Differential Localization of Two Histidine Kinases Controlling Bacterial Cell Differentiation  Robert T. Wheeler, Lucy Shapiro  Molecular Cell  Volume 4, Issue 5, Pages 683-694 (November 1999) DOI: 10.1016/S1097-2765(00)80379-2

Figure 1 Characterization of ΔdivJ;plec::Tn5 and ΔdivJ, plec::Tn5 (A) Schematic diagram of the Caulobacter crescentus cell cycle. The timing of developmental events is indicated with bars. (B) Electron micrographs of the wild-type (CB15N), ΔdivJ, plec::Tn5, and ΔdivJ;pleC::Tn5 strains. Arrows indicate elongated stalks, and arrowheads indicate misplaced stalks. (C) DNA replication in ΔdivJ mutant monitored by fluorescence-activated cell sorting (FACS). Wild-type and ΔdivJ strains were treated with rifampicin to block replication initiation; cells were allowed to complete ongoing rounds of replication, then were fixed and stained with chromomycin to assay chromosome number. (D) In vivo phosphorylation of DivK in wild-type, ΔdivJ, pleC::Tn5, and ΔdivJ;plec::Tn5 strains. Mid-log phase cells were collected for analysis by Western blot or were labeled with 32Pi. DivK was immunoprecipitated from 32Pi-labeled cells, separated by SDS-PAGE, and visualized on a phosphoimager. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 2 Cell Cycle Regulation of DivJ and PleC (A) Immunoblot with α-DivJ antibodies. Lane 1, wild type (CB15N); lane 2, ΔdivJ; lane 3, divJ–M2; lane 4, divJ–GFP. (B) Cell cycle abundance of PleC–M2 and DivJ. Synchronous cells were collected at the indicated times and immunoblotted with α-M2 antibodies (for PleC–M2) or α-DivJ antibodies. The schematic diagram indicates cell cycle progression as followed by morphology. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 3 Cell Cycle Coimmunolocalization of DivJ–M2 and McpA (A) divJ–M2 cells were synchronized and at given stages of the cell cycle were fixed and costained for DivJ–M2 (indicated in green) and McpA (indicated in red). DNA was visualized with DAPI (indicated in blue). Colocalization of DivJ–M2 and McpA is shown in yellow. The upper panels show cells representative of different stages of the cell cycle, while the lower panels indicate the dynamic localization of the two proteins schematically. (B) LS 3208 (CB15N + pHCPxJM2) cells were induced to express DivJ–M2 for 45 min, then were synchronized, and swarmer (left) and stalked/predivisional (right) populations were immediately fixed and stained for DivJ–M2 (in green) and DNA (DAPI; in blue). (C) An immunoblot with α-M2 antibodies of swarmer and stalked/predivisional cells induced to express DivJ–M2 from the xylose promoter for 45 min and separated by Ludox density centrifugation. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 4 Time-Lapse Microscopy of DivJ–GFP and PleC–GFP (A) Cells of strain divJ–GFP were placed on a glass slide with an agarose pad containing minimal medium and were allowed to progress through one cell cycle. Images of the cells were taken every 30 min. The DIC images are in the center column, and the GFP fluorescence images are in the right column. A schematic representation of DivJ–GFP localization (in red) in a cross section of the left cell is shown in the left column. (B) Swarmer cells of strain LS 3209 (pleC::Tn5 + PleC–GFP) were placed on an agarose pad and were allowed to progress through the cell cycle. Images of cells were taken every 30 min. The DIC images are in the right column and the GFP fluorescence images are in the center column. A schematic representation of PleC–GFP localization in a cross section of the left-most cell is shown in the left column. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 5 Cell Cycle Localization of PleC–YFP and DivJ–CFP (A) Localization of DivJ–CFP and PleC–YFP in the same cell. Asynchronous cells of strain divJ–CFP,pleC–YFP were imaged. The left panel shows a schematic representation of cross sections of the cells in the field of view. The center panel shows the overlay of DivJ–CFP fluorescence (in green) and PleC–YFP fluorescence (in red). The right panel shows the corresponding DIC image labeled with the different cell types (SW, swarmer cell; ST, stalked cell; PD, predivisional cell). (B) Schematic representation of PleC and DivJ localization during the cell cycle. In swarmer cells, PleC localizes to the flagellar pole, while DivJ is undetectable. In stalked cells and early predivisional cells, PleC is dispersed throughout the membrane and DivJ localizes to the stalked pole. In late predivisional cells, PleC again localizes to the nascent flagellar pole and DivJ remains at the stalked pole. The progeny swarmer cells retain PleC at the flagellar pole, and the progeny stalked cells retain DivJ at the stalked pole. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 6 DivJ–GFP Is Delocalized in the pleC::Tn5 Mutant (A) Strain divJ–GFPSpSt,pleC::Tn5 was placed on a glass slide with an agarose pad and was allowed to progress through the cell cycle. Images of cells were taken every 60 min. The DIC images are in the left column, and the GFP fluorescence images are in the center column. The right column depicts a schematic representation of DivJ–GFP localization in a cross section of the left cell(s). (B) Strains divJ–M2SpSt and pleC::Tn5,divJ–M2SpSt were fixed and stained with α-M2 antibodies (shown in green) and FM4–64 (in red; stalks indicated by arrowheads). (C) Immunoblot with α-DivJ antibody in strains with chromosomally encoded divJ–GFPSpSt (lanes 1 and 2) or divJ–M2SpSt (lanes 3 and 4) in the wild-type background (lanes 1 and 3) and the pleC::Tn5 background (lanes 2 and 4). Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)

Figure 7 Model of DivJ Regulation by Localization (A) In wild-type cells, DivJ is localized to the stalked pole. We propose that this excludes its activity (represented by bars with double arrowheads) from the swarmer cell and swarmer compartment of the predivisional cell and limits its activity to stalked cells, predivisional cells, and the stalked compartment of the predivisional cell. Upon division, DivJ activity is restricted to the progeny stalked cell. (B) In pleC::Tn5 mutant cells, DivJ is dispersed throughout the cell and DivK phosphorylation is increased. We propose that the delocalization of DivJ allows phosphotransfer from DivJ to DivK to occur in all cell types throughout the cell cycle, leading to aberrant polar morphogenesis and the loss of asymmetry. Molecular Cell 1999 4, 683-694DOI: (10.1016/S1097-2765(00)80379-2)