Control of cell division by sex factor F in Escherichia coli. II. Identification of genes for inhibitor protein and trigger protein on the 42.84-43.6 F segment

The genetic structure of the 42.84-43.6 F (BamHI-PstI) segment of the F plasmid, which contains all the F DNA sequences necessary for coupling cell division of F+ bacteria with plasmid DNA replication, was analyzed by isolating a series of amber mutants. Two cistrons were found in this region and they were designated letA and letD (an abbreviation for lethal mutation). The letA and letD cistrons were mapped on the 42.84-43.35 F (BamHI- XmaI ) segment and the 43.07-43.6 F (HincII-PstI) segment, respectively, and are presumed to correspond to the first (43.04-43.26 F) and second (43.26-43.57 F) open reading frames, respectively, which were found in this region by nucleotide sequencing. The letD gene product acts to inhibit cell division of the host bacteria and to induce prophages in lysogenic bacteria, whereas the letA gene product acts to suppress the activity of the letD gene product. Taking into consideration the fact that the 42.84-43.6 F segment carries all the F plasmid genes necessary for coupling cell division with plasmid DNA replication, and that the expression of the genes is likely to be controlled by plasmid DNA replication, we constructed the following hypothesis. Before completion of plasmid DNA replication, LetD protein acts to prevent cell division of the host bacteria. When plasmid DNA replication is completed, synthesis of LetA protein (and also LetD protein) takes place and the LetA protein synthesized acts to suppress the activity of LetD protein and make the cell ready for cell division. Actual cell division will take place when replication of both chromosomal and plasmid DNA is completed and the termination protein of the chromosome and the LetA protein of F plasmid are both synthesized. When cell division takes place LetA protein is consumed, and as a result LetD protein becomes active and prevents cell division until the next round of DNA replication is completed.     

Germ-soma differentiation in volvox.

Volvox carteri is a spherical green alga with a predominantly asexual mode of reproduction and a complete germ-soma division of labor. Its somatic cells are specialized for motility, incapable of dividing, and programmed to die when only a few days old, whereas its gonidia (asexual reproductive cells) are nonmotile, specialized for growth and reproduction, and potentially immortal. When a gonidium is less than 2 days old it divides to produce a juvenile spheroid containing all of the somatic cells and gonidia that will be present in an adult of the next generation. The first visible step in germ-soma differentiation is a set of asymmetric cleavage divisions in the embryo that set apart small somatic initials from their large gonidial-initial sister cells. Three types of genes have been found to play key roles in germ-soma specification. First a set of gls genes act in the embryos to shift cell-division planes, resulting in the asymmetric divisions that set apart the large-small sister-cell pairs. Then a set of lag genes act in the large cells to prevent somatic differentiation, while the regA gene acts in the small cells to prevent reproductive development. An inducible transposon was used to tag and recover some of these and other developmentally important genes. The glsA gene encodes a chaperone-like protein that, like another chaperone that is one of its putative binding partners, is associated with the cell division apparatus, although how this leads to asymmetric division remains to be elucidated. The regA gene encodes a somatic-cell-specific nuclear protein that appears to function by repressing genes required for chloroplast biogenesis, thereby preventing somatic cells from growing enough to reproduce. Somatic-cell-specific expression of regA is controlled by three intronic enhancers.     

Regulation by polyamines of ornithine decarboxylase activity and cell division in the unicellular green alga Chlamydomonas reinhardtii

Polyamines are required for cell growth and cell division in eukaryotic and prokaryotic organisms. In the unicellular green alga Chlamydomonas reinhardtii, biosynthesis of the commonly occurring polyamines (putrescine, spermidine, and spermine) is dependent on the activity of ornithine decarboxylase (ODC, EC 4.1.1.17) catalyzing the formation of putrescine, which is the precursor of the other two polyamines. In synchronized C. reinhardtii cultures, transition to the cell division phase was preceded by a 4-fold increase in ODC activity and a 10- and a 20-fold increase, respectively, in the putrescine and spermidine levels. Spermine, however, could not be detected in C. reinhardtii cells. Exogenous polyamines caused a decrease in ODC activity. Addition of spermine, but not of spermidine or putrescine, abolished the transition to the cell division phase when applied 7 to 8 h after beginning of the light (growth) phase. Most of the cells had already doubled their cell mass after this growth period. The spermine-induced cell cycle arrest could be overcome by subsequent addition of spermidine or putrescine. The conclusion that spermine affects cell division via a decreased spermidine level was corroborated by the findings that spermine caused a decrease in the putrescine and spermidine levels and that cell divisions also could be prevented by inhibitors of S-adenosyl-methionine decarboxylase and spermidine synthase, respectively, added 8 h after beginning of the growth period. Because protein synthesis was not decreased by addition of spermine under our experimental conditions, we conclude that spermidine affects the transition to the cell division phase directly rather than via protein biosynthesis.     

Cell division genes and proteins in bacterial cells

In this review, genes and proteins involved in cytokinesis and cell proliferation of cell-wall bacteria and mycoplasms are considered. We hope that this comparative analysis of genes and proteins of phylogenetically distant bacteria, including the minimal cells of mycoplasmas, can be useful for understanding the basic principles of prokaryotic cell division. The ftsZ gene was found among representatives of all bacterial groups. The recent data indicate that FtsZ protein plays the central role in the process of bacterial cell division. FtsZ protein was revealed in all Eubacterial groups (including mycoplasmas), in Archaebacteria and chloroplasts, All FtsZ proteins are able to form protofilaments as a result of polymerization in vitro and demonstrate GTF-ase activity. On the base of these properties and some similarities in amino acid sequences with tubulins, it has been suggested that FtsZ protein is an evolutionary ancestor of Eukaryotic tubulins. On the earliest stage of bacterial cytokinesis FtsZ protein assembles into a submembranous Z-ring which encircles bacterial cell in the predivisional site. Some other bacterial proteins take part in stabilization and contraction of the Z-ring, which is considered as a cytoskeleton-like bacterial structure.     

Cell division is antagonized by the activity of peptidoglycan endopeptidases that promote cell elongation

A peptidoglycan (PG) cell wall composed of glycans crosslinked by short peptides surrounds most bacteria and protects them against osmotic rupture. In Escherichia coli, cell elongation requires crosslink cleavage by PG endopeptidases to make space for the incorporation of new PG material throughout the cell cylinder. Cell division, on the contrary, requires the localized synthesis and remodeling of new PG at midcell by the divisome. Little is known about the factors that modulate transitions between these two modes of PG biogenesis. In a transposon-insertion sequencing screen to identify mutants synthetically lethal with a defect in the division protein FtsP, we discovered that mutants impaired for cell division are sensitive to elevated activity of the endopeptidases. Increased endopeptidase activity in these cells was shown to interfere with the assembly of mature divisomes, and conversely, inactivation of MepS was found to suppress the lethality of mutations in essential division genes. Overall, our results are consistent with a model in which the cell elongation and division systems are in competition with one another and that control of PG endopeptidase activity represents an important point of regulation influencing the transition from elongation to the division mode of PG biogenesis.     

The relation between protein synthesis and lipide accumulation in L strain cells and Ehrlich ascites cells

It has long been known that fat accumulates in old injured cells both in tissue culture and in many mammalian disease states. The use of L cells grown in suspension tissue culture permitted the opportunity to study conditions in which lipide accumulation could be retarded or accelerated. These cultures exhibit a three-phase growth curve which is similar to that previously found with bacteria and consists of a lag period, logarithmic growth period, and stationary period. Daily aliquots were removed from cultures going through these phases and protein and cholesterol content correlated with cell division. It was found that L cells gradually accumulated lipide in the cell concurrent with retardation of cell division and protein synthesis. Conversely old lipide-laden cells, placed in fresh media and encouraged to active division with net protein synthesis progressed from a high to a low lipide/cell ratio over a period of 2 to 4 days. An amino acid analogue p-fluorophenylalanine and a mitotic inhibitor, colchicine, also markedly increased the lipide/cell ratio. Similar results were found in in vitro experiments with Ehrlich ascites cells.     

Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.

Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division.     

Adjacent positioning of cellular structures enabled by a Cdc42 GTPase-activating protein-mediated zone of inhibition

Cells of the budding yeast Saccharomyces cerevisiae are born carrying localized transmembrane landmark proteins that guide the subsequent establishment of a polarity axis and hence polarized growth to form a bud in the next cell cycle. In haploid cells, the relevant landmark proteins are concentrated at the site of the preceding cell division, to which they recruit Cdc24, the guanine nucleotide exchange factor for the conserved polarity regulator Cdc42. However, instead of polarizing at the division site, the new polarity axis is directed next to but not overlapping that site. Here, we show that the Cdc42 guanosine triphosphatase–activating protein (GAP) Rga1 establishes an exclusion zone at the division site that blocks subsequent polarization within that site. In the absence of localized Rga1 GAP activity, new buds do in fact form within the old division site. Thus, Cdc42 activators and GAPs establish concentric zones of action such that polarization is directed to occur adjacent to but not within the previous cell division site.     

The Relation between Protein Synthesis and Lipide Accumulation in L Strain Cells and Ehrlich Ascites Cells

It has long been known that fat accumulates in old injured cells both in tissue culture and in many mammalian disease states. The use of L cells grown in suspension tissue culture permitted the opportunity to study conditions in which lipide accumulation could be retarded or accelerated. These cultures exhibit a three-phase growth curve which is similar to that previously found with bacteria and consists of a lag period, logarithmic growth period, and stationary period. Daily aliquots were removed from cultures going through these phases and protein and cholesterol content correlated with cell division. It was found that L cells gradually accumulated lipide in the cell concurrent with retardation of cell division and protein synthesis. Conversely old lipide-laden cells, placed in fresh media and encouraged to active division with net protein synthesis progressed from a high to a low lipide/cell ratio over a period of 2 to 4 days. An amino acid analogue p-fluorophenylalanine and a mitotic inhibitor, colchicine, also markedly increased the lipide/cell ratio. Similar results were found in in vitro experiments with Ehrlich ascites cells.