Department of Microbiology, University of Georgiagrid.213876.9, Athens, Georgia, USA.
mBio. 2022 Jun 28;13(3):e0107122. doi: 10.1128/mbio.01071-22. Epub 2022 Jun 2.
Pseudomonas aeruginosa encodes eight members of the Rid protein superfamily. PA5339, a member of the RidA subfamily, is required for full growth and motility of P. aeruginosa. Our understanding of RidA integration into the metabolic network of P. aeruginosa is at an early stage, with analyses largely guided by the well-established RidA paradigm in Salmonella enterica. A P. aeruginosa strain lacking RidA has a growth and motility defect in a minimal glucose medium, both of which are exacerbated by exogenous serine. All described mutant phenotypes are rescued by supplementation with isoleucine, indicating the primary generator of the reactive metabolite 2-aminoacrylate (2AA) in mutants is a threonine/serine dehydratase. However, the critical (i.e., phenotype determining) targets of 2AA leading to growth and motility defects in P. aeruginosa remained undefined. This study was initiated to probe the effects of 2AA stress on the metabolic network of P. aeruginosa by defining the target(s) of 2AA that contribute to physiological defects of a mutant. Suppressor mutations that restored growth to a P. aeruginosa mutant were isolated, including an allele of (encoding cysteine desulfurase). Damage to IscS was identified as a significant cause of growth defects of P. aeruginosa during enamine stress. A suppressing allele encoded an IscS variant that was less sensitive to damage by 2AA, resulting in a novel mechanism of phenotypic suppression of a mutant. 2-aminoacrylate (2AA) is a reactive metabolite formed as an intermediate in various enzymatic reactions. In the absence of RidA, this metabolite can persist where it attacks and inactivates specific PLP-dependent enzymes, causing metabolic defects and organism-specific phenotypes. This work identifies the cysteine desulfurase IscS as the critical target of 2AA in Pseudomonas aeruginosa. A single substitution in IscS decreased sensitivity to 2AA and suppressed growth phenotypes of a mutant. Here, we provide the first report of suppression of a mutant phenotype by altering the sensitivity of a target enzyme to 2AA.
铜绿假单胞菌编码了 8 种 Rid 蛋白超家族成员。RidA 亚家族的成员 PA5339 是铜绿假单胞菌充分生长和运动所必需的。我们对 RidA 整合到铜绿假单胞菌代谢网络的理解还处于早期阶段，分析主要是基于沙门氏菌中成熟的 RidA 范例。缺乏 RidA 的铜绿假单胞菌在最小葡萄糖培养基中生长和运动缺陷，这两种缺陷都因外源丝氨酸而加剧。所有描述的突变表型都可以通过补充异亮氨酸得到挽救，表明突变体中活性代谢物 2-氨基丙烯酸（2AA）的主要生成物是苏氨酸/丝氨酸脱水酶。然而，导致铜绿假单胞菌生长和运动缺陷的 2AA 的关键（即表型决定）靶标仍然未知。本研究旨在通过确定 2AA 应激对铜绿假单胞菌代谢网络的影响，从而确定导致铜绿假单胞菌 突变体生理缺陷的靶标。分离出了恢复铜绿假单胞菌 突变体生长的抑制突变，包括 （编码半胱氨酸脱硫酶）的一个等位基因。在烯胺应激下，发现 IscS 的损伤是铜绿假单胞菌生长缺陷的一个重要原因。一个抑制等位基因编码的 IscS 变体对 2AA 的损伤敏感性降低，导致一种新的抑制 突变体表型的机制。2-氨基丙烯酸（2AA）是一种在各种酶促反应中形成的中间活性代谢物。在缺乏 RidA 的情况下，这种代谢物可以持续存在，攻击并失活特定的 PLP 依赖性酶，导致代谢缺陷和特定于生物体的表型。这项工作确定了铜绿假单胞菌中的半胱氨酸脱硫酶 IscS 为 2AA 的关键靶标。IscS 中的一个单一取代降低了对 2AA 的敏感性，并抑制了 突变体的生长表型。在这里，我们首次报道了通过改变靶酶对 2AA 的敏感性来抑制 突变体表型。
