Coordinated regulation of size and number of PHB granules by core and accessory phasins in the facultative microsymbiont Sinorhizobium fredii NGR234.

The exact roles of various granule-associated proteins (GAPs) of polyhydroxybutyrate (PHB) are poorly investigated particularly in bacteria associated with plants. Here, four structural GAPs, named as phasins PhaP1-PhaP4, were identified and demonstrated as true phasins colocalized with PHB granules in Sinorhizobium fredii NGR234, a facultative microsymbiont of Vigna unguiculata and many other legumes. The conserved PhaP2 dominated in regulation of granule size under both free-living and symbiotic conditions. PhaP1, another conserved phasin, made a higher contribution than accessory PhaP4 and PhaP3 to PHB biosynthesis at stationary phase. PhaP3, with limited phyletic distribution on the symbiosis plasmid of Sinorhizobium, was more important than PhaP1 in regulating PHB biosynthesis in V. unguiculata nodules. In the test conditions, no significant symbiotic defects were observed for mutants lacking individual or multiple phaP genes. The mutant lacking two PHB synthases showed impaired symbiotic performance while mutations in individual PHB synthases or a PHB depolymerase had no symbiotic defects. This phenomenon is not related to either the number or size of PHB granules in test mutants within nodules. Distinct metabolic profiles and cocktail pools of GAPs of different phaP(s) mutants imply that core and accessory phasins can be differentially involved in regulating other cellular process in the facultative microsymbiont S. fredii NGR234. Importance Polyhydroxybutyrate (PHB) granules are a store of carbon and energy in bacteria and archaea, and play an important role in stress adaptation. Recent studies have highlighted distinct roles of several granule-associated proteins (GAPs) in regulating the size, number and localization of PHB granules in free-living bacteria, though our knowledge on the role of GAPs in bacteria associated with plants is still limited. Here we report distinct roles of core and accessory phasins associated with PHB granules of Sinorhizobium fredii NGR234, a broad host range microsymbiont of diverse legumes. Core phasins PhaP2 and PhaP1 are conserved major phasins in free living cells. PhaP2 and an accessory phasin PhaP3 encoded by an auxiliary gene on the symbiosis plasmid are major phasins in nitrogen fixing bacteroids in cowpea nodules. GAPs and metabolic profiles can vary in different phaP mutants. Contrasting symbiotic performance between mutants lacking PHB synthases, depolymerase or phasins were revealed.