7 (12.4) 0.03 ± 0.01 WT+mglBA T54A MxH2405 2.5 (16.2) 9.3 (14.4) 0.01 ± 0.0 WT+mglBA T78A MxH2425 1.7 (25.0)
8.2 (13.4) 30 ± 6 WT+mglBA T78S MxH2426 2.2 (21.4) 7.1 (15.5) < 0.01 WT+mglBA T78D MxH2428 NM 6.0 (12.6) 90 ± 5 WT+mglBA P80A MxH2356 2.0 (23.6) 2.3 (18.3) 40 ± 6 WT+mglBA Q82A MxH2404 1.6 (30.0) 7.5 (13.5) < 0.01 WT+mglBA Ibrutinib in vivo Q82R MxH2368 2.6 (22.1) 10.0 (22.2) 100 ± 18 WT+mglBA L117/L120A MxH2337 1.3 (15.6) 8.1 (18.4) 100 ± 18 WT+mglBA L124K MxH2278 2.4 (15.1) 3.5 (15.4) < 0.01 WT+mglBA N141A MxH2336 1.7 (NR) 2.1 (17.2) 0.2 ± 0.2 WT+mglBA K142A MxH2364 1.4 (21.3) 9.3 (17.6) 40 ± 6 WT+mglBA D144A MxH2366 1.6 (22.5) 2.4 (11.5) 4 ± 1 Time-lapse microscopy was performed to determine the rates of gliding cells. a Gliding and reversal rates for cells using A-motility were measured on 1.5% CTPM agarose pads as described in Methods. NM = Cells were nonmotile. NR = no reversals observed. b Gliding and reversal rates for cells using S-motility were measured in 0.5% methylcellulose plus 0.5× CTPM as described in Methods. NM = Cells were nonmotile.
Gliding speeds are represented as the average and range of 25 cells from two independent assays. cSporulation rates are given as a percentage relative to the WT and the standard deviation if available. The ability of MglA mutants to complement the sporulation defects of the ΔmglBA mutant was performed as described in Methods. mgl alleles were introduced into the WT background to determine MglA mutants could interfere with the function of normal MglA during sporulation. All three strains were examined for their ability to move as individual cells or in groups NVP-AUY922 at
the edge of a colony arising from a single cell. The colony edge morphology is illustrated in Figure 2C. A- and S-motility were restored (panel 3) to the ΔmglBA mutant when complemented with wild type mglBA, but addition of mglBA constructs with mglA-G19A, K25A and T26N failed to complement. To determine whether these mutants produced stable MglA, whole cell extracts were ifoxetine probed with α-MglA antibody. As shown in Figure 2D, MglA protein was not detected by Western blot analysis for any of the PM1 mutants relative to the loading control (sample Western with loading control is shown in Additional file 6: FigureS6 Western control). WT cells displayed a punctate distribution of MglA along the cells length as visible by immunofluoresence, as shown in Figure 3A. In contrast, the deletion parent mglBA did not produce MglA and showed no fluorescence relative to the background, Figure 3B. All PM1 mutations in conserved residues resembled the deletion parent as shown in Figure 3B. To investigate the possibility that lack of MglA was due to decreased transcription, we performed RT-PCR to obtain a quantitative measure of transcription from the mgl locus. Total mRNA was obtained from mid-log phase M.