Analysis of two polyhydroxyalkanoate (PHA) synthases in Bradyrhizobium japonicum USDA 110

QUELAS, J.I.; MONGIARDINI, E.J.; PÉREZ GIMÉNEZ, JULIETA; PARISI, G.; LODEIRO, A.R.

JOURNAL OF BACTERIOLOGY

AMER SOC MICROBIOLOGY

Lugar: Washington; Año: 2013 vol. 195 p. 3145 - 3155

0021-9193

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). 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 phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary phase cultures was impaired only in ΔphaC1. Meanwhile, ΔphaC2 produced more PHA than the wild-type in this condition and surprisingly, phaC3 transcript increased in the ΔphaC2 background. Consistently, a double mutant ΔphaC2/ΔphaC3 accumulated less PHA than ΔphaC2. PHA accumulation in nodule bacteroids followed a similar pattern as in liquid cultures, being prevented in ΔphaC1 and increased in ΔphaC2 in relation to the wild-type. Therefore we used these mutants, together with a ΔphaC1/ΔphaC2 doublemutant, to study the B. japonicum 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, ΔphaC1 and ΔphaC1/ΔphaC2occupied only 13-15 % of the nodules, while ΔphaC2 occupied 81 %, suggesting that thePHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.