Laboratory of Microbiology, Department of Biochemistry and Microbiology, Faculty of Sciences, Ghent Universitygrid.5342.0, Ghent, Belgium.
Laboratory of Pharmaceutical Microbiology, Department of Pharmaceutical Analysis, Faculty of Pharmaceutical Sciences, Ghent Universitygrid.5342.0, Ghent, Belgium.
Appl Environ Microbiol. 2021 Oct 28;87(22):e0116921. doi: 10.1128/AEM.01169-21. Epub 2021 Sep 15.
Burkholderia cepacia complex strain R-12632 produces ditropolonyl sulfide, an unusual sulfur-containing tropone, via a yet-unknown biosynthetic pathway. Ditropolonyl sulfide purified from a culture of strain R-12632 inhibits the growth of various Gram-positive and Gram-negative resistant bacteria, with MIC values as low as 16 μg/ml. In the present study, we used a transposon mutagenesis approach combined with metabolite analyses to identify the genetic basis for antibacterial activity of strain R-12632 against Gram-negative bacterial pathogens. Fifteen of the 8304 transposon mutants investigated completely lost antibacterial activity against Klebsiella pneumoniae LMG 2095. In these loss-of-activity mutants, nine genes were interrupted. Four of those genes were involved in assimilatory sulfate reduction, two were involved in phenylacetic acid (PAA) catabolism, and one was involved in glutathione metabolism. Via semipreparative fractionation and metabolite identification, it was confirmed that inactivation of the PAA degradation pathway or glutathione metabolism led to loss of ditropolonyl sulfide production. Based on earlier studies on the biosynthesis of tropolone compounds, the requirement for a functional PAA catabolic pathway for antibacterial activity in strain R-12632 indicated that this pathway likely provides the tropolone backbone for ditropolonyl sulfide. Loss of activity observed in mutants defective in assimilatory sulfate reduction and glutathione biosynthesis suggested that cysteine and glutathione are potential sources of the sulfur atom linking the two tropolone moieties. The demonstrated antibacterial activity of the unusual antibacterial compound ditropolonyl sulfide warrants further studies into its biosynthesis and biological role. bacteria are historically known for their biocontrol properties and have been proposed as a promising and underexplored source of bioactive specialized metabolites. Burkholderia cepacia complex strain R-12632 inhibits various Gram-positive and Gram-negative resistant pathogens and produces numerous specialized metabolites, among which is ditropolonyl sulfide. This unusual antimicrobial has been poorly studied and its biosynthetic pathway remains unknown. In the present study, we performed transposon mutagenesis of strain R-12632 and performed genome and metabolite analyses of loss-of-activity mutants to study the genetic basis for antibacterial activity. Our results indicate that phenylacetic acid catabolism, assimilatory sulfate reduction, and glutathione metabolism are necessary for ditropolonyl sulfide production. These findings contribute to understanding of the biosynthesis and biological role of this unusual antimicrobial.
洋葱伯克霍尔德菌复合菌株 R-12632 通过未知的生物合成途径产生二硝甲砜，这是一种不寻常的含硫三酮。从 R-12632 菌株的培养物中纯化的二硝甲砜抑制各种革兰氏阳性和革兰氏阴性耐药菌的生长，MIC 值低至 16μg/ml。在本研究中，我们使用转座子诱变方法结合代谢物分析来确定 R-12632 菌株对革兰氏阴性细菌病原体的抗菌活性的遗传基础。在所研究的 8304 个转座子突变体中，有 15 个完全丧失了对肺炎克雷伯菌 LMG 2095 的抗菌活性。在这些失活突变体中，有 9 个基因被中断。其中 4 个基因参与同化硫酸盐还原，2 个基因参与苯乙酸（PAA）分解代谢，1 个基因参与谷胱甘肽代谢。通过半制备级分分离和代谢物鉴定，证实 PAA 降解途径或谷胱甘肽代谢的失活导致二硝甲砜产量的丧失。基于先前关于三酮化合物生物合成的研究，R-12632 菌株中功能性 PAA 分解代谢途径对抗菌活性的要求表明，该途径可能为二硝甲砜提供三酮骨架。在同化硫酸盐还原和谷胱甘肽生物合成缺陷的突变体中观察到的活性丧失表明半胱氨酸和谷胱甘肽可能是连接两个三酮部分的硫原子的潜在来源。这种不寻常的抗菌化合物二硝甲砜表现出的抗菌活性表明，需要进一步研究其生物合成和生物学作用。
细菌因其生物防治特性而在历史上闻名，被认为是具有生物活性的特殊代谢物的有前途且尚未充分探索的来源。洋葱伯克霍尔德菌复合菌株 R-12632 抑制各种革兰氏阳性和革兰氏阴性耐药病原体，并产生多种特殊代谢物，其中包括二硝甲砜。这种不寻常的抗菌物质研究甚少，其生物合成途径仍不清楚。在本研究中，我们对 R-12632 菌株进行了转座子诱变，并对失活突变体进行了基因组和代谢物分析，以研究其抗菌活性的遗传基础。我们的结果表明，苯乙酸分解代谢、同化硫酸盐还原和谷胱甘肽代谢是二硝甲砜产生所必需的。这些发现有助于了解这种不寻常的抗菌物质的生物合成和生物学作用。
