Full induction of MshA^P31S is required to recover piliation in the absence of <i>pilT</i>.

(A) Representative images of piliated cells from strains under increasing induction of either Ptac-riboswitch-mshAparent or Ptac-riboswitch-mshAP31S. All mshA alleles in these strains (native and ectopic) contain the T70C mutation needed for AF488-mal labeling. Concentrations of inducer used from left to right: (1) 0 μM IPTG + 0 μM theophylline, (2) 5 μM IPTG + 75 μM theophylline, (3) 20 μM IPTG + 300 μM theophylline, and (4) 100 μM IPTG + 1.5 mM theophylline. Phase images (top) show cell boundaries and fluorescence images (bottom) show AF488-mal labeled pili. Scale bar = 2 μm. (B) Quantification of piliation in samples from A. Cells were categorized as either having no pili (white bars), 1–2 pili (light gray bars), or at least 3 pili (dark gray bars). n = 300 cells analyzed from three independent biological replicates for all samples. (C) Western blot quantification of cell-associated MshA in the indicated strains in the induction conditions used in A and B. Band intensities are normalized to the RpoA loading control. Data are from three independent biological replicates. All data are displayed as the mean ± SD. (TIFF)