Effect of R36 and R42 mutations on mini-F clustering.

<p>Derivatives of strain DLT2583 (<i>para-tetR</i>::<i>gfp</i>) carrying the <i>tetO</i>-array mini-F, pDAG848, and producing SopA and mutant SopBs <i>in trans</i> were grown in MGC medium (A) or LB broth (B), and the mini-F plasmid foci were visualized as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003956#s4" target="_blank">Materials and Methods</a> and counted. Total cells scored were: A - 756 (wt), 626 (R36A) 415 (R36A, R42A); 344 (Δ<i>sopA</i>); 368 (Δ<i>sopA</i>, R36A); B - 2118 (wt), 3966 (R36A), 1874 (R36K), 1585 (R42A), 1736 (R42K). The wt and R36A focus number distributions in panel A were shown by the Student t-test to be different at a confidence interval of 95%. Sop protein concentrations relative to wt mini-F were measured by immuno-blot assay to be 1.9- and 2.2-fold (SopA) and 1.7 and 2.4-fold (SopB) higher in cells grown in MGC and LB respectively. C. Brightness of the foci in LB-grown cells, measured using Microbe Tracker. The distributions are different at the 95% confidence level by the Student t-test. Because focus intensity responds non-linearly to plasmid content (as seen elsewhere; e.g. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003956#pgen.1003956-Burton1" target="_blank">[51]</a>), foci in R36A cells were on average 1.3-fold brighter than in SopB-wt cells while consisting of 2.4 times as many molecules. D. Localization of pDAG848 without SopA. Cells were grown in MGC. The shortest pole-focus distances in single-focus cells were sorted into 1/10 cell-length classes. Dotted-line bars show distributions of single foci in the presence of SopA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003956#pgen-1003956-g005" target="_blank">Figure 5B</a>). Distributions of focus brightness in Δ<i>sopA</i> cells containing wt and R36A SopB proteins were not significantly different at the 95% confidence level, by the same test as applied to the brightness data of <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003956#pgen-1003956-g002" target="_blank">Figure 2C</a> (not shown).