Spore formation inthrough the newly formed spore septum. We propose that translocation of the prespore chromosome occurs by a mechanism that is functionally related to the conjugative transfer of plasmid DNA.

Fluorescence microscopic methods have been used to characterize the cell cycle of Bacillus subtilis at four different growth rates. The data obtained have been used to derive models for cell cycle progression. Like that of Escherichia coli, the period required by B. subtilis for chromosome replication at 37°C was found to be fairly constant (although a little longer, at about 55 min), as was the cell mass at initiation of DNA replication. The cell cycle of B. subtilis differed from that ofE. coli in that changes in growth rate affected the average cell length but not the width and also in the relative variability of period between termination of DNA replication and septation. Overall movement of the nucleoid was found to occur smoothly, as in E. coli, but other aspects of nucleoid behavior were consistent with an underlying active partitioning machinery. The models for cell cycle progression in B. subtilis should facilitate the interpretation of data obtained from the recently introduced cytological methods for imaging the assembly and movement of proteins involved in cell cycle dynamics.

The Bacillus subtilis divIVA gene encodes a coiledcoil protein that shows weak similarity to eukaryotic tropomyosins. The protein is targeted to the sites of cell division and mature cell poles where, in B.subtilis, it controls the site speci®city of cell division. Although clear homologues of DivIVA are present only in Gram-positive bacteria, and its role in division site selection is not conserved in the Gram-negative bacterium, Escherichia coli, a DivIVA±green¯uores-cent protein (GFP) fusion was targeted accurately to division sites and retained at the cell pole in this organism. Remarkably, the same fusion protein was also targeted to nascent division sites and growth zones in the ®ssion yeast Schizosaccharomyces pombe, mimicking the localization of the endogenous tropomyosin-like cell division protein Cdc8p, and F-actin. The results show that a targeting signal for division sites is conserved across the eukaryote±prokaryote divide.

The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.Cell division has been of considerable interest as an antibacterial target because it comprises a group of well conserved proteins that are all essential for the viability of a wide range of bacteria, and their activities are completely different from those of the proteins involved in the division of mammalian cells. A number of compounds that act on components of the cell division machinery have been described (1-7). So far, most of the effort has been directed at the FtsZ protein because it has several biochemical activities that can be assayed in vitro. Here we describe a novel approach to the discovery of inhibitors of bacterial cell division using a cell-based reporter assay. We have used the assay to identify a novel class of antibacterial compounds with potential broadspectrum activity. We show that the compounds act on the highly conserved, essential cell division protein FtsZ in vitro and in vivo. These compounds represent a potential class of new antibiotics that act by a different mechanism than any of the antibiotics currently in clinical use. Molecular Cloning-B. subtilis was transformed by the method described by Anagnostopoulos and Spizizen (8) as modified by Jenkinson (9) or the method described by Kunst and Rapoport (10), except that 20 min after the addition of DNA the transformed cultures were supplemented with 0.66% casamino acids solution. Transformants were selected on Oxoid nutrient agar containing chloramphenicol (5 g/ml). Sporulation was induced by growth in a hydrolyzed casein medium followed by resuspension in a starvation medium. Starvation medium was as described by Karamata and Gross (11). DNA manipulations and E. coli transformations were carried out as described by Sambrook et al. (12). All cloning was done in E. coli DH5␣ (Invitrogen). EXPERIMENTAL PROCEDURESCell Division Dual Reporter Assay-B. subtilis PL16 was grown in hydrolyzed casein medium to exponential phase and centrifuged, and cell pellets were frozen at Ϫ80°C. Frozen cell aliquots of strain PL16 were resuspended in ...