Low doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism.IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.

中文翻译：

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多组学分析提供了低剂量抗生素治疗与泰国伯克霍尔德氏菌次级代谢诱导之间的联系。

低剂量的抗生素可以触发细菌的次级代谢产物生物合成，但其潜在机制通常是未知的。我们试图通过研究抗生素甲氧苄啶如何激活泰国伯克霍尔德氏菌中毒力因子 malleilactone 的合成来更好地理解这种现象。我们使用转录组学、定量多重蛋白质组学和初级代谢组学，系统地绘制了甲氧苄啶引起的变化。令人惊讶的是，即使是亚抑制剂量的抗生素也会导致广泛的转录和翻译改变，约 8.5% 的转录组和约 5% 的蛋白质组上调或下调 > 4 倍。遗传生化实验的后续研究表明，由于叶酸途径的抑制，甲硫氨酸生物合成前体（尤其是高丝氨酸）的积累可以充分解释马来内酯合成的诱导。高丝氨酸通过转录调节因子 MalR 激活马来内酯基因簇，并产生与甲氧苄啶产生的次级代谢组非常相似。我们的工作强调了低剂量甲氧苄氨嘧啶引起的细菌生理学的广泛变化，并提供了对其对次级代谢的刺激作用的见解。重要意义抗生素的发现是上个世纪最重要的成就之一。尽管几乎所有临床抗生素的靶标都是已知的，我们对它们的自然功能和亚抑制浓度的影响的理解还处于初级阶段。刺激而非抑制功能归因于低剂量抗生素。其中，我们之前发现抗生素可以激活沉默的生物合成基因，从而增强细菌的代谢输出。这种现象背后的调节电路是未知的。我们迈出了阐明这些回路的第一步，并表明低剂量的甲氧苄啶 (Tmp) 对腐生菌 Burkholderia thailandensis 具有全细胞范围的影响。最重要的是，Tmp 对单碳代谢过程的抑制导致高丝氨酸的积累，从而诱导通过 LuxR 型转录调节因子产生沉默的细胞毒素。