Regulation of polyhydroxybutyrate synthesis in the soil bacterium <i>Bradyrhizobium diazoefficiens</i>

Abstract

Polyhydroxybutyrate (PHB) is a carbon and energy reserve polymer in various prokaryotic species. We determined that, when grown with mannitol as the sole carbon source, <i>Bradyrhizobium diazoefficiens</i> 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 the regulation of PHB synthesis, we generated mutations in the regulator gene <i>phaR</i> and the phasin genes <i>phaP1</i> and <i>phaP4</i>. Under permissive conditions, mutation of <i>phaR</i> impaired PHB accumulation, and a <i>phaP1</i> <i>phaP4</i> double mutant produced more PHB than the wild type, which was accumulated in a single, large cytoplasmic granule. Moreover, PhaR negatively regulated the expression of <i>phaP1</i> and <i>phaP4</i> as well as the expression of <i>phaA1</i> and <i>phaA2</i> (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), <i>phaC1</i> and <i>phaC2</i> (encoding PHB synthases), and fixK<SUB>2</SUB> (encoding a cyclic AMP receptor protein [CRP]/fumarate and nitrate reductase regulator [FNR]-type transcription factor of genes for microoxic lifestyle). In addition to the depressed PHB cycling, <i>phaR</i> mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the <i>phaC1</i> mutant strain, which is defective in PHB synthesis. These results suggest that <i>phaR</i> not only regulates PHB granule formation by controlling the expression of phasins and biosynthetic enzymes but also acts as a global regulator of excess carbon allocation and symbiosis by controlling fixK<SUB>2</SUB>.