Regulation of polyhydroxybutyrate synthesis in Bradyrhizobium diazoefficiens

MONGIARDINI, EJ; MESA, S; LODEIRO, AR; QUELAS, JI; JENDROSSEK, D

Budapest

Congreso; 12th European Nitrogen Fixation Conference; 2016

Institute of Biochemistry BRC HAS, Hungary

Polyhydroxyalkanoates (PHA) are carbon and energy reserve polymers that accumulate in various prokaryotic species. Microorganisms synthesize and storePHA inside their cytoplasm as carbon and energy reserves when the carbon sourceis not limiting. There are several types of this polymer, being poly-3- hydroxybutyrate (P3HB or PHB) the most common.In this work, we determined that, when grown with mannitol as the sole carbonsource, Bradyrhizobium diazoefficiens produces a homopolymer composed only of 3-hydroxybutyrate units (PHB). Conditions of oxygen limitation (such as microoxia, oxic stationary phase and bacteroids inside legume nodules) were permissive for the synthesis of PHB, which was observed as cytoplasmic granules. To study theregulation of PHB synthesis in B. diazoefficiens, we generated mutants in theregulator gene phaR and the phasin genes phaP1 and phaP4. Under permissiveconditions, mutation of phaR impaired PHB accumulation, and a phaP1/phaP4double mutant produced around three times more PHB than the wildtype.Furthermore, PHB was accumulated in a single, large cytoplasmic granule in thephaP1/phaP4 mutant. In addition, PhaR behaved as a global regulator, because itrepressed the expression of several PHB biosynthetic genes such as phaP1, phaP4,phaA1, phaA2 (3-ketoacyl- CoA thiolase), phaC1, phaC2 (PHB synthases), as well as that of the CRP/FNR-type transcription regulator fixK 2 , which in turn positively regulates the expression of more than 200 genes related with the microoxic lifestyle.Accordingly, the phaR mutant not only produced less PHB, but also overproducedextracellular polysaccharides (EPS).In addition to the effects observed in the free-living bacterial state, in the symbiotic state phaR mutants promoted higher soybean shoot dry weight and competitiveness for nodulation than the wildtype. These effects were contrary to that of phaC1 mutant strains, which are also defective in PHB synthesis, and indicate that these effects are not due solely to defects in PHB accumulation.These results suggest that phaR not only regulates PHB granules formation bycontrolling the expression of phasins and biosynthetic enzymes, but also acts asglobal regulator of excess carbon allocation by controlling fixK 2 , which may also regulate several aspects of the symbiosis with soybean plants.