Burkholderia thailandensis is a soil saprophyte that is closely related to the pathogen Burkholderia pseudomallei, the etiological agent of melioidosis in humans. The environmental niches and infection sites occupied by these bacteria are thought to contain only limited concentrations of oxygen, where they can generate energy via denitrification. However, knowledge of the underlying molecular basis of the denitrification pathway in these bacteria is scarce. In this study, we employed a transposon sequencing (Tn-Seq) approach to identify genes conferring a fitness benefit for anaerobic growth of B. thailandensis. Of the 180 determinants identified, several genes were shown to be required for growth under denitrifying conditions: the nitrate reductase operon narIJHGK2K1, the aniA gene encoding a previously unknown nitrite reductase, and the petABC genes encoding a cytochrome bc₁, as well as three novel regulators that control denitrification. Our Tn-Seq data allowed us to reconstruct the entire denitrification pathway of B. thailandensis and shed light on its regulation. Analyses of growth behaviors combined with measurements of denitrification metabolites of various mutants revealed that nitrate reduction provides sufficient energy for anaerobic growth, an important finding in light of the fact that some pathogenic Burkholderia species can use nitrate as a terminal electron acceptor but are unable to complete denitrification. Finally, we demonstrated that a nitrous oxide reductase mutant is not affected for anaerobic growth but is defective in biofilm formation and accumulates N₂O, which may play a role in the dispersal of B. thailandensis biofilms.

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全基因组突变分析对泰国伯克霍尔德菌反硝化途径的定位

伯克霍尔德氏菌是一种土壤腐生植物，与人类鼻疽病的病原体-伯克霍尔德氏菌假病菌密切相关。这些细菌占据的环境生态位和感染部位被认为只含有有限浓度的氧气，它们可以通过反硝化作用产生能量。但是，对这些细菌中反硝化途径的潜在分子基础的了解很少。在这项研究中，我们采用了转座子测序（Tn-Seq）方法来鉴定赋予泰国双歧杆菌厌氧生长适度有益的基因。在确定的180个决定因素中，显示了在反硝化条件下生长所需的几个基因：硝酸还原酶操纵子narIJHGK2K1，所述阿尼亚编码先前未知的亚硝酸还原酶基因和petABC编码细胞色素的基因BC ₁，以及三种新型稳压器，控制反硝化。我们的Tn-Seq数据使我们能够重建泰国芽孢杆菌的整个反硝化途径并阐明其调控方法。对生长行为的分析以及对各种突变体反硝化代谢产物的测量结果表明，硝酸盐还原可为厌氧生长提供足够的能量，鉴于某些致病性伯克霍尔德氏菌这一事实，这是一个重要发现物种可以使用硝酸盐作为末端电子受体，但无法完成反硝化作用。最后，我们证明了一氧化二氮还原酶突变体不影响厌氧生长，但在生物膜形成中有缺陷并累积N ₂ O，这可能在泰国芽孢杆菌生物膜的扩散中起作用。