Functional characterization of asnC family transcriptional regulator in Pseudomonas aeruginosa PGPR2 during root colonization

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.