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Bradyrhizobium diazoefficiens USDA 110 accumulates poly-beta-hydroxybutyrate (PHB) in the cells grown with an excess of carbon sources. Previously we demonstrated that PhaP proteins (Phasins) stabilizing PHB granules played important roles in PHB accumulation in USDA110 (1). And PhaR repressor was suggested to regulate expression of phaP genes. In this study, we created and analyzed a phaR-deficient mutant strain to investigate changes in various phenotypes, including bacterial growth, PHB accumulation, and symbiotic nitrogen fixation. In addition, PhaR was produced and purified in Escherichia coli to analyze its DNA binding in vitro.
When the phaR-deficient mutant was cultured in PHB non-accumulating conditions, among the genes involved in PHB accumulation, transcription levels of phaP1, phaP4, and phbZ1 were elevated significantly, indicating that PhaR could be involved in transcriptional repression of the two phasins, PhaP1 and PhaP4, as well as the PHB degrading enzyme, PhbZ1. Indeed, in vitro analyses, including gel mobility shift and DNase I foot print analyses, revealed that PhaR specifically bound to the promoter regions of these genes, suggesting its function as transcriptional repressor.
When cultured in PHB accumulating conditions, growth of phaR-deficient mutant was impaired, and its intracellular PHB accumulation was reduced more pronouncedly than in the phaP4-deficient mutant. On the other hand, it secreted twice more EPS than both the parental and the phaP4-deficient strains. The results suggest that elevated expression of the two phasins and/or the PHB degrading enzyme might cause the less efficient PHB accumulation. Moreover, inactivation of phaR might switch the cellular metabolism from PHB accumulation to EPS secretion, and thus caused metabolic changes might be toxic to restrict the cell growth. Unexpectedly, the phaR-deficient mutant showed somehow elevated resistance to thermal stresses, although PHB non-accumulating mutants of other microorganisms reportedly exhibited less tolerance to various stresses.
When soybean was infected with phaR-deficient mutant, nodules were formed almost similarly as infected with the parental strain, and normal growth of soybean plant was observed without supplemental nitrogen sources. The results suggest that neither the less efficient PHB accumulation nor the enhanced EPS secretion might affect the establishment of symbiosis and nitrogen fixation. Since PHB may serve as energy storage compound in bacteroides, we are currently investigating effects of the phaR inactivation on efficiency in bacterial outgrowth from matured nodules. |