Fig. 6. Overexpression of folK (encoding HPPK) rescued cells from CD15-3 induced growth inhibition.
A Overexpression was induced with 0.1% arabinose and folK was expressed under pBAD promoter. folK rescued growth rate at all CD15-3 concentrations. B Overexpression of folA (encoding DHFR) was induced at 0.005% arabinose. folA overexpression had only partial effect in recovering growth rate in CD15-3 treated cells. Growth rates for control (pBAD empty plasmids) and each of the conditions tested (folk and folA overexpressions) were derived from at three biologically independent bacterial (WT Escherichia coli BW25113) cell cultures. C Distribution of cell lengths as derived from DIC imaging of WT (WT Escherichia coli BW25113) and WT cells overexpressing folK under control and CD15-3 treatment conditions. Untreated WT and cells overexpressing folK had comparable cell lengths with median cell lengths of 1.06 µm. (n.s. indicates the distribution of cell lengths were not significantly different, one-sided Mann-Whitney test, p-value = 0.952). Both WT and cells overexpressing folK were subjected to CD15-3 treatment at IC50 concentrations. WT treated cells had a filamentous morphology and the median cell length (2.41 µm) double of that of the untreated WT set. WT cells overexpressing folK after CD15-3 treatment had a median cell length of 1.252 µm which is slightly higher than that of untreated set. However, the cell size distribution of the cells overexpressing folK showed much less change after CD15-3 treatment compared to that observed for the WT (* indicates the distribution of cell lengths were significantly different, one-sided Mann-Whitney test, p value < 0.001). WT cells with empty p-BAD plasmid under no treatment had a minimum length of 0.5 µm and a maximum length of 2.27 µm. WT cells with p-BAD plasmid expressing folK under no treatment had a minimum length of 0.5 µm and a maximum length of 2.31 µm. WT cells with empty p-BAD plasmid under CD15-3 treatment had a minimum length of 0.5 µm and a maximum length of 7.48 µm. WT cells with p-BAD plasmid expressing folK under CD15-3 treatment had a minimum length of 0.5 µm and a maximum length of 2.78 µm. Imaging experiments were carried out for 4 times for each of the test conditions as well as the control. The statistics for cell length was derived after measuring cell length for >300 cells (300 to 400 cells) for each of the control and experimental conditions. The central band in the boxplots represents the median of the distribution, square-points inside the box represent the mean cell length of the distribution, the box ends represent the 25th and 75th percentiles, and the whiskers represent the 10th and 90th percentiles. D Inhibition of purified HPPK (encoded by folK) by CD15-3 in an in vitro assay (see text and Methods for details). Residual activity of HPPK at various concentrations of CD15-3 is shown. Inset shows the plot for the absolute chemiluminescence signals at various concentrations of CD15-3 in the HPPK assay buffer. At each of the CD15-3 concentration tested (from 0 to 400 µM) three individual chemiluminescence readouts were recorded. arb. units. in the inset figure refers to arbitrary unit. Activity was calculated from the chemiluminescence signals derived from three biologically independent repeats. All the data are presented as mean values +/- SEM. Source data are provided as a Source Data file.