Poly-β-hydroxybutyrate (PHB) is a polymer used for the storage of carbon and energy in a large variety of prokaryotes. It is accumulated in the cytoplasm if a carbon source is provided in excess and if any other essential nutrient is limited . PHB belongs to the polyesters class of polymers, which is of interest as an industrial plastic because of its biodegradability and origin from renewable resources. Microbial PHB synthesis is a promising strategy for the production of bioplastics and offers a promising opportunity to transition toward a future-oriented bioeconomy .
Most species of rhizobia synthesize PHB and accumulate it in intracellular granules . In some species, PHB accumulation can exceed 50% of the cell’s dry weight [4, 5]. Various ways that rhizobia can use PHB to benefit their plant hosts have been proposed. For instance, it was proposed that PHB utilization could sustain the oxygen demand of the bacteroids during darkness; thus, contributing to the preservation of nodule activity and the continuation of nitrogen fixation at high rates . PHB may also fuel the differentiation of rhizobia into nitrogen-fixing bacteroids . In addition, rhizobia may simply degrade PHB in ways that enhance their own fitness. PHB may provide the energy and carbon required for bacterial reproduction, or for stress tolerance required within senescing nodules or after symbiotic rhizobia escape into the soil and transition to the free-living state.
Biochemically, PHB synthesis can compete with nitrogen fixation . In addition, a negative correlation was observed between the rate of nitrogen fixation and PHB accumulation [8, 9]. A mutant of Rhizobium etli, that did not accumulate PHB, was shown to significantly fix more nitrogen than the isogenic wild type [10, 11], whereas non-fixing nifH mutants of R. etli and Bradyrhizobium japonicum accumulated more PHB than their isogenic nitrogen-fixing parental strains. There is a conflict between rhizobia and legumes over the rate of PHB accumulation, due to the metabolic tradeoff between nitrogen fixation and PHB accumulation. Therefore, PHB biosynthesis and accumulation in species of rhizobia may be controlled to balance the tradeoff, but the mechanism underlying this control has not yet been fully explained.
One of the best studied microorganisms with respect to PHB biosynthesis and accumulation is the Gram-negative bacterium Ralstonia eutropha. It synthesizes PHB using the three PHB synthetic genes: phbA, which encodes 3-ketoacyl-CoA thiolase; phbB, which encodes acetoacetyl CoA reductase; and phbC, which encodes the enzyme PHB synthase. PHB degradation, however, is performed by PHB depolymerase, which is encoded by phaZ. Phasins, encoded by phaP, are a class of low-molecular-mass amphipathic proteins that form a layer at the surface of the PHB granule and stabilize it . The R. eutropha possesses at least four phaP paralogs identified so far . Expression of the major phasin, encoded by phaP1, is regulated by the transcriptional repressor PhaR [17, 18]. Under conditions less favorable for PHB biosynthesis, PhaR binds to the phaP1 promoter region to repress transcription of this gene. After the onset of PHB biosynthesis, when the nascent PHB granules gradually form, PhaR leaves the promoter and binds to the granules so that phaP1 is transcribed and translated. During the later stages of PHB accumulation, PhaR is estimated to bind no longer to the granules as it is displaced by PhaP1 phasin. The displaced PhaR returns to bind to the phaP1 promoter and represses transcription again .
Most members of the Rhizobiaceae are known to possess single copies of the PHB biosynthesis genes. For instance, strains of Sinorhizobium meliloti, the symbionts of alfalfa, regarded as one of the model organisms to study symbiotic nitrogen fixation, are characterized to have a single set of the genes for PHB metabolism, namely phbA, phbB, phbC, and phaZ[19, 20], whereas two paralogous genes, phaP1 and phaP2, encode functional phasins . On the other hand, strains of B. japonicum, the symbionts of soybean, are known to accumulate a large amount of PHB , and the B. japonicum USDA110 genome was found to contain five paralogs of phbC, as well as two paralogs of phbAB. This genetic redundancy may suggest a functional importance that has not yet been fully elucidated. In this study, we examined the expression profiles of the paralogs relevant to PHB metabolism in free-living B. japonicum cells under PHB accumulating and non-accumulating conditions. Then, we identified the phasin-encoding paralogs from the genomic information , and used a variety of tools to investigate their involvement in PHB accumulation.