Figure 2: PAS domains regulate CckA localization and activity in vivo.

(a) A cell length and subcellular localization assay shows that deletion of CckA’s PAS domains impact both cell length and CckA’s subcellular distribution. In each strain, the chromosomal cckA locus was replaced with a gentamycin cassette, and the stalked-pole marker protein StpX was expressed with an mCherry tag. CckA-eYFP constructs were expressed from the cckA native promoter on a low-copy replicating plasmid. White arrowheads indicate the stalked pole in the eYFP images. Scale bar is the same for each field, 2 μm. (b) Deletion of either PAS domain causes an increased cell-length phenotype. Cell lengths were grouped in 0.5 μm bins, with cells >8 μm binned together, and represented as a percentage of the total population. N>1,000 cells for each strain. (c) PAS-A is necessary for localization to the cell poles, and PAS-B regulates new pole localization in vivo. Quantitation of the proportion of cells in which CckA-eYFP localized to both cell poles (black bar), formed a single focus at the stalked pole (grey bar) or remained diffuse over the membrane (white bar). A stpX-mCherry label was used to definitively assign the stalked pole CckA accumulations. Deletion of PAS-B specifically depleted the new-cell-pole population while maintaining stalk cell pole localization. Deletion of PAS-A totally abrogated subcellular accumulation at both poles. Fewer than 5% of cells in any strain had monopolar accumulations of CckA-eYFP at the new cell pole. Error bars represent the s.d. across individual fields of view, with at least 200 cells counted for each mutant. (d) RT-qPCR assays of the expression from CtrA-controlled promoters ccrM, sciP and divK show that deletion of either PAS domain results in decreased gene expression for all three promoters. Expression was measured for a separate amplicon within each of the three genes, and expression of each gene was normalized to an internal standard, an amplicon within Rho. Error bars represent the s.d. of three biological replicates, each composed of at least two technical replicates.