In the differentiating bacterium Caulobacter crescentus, the cell division initiation protein FtsZ is present in only one of the two cell types. Stalked cells initiate a new round of DNA replication immediately after cell division and contain FtsZ, whereas the progeny swarmer cells are unable to initiate DNA replication and do not contain FtsZ. We show that FtsZ expression is controlled by cell cycle-dependent transcription and proteolysis. Transcription of ftsZ is repressed in swarmer cells and is activated concurrently with the initiation of DNA replication. At the end of the DNA replication period, transcription of ftsZ decreases substantially. We show that the global cell cycle regulator CtrA is involved in the cell cycle control of ftsZ transcription. CtrA binds to a site that overlaps the ftsZ transcription start site. Removal of the CtrA-binding site results in transcription of the ftsZ promoter in swarmer cells. Decreasing the cellular concentration of CtrA increases ftsZ transcription and conversely, increasing the concentration of CtrA decreases ftsZ transcription. Because CtrA is present in swarmer cells, is degraded at the same time as ftsZ transcription begins, and reappears when ftsZ transcription decreases at the end of the cell cycle, we propose that CtrA is a repressor of ftsZ transcription. We show that proteolysis is an important determinant of cell type-specific distribution and cell cycle variation of FtsZ. FtsZ is stable when it is synthesized and assembles into the cytokinetic ring at the beginning of the cell cycle. After the initiation of cell division, the rate of FtsZ degradation increases as both the constriction site and the FtsZ ring decrease in diameter. When ftsZ is expressed constitutively from inducible promoters, the abundance of FtsZ still varies during the cell cycle. The coupling of transcription and proteolysis to cell division ensures that FtsZ is inherited only by the progeny cell that will begin DNA replication immediately after cell division.[Key Words: Caulobacter; FtsZ; cell division; proteolysis; cell cycle; differentiation] Received December 22, 1997; revised version accepted January 23, 1998.The mechanism by which cells coordinate DNA replication, cell growth, and cell division are not well understood (Donachie 1993;Vicente and Errington 1996). Research on cell division in Escherichia coli has pointed to the FtsZ protein as an essential determinant of the timing and the localization of cell division (Erickson 1995; Rothfield and Justice 1997). FtsZ is a tubulin-like GTPase that polymerizes and forms a cytokinetic ring associated with the cytoplasmic membrane at the site of cell division in bacteria (Bi and Lutkenhaus 1991) and archaea (Baumann and Jackson 1996;Margolin et al. 1996;Wang and Lutkenhaus 1996). Localization of FtsZ is likely to be the key event in assembly of the cell division apparatus. FtsZ recruits other cell division proteins to the site of division Ma et al. 1996) and may constrict, providing mechanical force for division. In E. coli, the concentr...

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SummaryThe cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N-terminal region of Ϸ320 amino acids; a variable spacer region; and a conserved C-terminal region of eight amino acids. We show that FtsZ mutants missing different C-terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C-terminal conserved region, the linker, and 40 amino acids from the end of the Nterminal conserved region (FtsZ⌬C281) prevents the localization or the polymerization of FtsZ. Because two-hybrid analysis indicates that FtsZ⌬C281 does not interact with FtsZ, we hypothesize that FtsZ⌬C281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C-terminus of FtsZ (FtsZ⌬C485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C-terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.

Endoglucanase A (CenA) from the bacterium Cellulomonas fimi is composed of a catalytic domain and a nonhydrolytic cellulose-binding domain that can function independently. The individual domains interact synergistically in the disruption and hydrolysis of cellulose fibers. This intramolecular synergism is distinct from the well-known intermolecular synergism between individual cellulases. The catalytic domain corresponds to the hydrolytic C. system and the cellulose-binding domain corresponds to the nonhydrolytic C, More than 40 years ago, Reese and his associates (1) postulated that the microbial conversion of native cellulose to soluble sugars involves two systems that act consecutively. The original model proposed that the C1 system acts first, making the substrate more accessible to the hydrolytic enzymes, or Cx systems (1). This model is illustrated in Fig. 1. The precise action of C1 was not made clear. Reese suggested that it might "be concerned with a splitting of the crosslinkages postulated in native cellulose"-i.e., it was nonhydrolytic. Subsequently, Reese's suggestion was misinterpreted: CQ was an endoglucanase that acted before C1, a cellobiohydrolase (2). Confirmation of Reese's hypothesis was difficult because of the complexity of cellulase systems and the need to have rigorously purified components. A strictly nonhydrolytic C1 component has never been identified. Molecular cloning techniques have provided pure enzymes and enzyme fragments. Consequently, much has been learned recently about the structures and functions of f3-1,4-glycanases. The results presented here suggest that, at least in the bacterium Cellulomonasfimi, C1 activity resides not in a system distinct from Cx but in discrete domains within the 3-1,4-glycanases themselves.The majority of cellulases are modular proteins (3). All of them have a catalytic domain; many of them have discrete, independently functioning cellulose-binding domains (CBDs) that are devoid of hydrolytic activity. Removal ofthe CBD by proteolysis or genetic manipulation reduces the hydrolytic activity of the catalytic domain on insoluble cellulose but not on soluble derivatives of cellulose (4-8). Discrete binding domains are also found in enzymes that hydrolyze other insoluble substrates, such as chitinases and amylases. Removal of the binding domains from some of these enzymes also decreases their activities against insoluble substrates (9, 10). These observations imply a general role for binding domains in the hydrolysis of insoluble polysaccharides.The CBD could have an active role in the hydrolysis of cellulose, or it could simply increase the effective concentration of the enzyme on the substrate. The CBD of endo-,B-1,4-glycanase A (CenA) from the bacterium C.fimi disrupts the surfaces of cotton and ramie cellulose fibers and releases small particles from them (11). The CBDs of CenA and Cex, an exo-P-1,4-glycanase from C. fimi, prevent flocculation of bacterial microcrystalline cellulose (BMCC; ref. 12); in other words, the CBDs disperse the su...

Many genes involved in cell division and DNA replication and their protein products have been identified in bacteria; however, little is known about the cell cycle regulation of the intracellular concentration of these proteins. It has been shown that the level of the tubulin-like GTPase FtsZ is critical for the initiation of cell division in bacteria. We show that the concentration of FtsZ varies dramatically during the cell cycle of Caulobacter crescentus. Caulobacter produce two different cell types at each cell division: (i) a sessile stalked cell that can initiate DNA replication immediately after cell division and (ii) a motile swarmer cell in which DNA replication is blocked. After cell division, only the stalked cell contains FtsZ. FtsZ is synthesized slightly before the swarmer cells differentiate into stalked cells and the intracellular concentration of FtsZ is maximal at the beginning of cell division. Late in the cell cycle, after the completion of chromosome replication, the level of FtsZ decreases dramatically. This decrease is probably mostly due to the degradation of FtsZ in the swarmer compartment of the predivisional cell. Thus, the variation ofFtsZ concentration parallels the pattern of DNA synthesis. Constitutive expression of FtsZ leads to defects in stalk biosynthesis suggesting a role for FtsZ in this developmental process in addition to its role in cell division.The cell division cycle and the DNA replication cycle must be well-coordinated for cell proliferation to proceed normally and for each progeny cell to inherit its complement of genetic material. In bacteria with no obvious differentiation, such as Escherichia coli during exponential growth, the task of coordinating the expression and function of a multitude of cell division and DNA replication genes is a formidable one (reviewed in ref.

SummarySwarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.

Cellulomonas fimi endo-beta-1,4-glucanase A (CenA) contains a discrete N-terminal cellulose-binding domain (CBDCenA). Related CBDs occur in at least 16 bacterial glycanases and are characterized by four highly conserved Trp residues, two of which correspond to W14 and W68 of CBDCenA. The adsorption of CBDCenA to crystalline cellulose was compared with that of two Trp mutants (W14A and W68A). The affinities of the mutant CBDs for cellulose were reduced by approximately 50- and 30-fold, respectively, relative to the wild type. Physical measurements indicated that the mutant CBDs fold normally. Fluorescence data indicated that W14 and W68 were exposed on the CBD, consistent with their participation in binding to cellobiosyl residues on the cellulose surface.

A genetic screen for cell division cycle mutants of Caulobacter crescentus identified a temperature-sensitive DNA replication mutant. Genetic complementation experiments revealed a mutation within the dnaE gene, encoding the ␣-catalytic subunit of DNA polymerase III holoenzyme. Sequencing of the temperature-sensitive dnaE allele indicated a single base pair substitution resulting in a change from valine to glutamic acid within the C-terminal portion of the protein. This mutation lies in a region of the DnaE protein shown in Escherichia coli, to be important in interactions with other essential DNA replication proteins. Using DNA replication assays and fluorescence flow cytometry, we show that the observed block in DNA synthesis in the Caulobacter dnaE mutant strain occurs at the initiation stage of replication and that there is also a partial block of DNA elongation.The initiation of DNA replication is a critical step in the cell cycle and requires the cooperative activities of a large number of proteins. The molecular events leading to the initiation of DNA replication have been clearly defined in Escherichia coli (15,23). The process begins with the DnaA protein binding to conserved regions within the oriC region (DnaA boxes), leading to the formation of an open complex (4,14,17,21). Upon formation of this single-stranded region, the helicase, DnaB, is recruited to the origin locus, followed by the primase, DnaG (2, 4, 16). This enzyme forms short ribonucleotide primers upon which DNA polymerase III (Pol III) holoenzyme units can assemble to drive bidirectional chromosomal replication.The E. coli Pol III holoenzyme is composed of 10 subunits that can be organized into three functional groups (24,32,36,39,41,45). The Pol III core consists of the ␣-catalytic subunit, encoded by the dnaE gene (35, 59); the 3Ј-5Ј exonuclease subunit, ε (52); and a third subunit, , which is not conserved in bacteria and whose function is unknown (6,41,54,56). The core enzyme associates with the ring-shaped ␤ 2 sliding clamp that holds the enzyme to the DNA and increases its polymerizing efficiency (7,18,25,31,44,49,55,57). The subunit associates with the DnaB helicase and the Pol III ␣ subunit, which results in the connection of two core polymerases at the replication forks (30, 62) and hence facilitates simultaneous leading-and lagging-strand DNA synthesis (7,29). It is proposed that during the initiation event, a ␤ dimer assembles onto primed DNA template sites and that its subsequent association with the Pol III core enzyme tethers the polymerases to the DNA (31, 57).The initiation step of DNA replication has not been as well characterized in the gram-negative, alpha-purple bacterium Caulobacter crescentus, where DNA replication is tightly linked to cell division and differentiation. In this organism, asymmetric cell division gives rise to a swarmer cell, with a single flagellum at one pole and a nonmotile stalked cell (5, 19). The stalked cell eventually forms a predivisional cell, synthesizing a flagellum at the pole opposite ...