Pseudomonas putida S12 is intrinsically solvent-tolerant and constitutes a promising platform for biobased production of aromatic compounds and biopolymers. The genome of P. putida S12 consists of a 5.8 Mbp chromosome, and a 580 kbp megaplasmid pTTS12 that carries several gene clusters involved in solvent tolerance. Removal of pTTS12 caused a significant reduction in solvent tolerance. In this study, we succeeded in restoring solvent tolerance in plasmid-cured P. putida S12 using adaptive laboratory evolution (ALE), underscoring the innate solvent-tolerance of this strain. Whole genome sequencing revealed several single nucleotide polymorphisms (SNPs) and a mobile element insertion, enabling ALE-derived strains to survive and sustain growth in the presence of a high toluene concentration (10% v/v). Mutations were identified in an RND efflux pump regulator arpR, resulting in constitutive upregulation of the multifunctional efflux pump ArpABC. SNPs were also found in the intergenic region and subunits of ATP synthase, RNA polymerase subunit B, global two-component regulatory system (GacA/GacS) and a putative AraC-family transcriptional regulator Afr. RNA-seq analysis further revealed a constitutive down-regulation of energy consuming activities in ALE-derived strains, including flagellar assembly, F0F1 ATP synthase, and membrane transport proteins. Out results indicate that constitutive expression of an alternative solvent extrusion pump in combination with high metabolic flexibility ensures restoration of solvent-tolerance in P. putida S12 lacking its megaplasmid.

中文翻译：

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实验室适应性进化可恢复质粒固化的恶臭假单胞菌S12的耐溶剂性。分子分析

恶臭假单胞菌（Pseudomonas putida）S12本质上是耐溶剂的，并且构成了基于生物的芳族化合物和生物聚合物生产的有希望的平台。恶臭假单胞菌S12的基因组由一个5.8 Mbp的染色体和一个580 kbp的大质粒pTTS12组成，该质粒带有几个涉及耐溶剂性的基因簇。去除pTTS12导致溶剂耐受性显着降低。在这项研究中，我们使用自适应实验室进化（ALE）成功地恢复了质粒固化的恶臭假单胞菌S12的耐溶剂性，强调了该菌株的固有耐溶剂性。全基因组测序显示了几个单核苷酸多态性（SNP）和一个移动元件插入，使ALE衍生的菌株能够在高甲苯浓度（10％v / v）存在的情况下存活并维持生长。在RND外排泵调节器arpR中鉴定出突变，导致多功能外排泵ArpABC的组成型上调。在ATP合酶的基因间区域和亚基，RNA聚合酶亚基B，全局两组分调节系统（GacA / GacS）和推定的AraC家族转录调节剂Afr中也发现了SNP。RNA-seq分析进一步揭示了ALE衍生菌株（包括鞭毛装配，F0F1 ATP合酶和膜转运蛋白）中能量消耗活动的组成性下调。结果表明，替代溶剂挤出泵的本构表达与高代谢灵活性相结合，可确保在缺乏大质粒的恶臭假单胞菌S12中恢复其耐溶剂性。导致多功能外排泵ArpABC的组成上调。在ATP合酶的基因间区域和亚基，RNA聚合酶亚基B，全局两组分调节系统（GacA / GacS）和推定的AraC家族转录调节剂Afr中也发现了SNP。RNA-seq分析进一步揭示了ALE衍生菌株（包括鞭毛装配，F0F1 ATP合酶和膜转运蛋白）中能量消耗活动的组成性下调。结果表明，替代溶剂挤出泵的本构表达与高代谢灵活性相结合，可确保缺乏大质粒的恶臭假单胞菌S12恢复耐溶剂性。导致多功能外排泵ArpABC的组成型上调。在ATP合酶的基因间区域和亚基，RNA聚合酶亚基B，全局两组分调节系统（GacA / GacS）和推定的AraC家族转录调节剂Afr中也发现了SNP。RNA-seq分析进一步揭示了ALE衍生菌株（包括鞭毛装配，F0F1 ATP合酶和膜转运蛋白）中能量消耗活动的组成性下调。结果表明，替代溶剂挤出泵的本构表达与高代谢灵活性相结合，可确保缺乏大质粒的恶臭假单胞菌S12恢复耐溶剂性。全球两部分调节系统（GacA / GacS）和推定的AraC家族转录调节剂Afr。RNA-seq分析进一步揭示了ALE衍生菌株（包括鞭毛装配，F0F1 ATP合酶和膜转运蛋白）中能量消耗活动的组成性下调。结果表明，替代溶剂挤出泵的本构表达与高代谢灵活性相结合，可确保缺乏大质粒的恶臭假单胞菌S12恢复耐溶剂性。全球两部分调节系统（GacA / GacS）和推定的AraC家族转录调节剂Afr。RNA-seq分析进一步揭示了ALE衍生菌株（包括鞭毛装配，F0F1 ATP合酶和膜转运蛋白）中能量消耗活动的组成性下调。结果表明，替代溶剂挤出泵的本构表达与高代谢灵活性相结合，可确保缺乏大质粒的恶臭假单胞菌S12恢复耐溶剂性。