Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803

Abstract

Cyanobacteria are photoautotrophic microorganisms which fix atmospheric carbon dioxide via the Calvin-Benson cycle to produce carbon backbones for primary metabolism. Fixed carbon can also be stored as intracellular glycogen, and in some cyanobacterial species like Synechocystis sp. strain PCC 6803, polyhydroxybutyrate (PHB) accumulates when major nutrients like phosphorus or nitrogen are absent. So far only three enzymes which participate in PHB metabolism have been identified in this organism, namely, PhaA, PhaB, and the heterodimeric PHB synthase PhaEC. In this work, we describe the cyanobacterial PHA surface-coating protein (phasin), which we term PhaP, encoded by ssl2501. Translational fusion of Ssl2501 with enhanced green fluorescent protein (eGFP) showed a clear colocalization to PHB granules. A deletion of ssl2501 reduced the number of PHB granules per cell, whereas the mean PHB granule size increased as expected for a typical phasin. Although deletion of ssl2501 had almost no effect on the amount of PHB, the biosynthetic activity of PHB synthase was negatively affected. Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two ␣-helices, both of them associating with PHB granules. Purified PhaP forms oligomeric structures in solution, and both ␣-helices of PhaP contribute to oligomerization. Together, these results support the idea that Ssl2501 encodes a cyanobacterial phasin, PhaP, which regulates the surface-to-volume ratio of PHB granules. C yanobacteria are photosynthetic microorganisms capable of oxygenic photosynthesis. ATP and reduction equivalents derived from photosynthetic electron flow are utilized to fix carbon dioxide and generate 3-phosphoglycerate (1). This metabolite can be utilized for either gluconeogenesis or glycolysis, providing the necessary carbon skeletons for biosynthesis of amino acids and other metabolites required for cell growth (2), when growth conditions are suitable and nutrients are abundant. In fact, carbon flux is greatly affected by the availability of macronutrients like nitrogen (3-5) and phosphorus, which may limit growth (6). Under nutrient-limiting conditions, cyanobacteria undergo a stress adaptation process termed chlorosis (7). This process leads to the degradation of light-harvesting complexes, causing reduced photosynthetic activity and thereby reduced metabolic activity (8). Furthermore, carbon flux is redirected toward glycogen synthesis upon macronutrient starvation (5). In addition, some cyanobacterial strains like Synechocystis sp. strain PCC 6803 (referred to here as Synechocystis) accumulate polyhydroxybutyrate (PHB) as a carbon and redox storage compound (9). PHB is synthesized in three biosynthetic steps, and all three enzymes catalyzing the reactions are known (10, 11). The first step involves a condensation of two acetyl coenzyme A (acetyl-CoA) groups to acetoacetyl-CoA by PhaA (slr1993). In the second step, PhaB (slr1994) reduces acetoacetyl-CoA to hydroxybutyryl-CoA, utilizing NADPH...

Phasins, Multifaceted Polyhydroxyalkanoate Granule-Associated Proteins

“…PCC 6803, and archaeal phasins. Analysis of PhaP from Synechocystis revealed that it is similar to thylakoidassociated proteins from different algal species and to a protein from Arthrospira platensis that contains a Phasin_2 motif (12). Many phasins have been identified in Archaea, but to date, only that of H. mediterranei has been experimentally studied.…”

“…Secondary-structure in silico predictions for phasins PhaP1 to PhaP4 from R. eutropha, PhaP1 from Ralstonia solanacearum, PhaP from Ralstonia metallidurans (17), PhaP Az (18), and PhaP Sp (12) revealed that these phasins have a high percentage (close to 90%) of amino acids in an ␣-helix conformation. This was found to be a general characteristic of phasins (18)(19)(20).…”

“…strain FA8 (PhaP Az ) (18), PhaF from P. putida (PhaF Pp ) (19), PhaP from Aeromonas hydrophila (PhaP Ah ) (20), and PhaP from Synechocystis sp. PCC 6803 (PhaP Sp ) (12).…”

“…Several phasins were identified in many PHA-producing bacteria, such as Ralstonia eutropha (also known as Cupriavidus necator) (5-7), Bacillus megaterium (8), R. ruber (4), Paracoccus denitrificans (9), Pseudomonas putida (10), Azotobacter vinelandii (11), and Synechocystis sp. strain PCC 6803 (12), among many others. Phasins have also been identified in Archaea such as Haloferax mediterranei (13).…”

Identification of Four Polyhydroxyalkanoate Structural Genes in Synechocystis cf. salina PCC6909: In silico Evidences

“…PCC6803, PHAs synthesis occurs in the presence of light and starts with the condensation of two Acetyl-CoA molecules by PhaA (acetyl-CoA-acetyltransferase), generating Acetoacetyl-CoA, then reduced by PhaB [3-oxacyl-(acylcarrier-protein) reductase 2] to (R)-3-hydroxybutyryl-CoA. At this stage, a heterodimer complex composed of PhaE [poly (3-hydroxyalkanoate) synthase component] and PhaC [poly(3-hydroxyalkanoate) synthase] polymerizes the Hydroxybutyryl-CoA to Polyhydroxybutyrate [21]. Recently, the activity of the co-expressed PhaE and PhaC in a cell-free system was determined and the values obtained were comparable to those of PHA synthases belonging to class I [22].…”