Analysis of two polyhydroxyalkanoate synthases in Bradyrhizobium japonicum USDA 110

Abstract

<i>Bradyrhizobium japonicum</i> USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: <i>bll4360</i> (<i>phaC1</i>), <i>bll6073</i> (<i>phaC2</i>), <i>blr3732</i> (<i>phaC3</i>), <i>blr2885</i> (<i>phaC4</i>), and <i>bll4548</i> (<i>phaC5</i>). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only <i>phaC1</i> and <i>phaC2</i> were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the Δ<i>phaC1</i> mutant. Meanwhile, the Δ<i>phaC2</i> mutant produced more PHA than the wild type under this condition, and surprisingly, the <i>phaC3</i> transcript increased in the Δ<i>phaC2</i> background. A double mutant, the Δ<i>phaC2</i> Δ<i>phaC3</i> mutant, consistently accumulated less PHA than the Δ<i>phaC2</i> mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the Δ<i>phaC1</i> mutant and increased in the Δ<i>phaC2</i> mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a Δ<i>phaC1</i> Δ<i>phaC2</i> double mutant, to study the <i>B. japonicum</i> PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the Δ<i>phaC1</i> and Δ<i>phaC1</i> Δ<i>phaC2</i> mutants occupied only 13 to 15% of the nodules, while the Δ<i>phaC2</i> mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.