Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
J Bacteriol. 2021 Jun 22;203(14):e0017921. doi: 10.1128/JB.00179-21.
The P. aeruginosa reference strain PAO1 has been used to delineate much of the physiology, metabolism, and fundamental biology of the species. The wild-type parent of PAO1 was lost, and PAO1 carries a regulatory mutation introduced for positive genetic selection that affects antibiotic resistance, virulence, quorum sensing, and other traits. The mutation is a loss-of-function change in an oxidoreductase gene (), which constitutively activates a stress response controlled by a positive regulator (MexT). Fitness defects associated with the constitutive response have led to the inadvertent selection of -minus suppressor mutations, creating genetic heterogeneity in PAO1 sublines studied in different laboratories. To help circumvent complications due to the -minus phenotypes, we created a wild-type version of PAO1 (called LPAO) by "reverting" its to the functional allele likely to have been in its parent. Phenotypic analysis revealed that the -minus allele in PAO1 makes growth sensitive to salt (NaCl) and is lethal when combined with mutations inactivating the major sodium antiporter (ShaABCDEF). The salt sensitivity of PAO1 may underlie some complex -minus phenotypes and help explain the selection of -minus suppressor mutations. To facilitate genetic comparisons of PAO1, LPAO, and other P. aeruginosa strains, we developed a transformation procedure to transfer selectable alleles, such as transposon insertion alleles, between strains. Overall, the study helps explain phenotypic heterogeneity of PAO1-derived strains and provides resources to help recognize and eliminate difficulties due to it. The P. aeruginosa reference strain PAO1 carries a regulatory mutation that may affect processes characterized in it. To eliminate complications due to the mutation, we constructed a version of the missing wild-type parent strain and developed methods to transfer mutations between PAO1 and the new strain. The methods are likely to be applicable to other isolates of P. aeruginosa as well.
铜绿假单胞菌参考菌株 PAO1 已被用于描绘该物种的许多生理学、代谢和基础生物学特性。PAO1 的野生型亲本丢失了，PAO1 携带了一个引入的调控突变，用于正向遗传选择，该突变影响抗生素抗性、毒力、群体感应和其他特性。该突变是一种氧化还原酶基因 () 的功能丧失变化，该基因持续激活受正调控因子 (MexT) 控制的应激反应。与组成型反应相关的适应性缺陷导致了 - 缺失抑制突变的意外选择，在不同实验室研究的 PAO1 亚系中造成了遗传异质性。为了帮助规避由于 - 缺失表型引起的并发症，我们通过“回复”其到可能存在于亲本中的功能等位基因，创建了 PAO1 的野生型版本 (称为 LPAO)。表型分析表明，PAO1 中的 - 缺失等位基因使生长对盐 (NaCl) 敏感，并且当与失活主要钠离子反向转运蛋白 (ShaABCDEF) 的突变结合时是致命的。PAO1 的盐敏感性可能是一些复杂 - 缺失表型的基础，并有助于解释 - 缺失抑制突变的选择。为了促进 PAO1、LPAO 和其他铜绿假单胞菌菌株的遗传比较，我们开发了一种转化程序，用于在菌株之间转移可选择的等位基因，例如转座子插入等位基因。总的来说，该研究有助于解释源自 PAO1 的菌株的表型异质性，并提供资源来帮助识别和消除由于该突变引起的困难。铜绿假单胞菌参考菌株 PAO1 携带一个可能影响其中所描述过程的调控突变。为了消除由于该突变引起的并发症，我们构建了缺失的野生型亲本菌株的一个版本，并开发了在 PAO1 和新菌株之间转移突变的方法。这些方法可能也适用于其他铜绿假单胞菌分离株。
