FIGURE 6.
Identification and comparison of PilM and PilM·PilN(1–12) interactions with PilB, PilT, and PilC. A, qualitative BACTH results from MacConkey-maltose agar assay. Colonies that turned red are indicated with a plus sign to denote an interaction; colonies that remained white are indicated with a minus sign to denote no interaction; and colonies that displayed an intermediate phenotype (speckled or inconsistently red colonies) are indicated with a plus/minus sign. B, P. aeruginosa BACTH results represented as solid lines for positive (+) results and dotted lines for intermediate (+/−) results. C, quantitative BACTH β-galactosidase assay results. pKT25 with a PilM-M::N1–12/N/O/C/B/T/U/A insert was co-transformed with pUT18C:PilM (black), pUT18C:PilM·PilN(1–12) (gray), or empty pUT18C (white). Data represent the mean, and the error bars represent the S.E. The assay was performed in eight replicates. *, p < 0.05, Student's t test. D, in vitro PilM and PilM·PilN(1–12) pull-down experiments with His-tagged PilB and PilT. The His-tagged proteins were pulled down using nickel affinity chromatography and visualized by Western blot analysis as indicated. Two PilT bands are evident and may represent partial breakdown. †, a band non-specifically bound by the PilB antibody.