Nitrogen Fixation Laboratory, Department of Biochemistry and Molecular Biology, Federal University of Paraná (UFPR), Curitiba, Brazil.
Nuclear Magnetic Resonance Centre, Department of Biochemistry and Molecular Biology, Federal University of Paraná (UFPR), Curitiba, Brazil.
Appl Environ Microbiol. 2020 Aug 18;86(17). doi: 10.1128/AEM.01265-20.
Under conditions of carbon starvation or thermal, osmotic, or oxidative shock, mutants affected in the synthesis or mobilization of poly-3-hydroxybutyrate (PHB) are known to survive less well. It is still unclear if the synthesis and accumulation of PHB are sufficient to protect bacteria against stress conditions or if the stored PHB has to be mobilized. Here, we demonstrated that mobilization of PHB in SmR1 was heat-shock activated at 45°C. proton (H) nuclear magnetic resonance spectroscopy (i.e., H-nuclear magnetic resonance) showed that heat shock increased amounts of 3-hydroxybutyrate (3HB) only in strains able to synthesize and mobilize PHB. SmR1 mutants unable to synthesize or mobilize PHB were more susceptible to heat shock and survived less well than the parental strain. When 100 mM 3-hydroxybutyrate was added to the medium, the Δ strain (an mutant unable to synthesize PHB) and the double mutant with deletion of both and (i.e., Δ) (unable to mobilize PHB) showed partial rescue of heat adaptability (from 0% survival without 3HB to 40% of the initial viable population). Addition of 200 mM 3HB before the imposition of heat shock reduced protein aggregation to 15% in the Δ mutant and 12% in the Δ mutant. We conclude that SmR1 is naturally protected by 3HB released by PHB mobilization, while mutants unable to generate large amounts of 3HB under heat shock conditions are less able to cope with heat damage. Bacteria are subject to abrupt changes in environmental conditions affecting their growth, requiring rapid adaptation. Increasing the concentration of some metabolites can protect bacteria from hostile conditions that lead to protein denaturation and precipitation, as well as damage to plasma membranes. In this work, we demonstrated that under thermal shock, the bacterium depolymerized its intracellular stock polymer known as poly-3-hydroxybutyrate (PHB), rapidly increasing the concentration of 3-hydroxybutyrate (3HB) and decreasing protein precipitation by thermal denaturation. Mutant strains unable to produce or depolymerize PHB suffered irreparable damage during thermal shock, resulting in fast death when incubated at 45°C. Our results will contribute to the development of bacteria better adapted to high temperatures found either in natural conditions or in industrial processes. In the case of and other bacteria that interact beneficially with plants, the understanding of PHB metabolism can be decisive for the development of more-competitive strains and their application as biofertilizers in agriculture.
在碳饥饿或热、渗透或氧化冲击的条件下，人们已经知道，在聚-3-羟基丁酸（PHB）的合成或动员方面受到影响的突变体的存活能力较差。目前尚不清楚 PHB 的合成和积累是否足以保护细菌免受应激条件的影响，或者储存的 PHB 是否必须动员。在这里，我们证明了 SmR1 中的 PHB 动员在 45°C 时被热休克激活。质子（H）核磁共振波谱（即 H-核磁共振）表明，热休克仅在能够合成和动员 PHB 的菌株中增加 3-羟基丁酸（3HB）的量。不能合成或动员 PHB 的 SmR1 突变体对热冲击更敏感，存活能力比亲本菌株差。当向培养基中添加 100mM 3-羟基丁酸时，Δ 菌株（一种不能合成 PHB 的 突变体）和缺失 和 （即 Δ）的双突变体（不能动员 PHB）显示出对热适应性的部分挽救（从没有 3HB 的 0%存活增加到初始活菌数的 40%）。在施加热冲击之前添加 200mM 3-羟基丁酸可将 Δ 突变体中的蛋白聚集减少到 15%，将 Δ 突变体中的蛋白聚集减少到 12%。我们得出的结论是，自然状态下 SmR1 受到 PHB 动员释放的 3HB 的保护，而在热冲击条件下不能产生大量 3HB 的突变体则更难应对热损伤。细菌会受到影响其生长的环境条件的突然变化的影响，需要快速适应。增加某些代谢物的浓度可以保护细菌免受导致蛋白质变性和沉淀以及质膜损伤的恶劣条件的影响。在这项工作中，我们证明了在热冲击下，细菌将其称为聚-3-羟基丁酸（PHB）的细胞内库存聚合物解聚，迅速增加 3-羟基丁酸（3HB）的浓度，并通过热变性降低蛋白质沉淀。不能产生或解聚 PHB 的突变体菌株在热冲击过程中遭受不可逆转的损伤，在 45°C 孵育时迅速死亡。我们的研究结果将有助于开发更能适应自然条件或工业过程中发现的高温的细菌。就 和其他与植物有益相互作用的细菌而言，对 PHB 代谢的理解对于开发更具竞争力的菌株及其在农业中的生物肥料应用至关重要。
