PhaP phasins play a principal role in poly-β-hydroxybutyrate accumulation in free-living Bradyrhizobium japonicum

Abstract

BackgroundBradyrhizobium japonicum USDA110, a soybean symbiont, is capable of accumulating a large amount of poly-β-hydroxybutyrate (PHB) as an intracellular carbon storage polymer during free-living growth. Within the genome of USDA110, there are a number of genes annotated as paralogs of proteins involved in PHB metabolism, including its biosynthesis, degradation, and stabilization of its granules. They include two phbA paralogs encoding 3-ketoacyl-CoA thiolase, two phbB paralogs encoding acetoacetylCoA reductase, five phbC paralogs encoding PHB synthase, two phaZ paralogs encoding PHB depolymerase, at least four phaP phasin paralogs for stabilization of PHB granules, and one phaR encoding a putative transcriptional repressor to control phaP expression.ResultsQuantitative reverse-transcriptase PCR analyses of RNA samples prepared from cells grown using three different media revealed that PHB accumulation was related neither to redundancy nor expression levels of the phbA, phbB, phbC, and phaZ paralogs for PHB-synthesis and degradation. On the other hand, at least three of the phaP paralogs, involved in the growth and stabilization of PHB granules, were induced under PHB accumulating conditions. Moreover, the most prominently induced phasin exhibited the highest affinity to PHB in vitro; it was able to displace PhaR previously bound to PHB.ConclusionsThese results suggest that PHB accumulation in free-living B. japonicum USDA110 may not be achieved by controlling production and degradation of PHB. In contrast, it is achieved by stabilizing granules autonomously produced in an environment of excess carbon sources together with restricted nitrogen sources.

Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

“…However, this cycle of synthesis and degradation must be regulated because if the two pathways occur simultaneously (8), the net result would be consumption of energy and reducing power. The proteins involved in the different steps of the PHB cycle are well-characterized (9-12), and all of them are present in B. diazoefficiens (3,13,14); however, regulation of the cycle in this bacterium was not yet studied. In other rhizobium species, such as Rhizobium etli and Ensifer meliloti, the gene product of phaR (PHA [polyhydroxyalkanoate] regulator, previously known as aniA for anaerobically induced gene A) was reported to control, at least in part, PHB synthesis (15, 16).…”

“…The expression of these genes depends on the culture conditions. In particular, conditions permissive for PHB synthesis, such as microoxia (26) and growth in yeast extract-mannitol (YM) (14), increase the expression of phaP1 and phaP4, the last being the paralog with the highest expression in YM. PhaR and the four PhaP paralogs bind to PHB in vitro (14), with PhaP4 being the phasin with the highest affinity.…”

“…In particular, conditions permissive for PHB synthesis, such as microoxia (26) and growth in yeast extract-mannitol (YM) (14), increase the expression of phaP1 and phaP4, the last being the paralog with the highest expression in YM. PhaR and the four PhaP paralogs bind to PHB in vitro (14), with PhaP4 being the phasin with the highest affinity. Further, an increasing concentration of PhaP4 triggered a competitive displacement of PhaR bound to PHB fine powder in suspension (14).…”

Genomics of Specificity in the Symbiotic Interaction between Rhizobium leguminosarum and Legumes

“…Phasins were expressed during PHB (poly-ß-hydroxybutyrate, the most common PHA) synthesis in Bradyrhizobium japonicum (Yoshida et al, 2013). Moreover, the expression of pRL100120 is 9-fold higher in bacteroids than in free-living bacteria for R. leguminosarum bv.…”

“…Phasins are granule-associated proteins found covering PHA (polyhydroxyalkanoate) storage granules in bacteria; a layer of phasins stabilises the granules of PHA and prevents coalescence of separated granules in the cytoplasm (de Almeida et al, 2007). Phasins were expressed during PHB (poly-ß-hydroxybutyrate, the most common PHA) synthesis in Bradyrhizobium japonicum (Yoshida et al, 2013). Moreover, the expression of pRL100120 is 9-fold higher in bacteroids than in free-living bacteria for R. leguminosarum bv.…”