binding with the cytoplasmic membrane and the growing PHB chain. The polymer interacts with the cytoplasmic membrane by hydrophobic bonds. We observe the formation of PHB molecules in the cytoplasmic membrane. Later, the granules detach from the membrane, forming structures like “buds”, and the other proteins of the granule (phasins, PhaR) bind to the growing surface. Usually, the diameter of the granule range between 100 and 500nm. Various granules are synthesized in the bacteria. The number seems to be controlled by phasins too, but again, no publication can confirm it. On the pictures below, we can follow the synthesis of various granules. The formation of this structure seems to be fast. Indeed, the first granule appears after 10minutes (not shown in the picture). Figure 4 : Polyhydroxyalkanoate Granules Are Complex Subcellular Organelles (Carbonosomes), Jendrossek, 2009 In our experiments, after 24h of growth in an appropriate medium, we observed various granules in our bacteria. But the number was very different in each bacterium. The regulation of the size and the number of granules seems to be the result of various factors, like the protein regulation by phaP and PhaR, the medium, the culture conditions… On others pictures, we could see that some granules were in the medium, freed from the bacteria envelop. So, even the lysis of the cell caused by an excess of granules seems not to occur at the same time for each bacterium. Synthesis of the granules