Department of Agrobioscience, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657 8501, Japan.
Department of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657 8501, Japan.
BMC Microbiol. 2018 Oct 24;18(1):156. doi: 10.1186/s12866-018-1317-2.
Bradyrhizobium diazoefficiens USDA110 nodulates soybeans for nitrogen fixation. It accumulates poly-3-hydroxybutyrate (PHB), which is of physiological importance as a carbon/energy source for survival during starvation, infection, and nitrogen fixation conditions. PHB accumulation is orchestrated by not only the enzymes for PHB synthesis but also PHB-binding phasin proteins (PhaPs) stabilizing the PHB granules. The transcription factor PhaR controls the phaP genes.
Inactivation of phaR led to decreases in PHB accumulation, less cell yield, increases in exopolysaccharide (EPS) production, some improvement in heat stress tolerance, and slightly better growth under microaerobic conditions. Changes in the transcriptome upon phaR inactivation were analyzed. PhaR appeared to be involved in the repression of various target genes, including some PHB-degrading enzymes and others involved in EPS production. Furthermore, in vitro gel shift analysis demonstrated that PhaR bound to the promoter regions of representative targets. For the phaP1 and phaP4 promoter regions, PhaR-binding sites were determined by DNase I footprinting, allowing us to deduce a consensus sequence for PhaR-binding as TGCRNYGCASMA (R: A or G, Y: C or T, S: C or G, M: A or C). We searched for additional genes associated with a PhaR-binding sequence and found that some genes involved in central carbon metabolism, such as pdhA for pyruvate dehydrogenase and pckA for phosphoenolpyruvate carboxykinase, may be regulated positively and directly by PhaR.
These results suggest that PhaR could regulate various genes not only negatively but also positively to coordinate metabolism holistically in response to PHB accumulation.
慢生根瘤菌 USDA110 能够为固氮作用结瘤大豆。它会积累聚-β-羟基丁酸（PHB），这是一种在饥饿、感染和固氮条件下作为生存的碳/能量源的生理重要物质。PHB 的积累不仅由 PHB 合成酶协调，还由稳定 PHB 颗粒的 PHB 结合相蛋白（PhaP）协调。转录因子 PhaR 控制着 phaP 基因。
phaR 的失活导致 PHB 积累减少、细胞产量降低、胞外多糖（EPS）产量增加、耐热能力略有提高、微需氧条件下生长略好。分析了 phaR 失活后转录组的变化。phaR 似乎参与了各种靶基因的抑制，包括一些 PHB 降解酶和其他参与 EPS 生产的基因。此外，体外凝胶迁移分析表明 PhaR 结合到代表性靶基因的启动子区域。对于 phaP1 和 phaP4 启动子区域，通过 DNase I 足迹法确定了 PhaR 结合位点，使我们能够推断出 PhaR 结合的共有序列为 TGCRNYGCASMA（R：A 或 G，Y：C 或 T，S：C 或 G，M：A 或 C）。我们搜索了与 PhaR 结合序列相关的其他基因，发现一些参与中心碳代谢的基因，如丙酮酸脱氢酶的 pdhA 和磷酸烯醇丙酮酸羧激酶的 pckA，可能受到 PhaR 的正向直接调控。
这些结果表明 PhaR 不仅可以负调控，还可以正调控各种基因，以协调 PHB 积累时的整体代谢。
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