Fig 6. Analysis of loss-of-function and gain-of-function associated with chromosomal deletion and in-trans complementation of Bucl8-pump components.
(A) Changes in sensitivity/resistance patterns in bacterial strains. MIC was determined by plating bacteria on LA containing each substrate. FA, fusaric acid; pHBA, para-hydroxybenzoic acid; CHL, chloramphenicol. (B) Relative expression of fusC genes. RT-qPCR was performed on total mRNA isolated from non-treated and FA-treated (1000 μM, 1 hour) Bp82 cultures (OD600 ~0.4). Graph shows fold change of relative gene expression compared to untreated cultures and normalized to 16S rRNA. Technical and experimental replicates were done in triplicate. (C-D) Effect of chromosomal deletion on growth. Parental strain Bp82 and its bucl8-fusE deletion mutant (Bp82Dbucl8-fusE) were grown in LBM broth at 37°C with shaking. Changes in OD600 (C) were recorded and CFU numbers (D) by plating on LA medium every hour. Data represents the average of three biological replicates. 2-way AVOVA with Tukey multiple comparison test, ***p < 0.001. Error bars represent ±SEM.