After nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis, two Bacillus subtilis mutants (div IV-Al and div IV-B1) were isolated that are defective in the location of division site along cell length. Both mutations were transferred into strain CU403 by transformation, and their properties were studied in the CU403 genetic background. Location of divisions in close proximity to cell pole regions in both mutants results in minicell production. Purified minicells contain a ratio of ribonucleic acid to protein comparable to that found in the parent cells. Autoradiographs of 3H-thymine incorporation into deoxyribonucleic acid (DNA), thymine-2-14C incorporation into DNA, electron micrographs, and chemical analyses for DNA all fail to demonstrate DNA in the minicells. Minicells produced by both mutants are highly motile, an indication of functional energy metabolism. Electron micrographs reveal that minicells are produced by a structurally normal division mechanism and that minicells contain a normal cell surface. The div IV-Al mutation has been mapped by PBS1 transduction linked to ura. The div IV-Bl mutation is closely linked to pheA by both PBS1 transduction and by co-transformation. on October 10, 2020 by guest http://jb.asm.org/ Downloaded from MINICELLS OF B. SUBTILIS MATERIALS AND METHODS Strains of B. subtilis and bacteriophage. All bacterial strains used (Table 1) were derivatives of B. subtilis 168. The mutations involved in cell division, which are described in this paper, were transferred to strain CU403 by transduction and transformation for analysis in an isogenic background. Bacteriophage PBS1 was used for transduction. Media. Liquid cultures were grown with aeration at 30 C in Trypticase soy broth (TSB; BBL, Cockeysville, Md.) supplemented with 20 Mg of thymine/ml. The concentration of thymine was lowered to 2 Mg per/ml when thymine incorporation was being studied. Increase in cell mass was followed with a Spectronic 20 colorimeter (Bausch and Lomb, Inc. Rochester, N.Y.) at A6s0o nm.

Multicellular rosettes are transient epithelial structures that serve as intermediates during diverse organ formation. We have identified a unique contributor to rosette formation in zebrafish Kupffer's vesicle (KV) that requires cell division, specifically the final stage of mitosis termed abscission. KV utilizes a rosette as a prerequisite before forming a lumen surrounded by ciliated epithelial cells. Our studies identify that KV-destined cells remain interconnected by cytokinetic bridges that position at the rosette's center. These bridges act as a landmark for directed Rab11 vesicle motility to deliver an essential cargo for lumen formation, CFTR (cystic fibrosis transmembrane conductance regulator). Here we report that premature bridge cleavage through laser ablation or inhibiting abscission using optogenetic clustering of Rab11 result in disrupted lumen formation. We present a model in which KV mitotic cells strategically place their cytokinetic bridges at the rosette center, where Rab11associated vesicles transport CFTR to aid in lumen establishment.

Spores of the Bacillus subtilis minicell-producing mutant div IV-B1 were germinated and grown to microcolonies in chambers which facilitate continuous observation of the developing clones with a phase-contrast microscope. Time lapse photographs were taken of 46 clones, covering the period from the beginning of outgrowth until at least two rounds of cell division had been completed. Cell lineages were constructed from contour length measurements of the photographs. These data include cell lengths, division site locations, and cell numbers in clones of various ages. From these data we have determined that the probability of a minicell being produced at any division by the div IV-B1 mutant is 0.31. The location of the abnormal division site which generates the first minicell produced in the outgrowing clone appears to be random with respect to the existing cell poles. In contrast, the location of the second abnormal division site, and hence the second minicell, is not random but rather occurs preferentially in proximity to the first minicell. This clustering of abnormal events suggests that division site location is related to pole age (generations), although other influences on minicell clustering cannot be ruled out at present.

Purified minicells produced by Bacillus subtilis div IV-B1 mutants were used to immunize rabbits. Immune serum was obtained that agglutinated minicells and was able to form precipitin lines when reacted with minicell antigens in double-diffusion or immunoelectrophoretic procedures. Antiminicell serum agglutinated rod-shaped B. subtilis cells to long filaments produced by growth of a cell division-defective mutant at restrictive temperature. These findings indicate that minicells are immunogens capable of eliciting the production of antibodies that cross-react with the lateral, cylindrical regions of B. subtilis rods. It appears, therefore, that poles share a common antigen(s) with cylindrical regions of the cell.Rod-shaped bacteria consist of two geometrically distinct regions: cylindrical sides and hemispherical poles. During the growth cycle, cells elongate by the extension of the cylindrical region and then compartmentalize into two daughter cells by the construction of a septum. Division is completed by the differentiation of the septum into two poles. Each daughter cell consists of two poles, one of which is a generation older than the other, connected by a cylindrical region. Septum formation, and consequently pole production, is known to be under specific genetic control distinct from that which regulates cell growth by cylindrical length extension (7). It is of interest to determine whether the composition of the cell surface in the cylindrical portions of the cell is identical to that found in the poles. It has been demonstrated that wall regions isolated from the poles ofBacillus subtilis are more resistant to autolytic degradation than are wall regions from the cylindrical portion of the cell (3, 5). The reasons for this resistance are not clear since it has been reported that cell surface turnover occurs in cell pole regions, as well as in cylindrical portions of the cell (4).We have reported the isolation of a minicellproducing mutant of B. subtilis and have described the properties of purified minicells (8,9). Minicells consist entirely of cell pole surface and, thus, offer a system for investigating differences between poles and sides of rod-shaped bacteria (9). In this paper we report that mini-1 Present address: Department of Microbiology, University of Alabama in Birmingham, Birmingham, Ala. 35294. cells share a common antigen(s) found on cell cylindrical surfaces. MATERIALS AND METHODS Bacteria. Strain of B. subtilis and Escherichia coli used in these experiments are listed in Table 1. Media. Minimal medium consisted of: 0.0151 M (NH4)2S04, 0.0613 M K2HPO4-3H20, 0.0441 M KH2PO4, 0.0034 M sodium citrate 2H2O, 0.00081 M MgSO4-7H2O, and 0.0277 M glucose. Sterile supplements (20 ,ug/ml) were added as follows: thymine, isoleucine, and methionine for growth of CU403 cultures; thymine and methionine for growth of CU403 div IV-Bl and CU403 tms-12 cultures; and leucine, methionine, and adenine for growth of BC-101 cultures. Trypticase soy broth (BBL, Cockeysville, Md.; 30 g/liter) was supplemented with ...