Localizing the Subunit Pool for the Temporally Regulated Polar Pili of Caulobacter crescentus

ter crescentus are assembled at a specific time in the life cycle at one pole of the cell and are composed of the monomer protein, pilin. A previous study demonstrated that the onset of pilin synthesis occurs well before pili appear on the surface, suggesting that pilin accumulates within the cell. In the present study, an electron microscope immunocytochemistry assay was used to determine the subcellular location of this unassembled pilin and its fate during pilus assembly and cell division. Populations of synchronously growing cells were embedded in epoxy resin at selected times during the cell cycle. Ultrathin sections were treated with pilin-specific antibody, followed by protein A coupled to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear material were poorly labeled. Quantitation demonstrated that label density increased during the period of pilin synthesis and declined during the period of pilus assembly and maintenance. The pilin pool was not unequally segregated at division; e.g., to the daughter cell that is elaborating pili. Mutants which have simultaneously lost the ability to produce flagella, pili, and other polar organdies, possibly due to alterations in the specialized region of polar organelle assembly, were also examined by the immunocytochemistry technique. There was no significant difference in the pilin pool size relative to the wild type, indicating that pilin synthesis continues in the absence of a functioning assembly site. This pattern of synthesis and assembly for the pilus is significantly different from that of the polar flagellum which is produced at the same time and location on the cell surface. These findings are discussed in relation to the hypothesized organization center at the cell pole which may have a major role in directing the assembly of all the polar structures. URING its life cycle, the bacterium Caulobacter crescentus produces several surface structures at a specific location on the cell surface (18, 23). Among the structures produced are a single flagellum, several pili, an adhesive substance (termed a holdfast), and a stalk, fashioned from the membranes that constitute the cell wall of this gram-negative bacterium. In addition, these structures are expressed at a specific time during the life cycle; the flagellum, pili, and holdfast appear simultaneously at the cell pole while the stalk is elaborated at the same position later in the cell cycle. Thus this bacterium provides a distinctive opportunity to study molecular details of gene regulation, morphogenesis, and the mechanisms of directing the assembly of structures to specific sites in a cell. The differentially expressed structures are quite different from one another. The pilus filament is a relatively simple structure, composed of a single protein, pilin (29). Whether there is a basal structure containing additional components is unknown. The flagellum is more complex, containing, in addition to two filament proteins (12), a protein that polymerJohn Smit's current address is Department of Microbiology, University of British Columbia, #300-6174 University Boulevard, Vancouver, B.C. V6T lW5 izes to form a hook (13, 14) and numerous proteins .that comprise the motor apparatus at the base of the flagellum (10). The adhesive holdfast is a polysaccharide (Smit, J., unpublished studies), which presumably requires the presence of several enzymes and activated substrates at the localized region of expression. One approach in studying these structures is to address the complexity of temporal and spatial control of their coordinated assembly; i.e., are the mechanisms of regulated synthesis and assembly similar for each of the polar organelles or is the cell capable of focusing diverse processes simultaneously at one site in the cell? Analysis of pilus production was begun in a previous study in part to compare findings with what is known about production of the flagellum. Differences in the production of the two organelles were noted (25). The most significant difference was that synthesis of pilin begins much earlier in the cell cycle than the time of appearance of the assembled filament on the surface (Fig. 1). It may be that a cycle of pilin synthesis is complete before pilus assembly begins. Thus, a pool of pilin likely develops in the cell. This contrasts with the time of flagellin and flagellar hook protein synthesis, which occurs just before these proteins are assembled (6, 16). 9 The Rockefeller University Press, 0021-9525/87/10/1821/8 $2.00 The Journal of Cell Biology, Volume 105, October 1987 1821-1828 1821 on M ay 8, 2017 D ow nladed fom Published October 1, 1987