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

Phosphorelay is a central mechanism of signal transduction in prokaryotic cells. The phosphotransfer proteins originally described consist of two components, a sensor kinase, or histidine protein kinase, and a response regulator (34). Many response regulators are transcription factors whose activities are modulated in response to phosphorylation. These His-Asp proteins are also organized in multicomponent phosphorelays containing a histidine kinase (H1), a response regulator (D1), a histidine phosphotransferase (H2), and a second response regulator (D2) in which phosphate transfer to the conserved His and Asp residues occurs in the sequence H1 → D1 → H2 → D2 (3). Histidine kinase-mediated signal transduction is important in the control of cellular responses to changes in growth conditions, stress, osmolarity, cell density, and other environmental conditions (15). In the dimorphic bacterium Caulobacter crescentus, His-Asp proteins also respond to internal cell cycle cues and are essential for the regulation of cell cycle progression and polar morphogenesis. Because asymmetric cell division generates two different cell types in C. crescentus, the mother stalked cell and the new swarmer cell, these signal transduction pathways must regulate developmental events spatially as well as temporally (28, 40). The stalked and swarmer cells inherit distinctive developmental programs. During the stalked-cell cycle, the structures required to build a new swarmer cell, including the flagellum and bacteriophage receptor sites, are assembled at the stalk-distal pole. Immediately before cell division, the cell gains motility with the start of flagellum rotation. After cell separation is completed, the old stalked cell continues to divide asymmetrically while the new swarmer cell undergoes pilus assembly at the base of the flagellum, loss of motility, flagellum ejection, and stalk formation during its differentiation into a stalked cell (see below and Fig. ​Fig.1A1A). 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 ... C. crescentus contains 61 histidine kinases and 43 response regulators (29). Four of these kinases, DivJ, PleC, CckA, and DivL, and the response regulators DivK, CtrA, and PleD have been implicated in aspects of cell cycle regulation. The sensor kinases DivJ, CckA, and DivL have been shown to control the cell division cycle by regulating activity of the global transcription regulator CtrA (reviewed in references 17 and 30). At least in the case of DivJ, the activity of CtrA is controlled by a multicomponent phosphorelay pathway mediated by the essential response regulator DivK (52). The DivJ, PleC, and CckA kinases and the essential response regulator DivK are differentially localized to the cell poles of C. crescentus cells during cell cycle progression (18, 19, 50). PleC is required for the gain of cell motility (44), and its localization to the flagellated pole of the predivisional cell coincides with the initiation of flagellar rotation and the gain of motility late in the cell cycle (Fig. ​(Fig.1A)1A) (50). PleC is then segregated with the swarmer cell at division and remains at the flagellated pole of the new swarmer cell until the end of the G1 phase. At the G1-to-S-phase transition, transcription (31) and translation (50) of the divJ gene are initiated and the DivJ kinase is localized to the new stalked-cell pole concomitant with the loss of PleC from this cell pole (Fig. ​(Fig.1A)1A) (50). DivJ function is required for an early cell division step in the stalked-cell cycle (14, 31). In the studies reported here, we have analyzed both the sequences within DivJ and the cellular factors that are required for the polar localization of the DivJ kinase in C. crescentus. The effects of overlapping C-terminal deletions of DivJ identify an N-terminal sequence which does not contain the kinase catalytic domain and which is sufficient for polar localization of the fusion protein. We also present evidence that a short cytoplasmic linker between the transmembrane region and catalytic domain is required for efficient polar localization. An examination of mutants defective in polar morphogenesis and cell division progression indicate that developmental events associated with gain of motility and cell separation are required to establish the correct subcellular address for polar localization.