Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

Abstract

bBradyrhizobium 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 the ⌬phaC1 mutant. Meanwhile, the ⌬phaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ⌬phaC2 background. A double mutant, the ⌬phaC2 ⌬phaC3 mutant, consistently accumulated less PHA than the ⌬phaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ⌬phaC1 mutant and increased in the ⌬phaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ⌬phaC1 ⌬phaC2 double mutant, 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, the ⌬phaC1 and ⌬phaC1 ⌬phaC2 mutants occupied only 13 to 15% of the nodules, while the ⌬phaC2 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.

Lipogenesis and Redox Balance in Nitrogen-Fixing Pea Bacteroids

“…Importantly, this observation is supported by the work of Miller and Tremblay (53), who showed that S. meliloti bacteroids from alfalfa nodules contain 34% of the total neutral lipid fraction as di-and triglycerides whereas these lipids were undetected in free-living S. meliloti. Moreover, the extraordinary deposition of PHB in bacteroids from common bean and soybean is an extreme example of carbon storage and redox balancing that has hitherto lacked a coherent explanation, particularly since preventing synthesis in these symbioses does not prevent N 2 fixation (19)(20)(21). Here we show that bacteroids of some strains of R. leguminosarum, such as Rlv3841, make PHB via a putative nifA-dependent type III PHB synthase.…”

“…apparently do not (18). While abolishing PHB synthesis does not adversely affect N 2 fixation rates in soybean and common bean (19)(20)(21), in Azorhizobium caulinodans, mutation of PHB synthase prevents N 2 fixation in both free-living and symbiotic forms (22), implying a fundamental role for PHB synthesis in at least some N 2 -fixing rhizobia.…”

Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

“…Cloning procedures, including DNA isolation, restriction digestion, ligation, and transformation, were performed as described previously (29). Tri-or biparental matings were performed with E. coli DH5␣ or S17-1 strains as described previously (3). Electroporation was performed with a Gene Pulser system (Bio-Rad, Hercules, CA) at 1.5 V, 25 F, and 200 ⍀ in a 0.1-cm-gap-width electroporation cuvette.…”

“…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).…”

“…In addition to its agricultural relevance, B. diazoefficiens may be of industrial interest because it produces significant quantities of polyhydroxybutyrate (PHB), a polymer that accumulates as granules in the cytoplasm and has potential use as biodegradable plastic (2)(3)(4)(5)(6). PHB granules are synthesized as sinks of excess carbon and reducing power and are used as carbon and energy reserves when bacteria face starvation conditions (7).…”