Regulation of Polyhydroxybutyrate Synthesis in the Soil Bacterium Bradyrhizobium diazoefficiens

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, 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 the regulation of PHB synthesis, we generated mutations in the regulator gene phaR and the phasin genes phaP1 and phaP4. Under permissive conditions, mutation of phaR impaired PHB accumulation, and a phaP1 phaP4 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 phaP1 and phaP4 as well as the expression of phaA1 and phaA2 (encoding a 3-ketoacyl coenzyme A [CoA] thiolases), phaC1 and phaC2 (encoding PHB synthases), and fixK 2 (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, phaR mutants accumulated more extracellular polysaccharides and promoted higher plant shoot dry weight and competitiveness for nodulation than the wild type, in contrast to the phaC1 mutant strain, which is defective in PHB synthesis. These results suggest that phaR 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 2 . IMPORTANCEIn this work, we investigated the regulation of polyhydroxybutyrate synthesis in the soybean-nodulating bacterium Bradyrhizobium diazoefficiens and its influence in bacterial free-living and symbiotic lifestyles. We uncovered a new interplay between the synthesis of this carbon reserve polymer and the network responsible for microoxic metabolism through the interaction between the gene regulators phaR and fixK 2 . These results contribute to the understanding of the physiological conditions required for polyhydroxybutyrate biosynthesis. The interaction between these two main metabolic pathways is also reflected in the symbiotic phenotypes of soybeans inoculated with phaR mutants, which were more competitive for nodulation and enhanced dry matter production by the plants. Therefore, this knowledge may be applied to the development of superior strains to be used as improved inoculants for soybean crops. Bradyrhizobium diazoefficiens is an important soil bacterium that fixes atmospheric N 2 in symbiosis with soybean plants, a key crop for food production worldwide (1). 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-...

Polyhydroxyalkanoate‐associated phasins as phylogenetically heterogeneous, multipurpose proteins

“…Structurally, PhaP1 Bd ‐PhaP4 Bd are predicted to be predominantly alpha‐helical but only PhaP4 Bd contains additionally a C‐terminal region very rich in alanine residues (13 out of 34 amino acids) (Yoshida et al ., ). Besides, they are all proposed to oligomerize (Quelas et al ., ).…”

“…Transcription of phaP 3 seems to be low and constant during growth, suggesting that this phasin does not have a relevant role in PHA metabolism (Yoshida et al ., ). On the other hand, the study of single and double mutants has revealed that the combined role of PhaP1 Bd and PhaP4 Bd must be crucial in determining the number and size of the granules (Quelas et al ., ).…”

“…Four phasins have been identified in PHA granules from B. diazoefficiens , namely PhaP1 Bd ‐PhaP4 Bd (Yoshida et al ., ). None of them are involved in the bacterial growth kinetics (Quelas et al ., ), but they are all expressed in levels that correlate with the accumulated PHA (Yoshida et al ., ). In any case, expression of PhaP4 Bd is favoured when using yeast extract‐mannitol (YM) medium, and it presents the highest affinity to PHA granules in vitro (Yoshida et al ., ).…”

PhaR, a Negative Regulator of PhaP, Modulates the Colonization of a Burkholderia Gut Symbiont in the Midgut of the Host Insect, Riptortus pedestris

“…In Rhizobium etli, which is a symbiotic bacterium of plants, the ΔaniA (same as ΔphaR) mutant accumulated only 40% of polyhydroxybutyrate (PHB) compared to the wild-type strain by disappearance of PhaB protein expression (30). Also, in the case of the soil bacterium Bradyrhizobium diazoefficiens, when the phaR gene was knocked out, the expression of phaC2 became uncontrolled, and this led to rapid inhibition of PHB synthesis (32). Thus, PhaR is important to PHA biosynthesis, and it seems reasonable that the PHA synthetic mechanism has been primarily selected for survival at the free-living stage of the Burkholderia symbiont and secondarily coopted for survival under the symbiotic condition within the host insect (25).…”

“…PhaR is also identified as a negative regulator of PhaP in Paracoccus denitrificans (27). Also, PhaR was reported to control PHA synthesis in Rhizobia species (30)(31)(32). Although one phaP gene was characterized in our previous study (25), the functions of four other phaP family genes and one phaR gene have not been determined in the Burkholderia genome.…”