Department of Genetics, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India.
VIT Bhopal University, Sahore, India.
Mol Biol Rep. 2020 Oct;47(10):7941-7957. doi: 10.1007/s11033-020-05872-y. Epub 2020 Oct 4.
Transcriptional regulators in bacteria are the crucial players in mediating communication between environmental cues and DNA transcription through a complex network process. Pseudomonas aeruginosa PGPR2 is an efficient root colonizer and a biocontrol strain. Previously, we identified that the transcriptional regulator, asnC, negatively regulates the corn root colonization of P. aeruginosa PGPR2. In a transposon insertion sequencing (INSeq) screen, the asnC insertion mutant was positively selected during root colonization, meaning the disruption of asnC improves the fitness of the P. aeruginosa PGPR2 strain for the root colonization. In this study, we constructed isogenic mutant of asnC family transcriptional regulator encoded by PGPR2_17510 by allele exchange mutagenesis. The ΔasnC mutant was able to efficiently colonize corn roots with a twofold increase in population when compared to the wild-type strain. Similarly, the mutant strain outcompeted the wild-type strain in a competition assay, where the mutant strain represented 90% of the total population recovered from the root. We compared the whole transcriptome of the wild-type and the ΔasnC mutant of P. aeruginosa PGPR2 when exposed to the corn root exudates. The RNA-Seq revealed that a total of 360 genes were differentially expressed in the ΔasnC strain of P. aeruginosa PGPR2. Inactivation of asnC transcriptional regulator resulted in the up-regulation of several genetic factors implicated in metabolism, uptake of nutrients, motility, stress response, and signal transduction, which could play crucial roles in root colonization. This notion was further validated by phenotypic characterization and quantification of transcription pattern of selected genes associated with metabolism, motility, and carbon catabolite repression between wild type and mutant strain, which was in agreement with transcriptome data. Similarly, ΔasnC strain formed increased biofilm on abiotic surface validating our RNA-seq analysis, where transcript levels of several genes associated with biofilm formation were up-regulated in the mutant strain. We report that the inactivation of an asnC family transcriptional regulator encoded by PGPR2_17510 enhances the root colonization and biofilm-forming ability of P. aeruginosa PGPR2. Together, our results provide evidence for the molecular adaptations that enable ΔasnC mutant strain to colonize on the corn roots and to form a biofilm.
细菌中的转录调节剂是在通过复杂的网络过程介导环境线索与 DNA 转录之间的通讯的关键参与者。铜绿假单胞菌 PGPR2 是一种有效的根定植菌和生物防治菌株。以前，我们发现转录调节剂 asnC 负调控铜绿假单胞菌 PGPR2 对玉米根的定殖。在转座子插入测序 (INSeq) 筛选中，asnC 插入突变体在根定植过程中被正向选择，这意味着 asnC 的破坏提高了铜绿假单胞菌 PGPR2 菌株对根定植的适应性。在这项研究中，我们通过等位基因交换诱变构建了 PGPR2_17510 编码的 asnC 家族转录调节剂的同基因突变体。与野生型菌株相比，ΔasnC 突变体能够有效地定植玉米根，种群增加了两倍。同样，在竞争测定中，突变株与野生型菌株竞争，从根中回收的总群体中，突变株代表 90%。当铜绿假单胞菌 PGPR2 暴露于玉米根分泌物时，我们比较了野生型和 ΔasnC 突变体的全转录组。RNA-Seq 显示，PGPR2 中的总共 360 个基因在 ΔasnC 菌株中差异表达。asnC 转录调节剂的失活导致参与代谢、营养吸收、运动性、应激反应和信号转导的几个遗传因子的上调，这可能在根定植中发挥关键作用。通过表型特征和与代谢、运动性和碳分解代谢抑制相关的选定基因的转录模式的量化来验证这一观点，这与转录组数据一致。同样，ΔasnC 菌株在非生物表面上形成增加的生物膜，验证了我们的 RNA-seq 分析，其中与生物膜形成相关的几个基因的转录水平在突变株中上调。我们报告说，PGPR2_17510 编码的 asnC 家族转录调节剂的失活增强了铜绿假单胞菌 PGPR2 的根定植和生物膜形成能力。总之，我们的结果为使 ΔasnC 突变株能够在玉米根上定植并形成生物膜的分子适应提供了证据。
