Plant growth-promoting bacteria are a promising alternative to improve agricultural sustainability. Gluconacetobacter diazotrophicus is an osmotolerant bacterium able to colonize several plant species, including sugarcane, coffee, and rice. Despite its biotechnological potential, the mechanisms controlling such osmotolerance remain unclear. The present study investigated the key mechanisms of resistance to osmotic stress in G. diazotrophicus. The molecular pathways regulated by the stress were investigated by comparative proteomics, and proteins essential for resistance were identified by knock-out mutagenesis. Proteomics analysis led to identify regulatory pathways for osmotic adjustment, de novo saturated fatty acids biosynthesis, and uptake of nutrients. The mutagenesis analysis showed that the lack of AccC protein, an essential component of de novo fatty acid biosynthesis, severely affected G. diazotrophicus resistance to osmotic stress. Additionally, knock-out mutants for nutrients uptake (Δtbdr and ΔoprB) and compatible solutes synthesis (ΔmtlK and ΔotsA) became more sensitive to osmotic stress. Together, our results identified specific genes and mechanisms regulated by osmotic stress in an osmotolerant bacterium, shedding light on the essential role of cell envelope and extracytoplasmic proteins for osmotolerance.

植物生长促进细菌是提高农业可持续性的有前途的替代品。固氮葡萄糖乙酸杆菌是一种耐渗透细菌，能够在多种植物物种中定殖，包括甘蔗、咖啡和水稻。尽管具有生物技术潜力，但控制这种渗透压的机制仍不清楚。本研究调查了固氮菌抗渗透胁迫的关键机制。通过比较蛋白质组学研究了应激调节的分子途径，并通过敲除诱变鉴定了抗性所必需的蛋白质。蛋白质组学分析导致​​确定渗透调节的调控途径，从头饱和脂肪酸的生物合成和营养物质的吸收。诱变分析表明，缺乏从头脂肪酸生物合成的重要组成部分 AccC 蛋白严重影响了固氮菌对渗透胁迫的抗性。另外，对于摄取营养物（Δ敲除的突变体TBDR和Δ oprB）和相容性溶质合成（Δ MTLK和Δ奖励计划）成为对渗透胁迫更加敏感。总之，我们的结果确定了在耐渗透细菌中受渗透胁迫调节的特定基因和机制，揭示了细胞包膜和胞质外蛋白对渗透压的重要作用。