Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus - PubMed (original) (raw)

Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus

Stephen A Sciochetti et al. J Bacteriol. 2002 Nov.

Abstract

The Caulobacter crescentus sensor kinase DivJ is required for an early cell division step and localizes at the base of the newly formed stalk during the G1-to-S-phase transition when the protein is synthesized. To identify sequences within DivJ that are required for polar localization, we examined the ability of mutagenized DivJ sequences to direct localization of the green fluorescent protein. The effects of overlapping C-terminal deletions of DivJ established that the N-terminal 326 residues, which do not contain the kinase catalytic domain, are sufficient for polar localization of the fusion protein. Internal deletions mapped a shorter sequence between residues 251 and 312 of the cytoplasmic linker that are required for efficient localization of this sensor kinase. PleC kinase mutants, which are blocked in the swarmer-to-stalked-cell transition and form flagellated, nonmotile cells, also fail to localize DivJ. To dissect the cellular factors involved in establishing subcellular polarity, we have examined DivJ localization in a pleC mutant suppressed by the sokA301 allele of ctrA and in a pleD mutant, both of which display a supermotile, stalkless phenotype. The observation that these Mot(+) strains localize DivJ to a single cell pole indicate that localization may be closely coupled to the gain of motility and that normal stalk formation is not required. We have also observed, however, that filamentous parC mutant cells, which are defective in DNA segregation and the completion of cell separation, are motile and still fail to localize DivJ to the new cell pole. These results suggest that formation of new sites for DivJ localization depends on events associated with the completion of cell separation as well as the gain of motility. Analysis of PleC and PleD mutants also provides insights into the function of the His-Asp proteins in cell cycle regulation. Thus, the ability of the sokA301 allele of ctrA to bypass the nonmotile phenotype of the pleC null mutation provides evidence that the PleC kinase controls cell motility by initiating a signal transduction pathway regulating activity of the global response regulator CtrA. Analysis of the pleD mutant cell cycle demonstrates that disruption of the swarmer-to-stalked-cell developmental sequence does not affect the asymmetric organization of the Caulobacter cell cycle.

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Figures

FIG. 1.

C. crescentus cell cycle and polar localization of the DivJ kinase. (A) Sequence of developmental events in the cell cycle. The vertical arrows at the top of figure indicate the points at which the PleC and PleD functions are required in the sequence of morphological events. (B) Photomicrographs showing immunogold localization of the DivJ protein in thin sections of cells of parent strain CB15 (15-nm-diameter gold particles) and pleD mutant strain PC5375 (5-nm-diameter gold particles). Insert panel shows an enlargement with enhanced contrast of the pleD mutant cell pole in the lower right panel. The bars represent 1 μm.

FIG. 2.

Organization of DivJ protein sequence and location of C-terminal deletions. (A) DivJ sequence and conserved histidine kinase sequence motifs. TM, transmembrane regions. (B) C-terminal deletions in DivJ. (C) Internal deletions in the cytoplasmic domain of DivJ. The efficiency of localization as measured by the percentage of stalked cells observed with stalk pole-localized DivJ-GFP is indicated in panels B and C.

FIG. 3.

Localization of DivJ-GFP fusion proteins in various C. crescentus strains. (A) Strain PC9810, full-length DivJ-GFP in a divJ::Ω background; (B) PC9816, DivJ-GFP in a pleC mutant; (C) PC9861, full-length DivJ-GFP in a pleC sokA double mutant; (D) PC9818, full-length DivJ-GFP in a pleD divJ::Ω background; (E) PC9839, full-length DivJ-GFP in a synchronizable pleD divJ::Ω background, high-density fraction; (F) PC9839, low-density fraction; (G) PC9817, full-length DivJ-GFP in a ftsA(Ts) background; (H) PC9819, full-length DivJ-GFP in a ftsI(Ts) background; (I) PC9820, full-length DivJ-GFP in a ftsW(Ts) background; (J) PC9822, full-length DivJ-GFP in a parC(Ts) background. Cells in panels A through D and G through J were photographed during exponential-phase growth. Cells in panels G through J were shifted to 35°C for ∼7 h prior to photography. Cells in panels E and F were isolated from fractions resulting from centrifugation in a Ludox solution, washed, and then resuspended in M2 medium prior to photographing. Arrows point to stalk structures. The stalk pole DivJ-GFP localization observed in the low-density fraction isolated from a Ludox solution was lower than that seen in exponential-phase cultures (compare with Fig. 2B). This difference is probably due to the fact that the low-density fraction contains a mixture of stalked cells and predivisional cells, some of which had divided to give swarmer cell progeny by the time of collection and observation.

FIG. 4.

CtrA is a target of the PleC kinase in the regulation of cell motility. (A) Electron micrographs of negatively stained cells (52) from the strains indicated. Bar, 1 μm. (B) Suppression of the Mot− phenotype of pleC by the sokA301 mutation as assayed in motility agar.

FIG. 5.

Levels of DivJ detected on Western blots of synchronously dividing cells. (A) Wild-type strain CB15F; (B) pleD301F strain PC5375. Cell division, which occurred at 160 min in each culture, was monitored by phase-contrast microscopy. H and L in panel A and panel B correspond to cells isolated from the high- and low-density fractions, respectively, after centrifugation on Ludox gradients (see Materials and Methods).

FIG. 6.

Cell cycle analysis of the synchronous pleD mutant strain PC9839. DNA content in synchronous cells was determined by flow cytometry at various times after collection from Ludox gradients and resuspension in fresh medium. (A) Cells from the high-density fraction display a G1 arrest in DNA replication (T0 and T45). The G1 period was followed by entry into S phase (T75) and then G2 phase (T120 and 135). By T165, many of the cells had divided as indicated by the large 1n peak in the corresponding panel. (B) Cells from the high-density fraction were allowed to grow to the midpoint of division, as approximated by phase-contrast microscopy, and then fractionated a second time. Cells collected from the low-density cell fraction (T0) were predominantly in S phase, as were those at T45. S phase was followed by entrance into G2 phase, which occurred between T75 (data not shown) and T105. By T105, a large portion of the cells had divided, as indicated by the large 1n peak in the corresponding panel. T, time (minutes) after resuspension. See Materials and Methods for details of cell fractionation and analysis.

FIG. 7.

Model for the regulation of DivJ localization and asymmetric DNA initiation in wild-type C. crescentus and pleD mutant cells. (A) Strain CB15 cell cycle. DivJ regulates a phosphorelay pathway controlling CtrA activity early in the stalked-cell cycle, as shown (52). Localization of DivJ at the stalked-cell pole is dependent on events occurring late in the cell cycle, presumably at the new cell pole (▵). Subsequent developmental events at this pole during the G1-to-S-phase transition make the pole competent as a target of DivJ localization (see Discussion). Motility in the predivisional cell requires PleC (44), which is located in the swarmer-cell compartment (50), and initiates a signal transduction pathway (PleC → DivK → Hpt → CtrA) controlling CtrA activity and the gain of motility, as shown in Fig. 4. (B) Asymmetric control of DNA replication in the pleD mutant cell cycle. The localization of DivJ uniquely to one cell pole of the old, S-phase progeny cell at division (Fig. 6) indicates that DivJ localization is regulated by asymmetrically distributed factors, as described above for CB15.

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Protein sequences and cellular factors required for polar localization of a histidine kinase in Caulobacter crescentus - PubMed (original) (raw)