Organisms respond to perturbations in DNA replication. We characterized the global transcriptional response to inhibition of DNA replication in Bacillus subtilis. We focused on changes that were independent of the known recA-dependent global DNA damage (SOS) response. We found that overlapping sets of genes are affected by perturbations in replication elongation or initiation and that this transcriptional response serves to inhibit cell division and maintain cell viability. Approximately 20 of the operons (>50 genes) affected have potential DnaA-binding sites and are probably regulated directly by DnaA, the highly conserved replication initiation protein and transcription factor. Many of these genes have homologues and recognizable DnaA-binding sites in other bacteria, indicating that a DnaA-mediated response, elicited by changes in DNA replication status, may be conserved.DNA replication ͉ transcription ͉ DNA microarrays ͉ Bacillus subtilis

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexAregulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5-CGAACATATGTTCG-3. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.Exposure of prokaryotes to DNA-damaging agents results in the induction of a diverse set of physiological responses collectively called the SOS response (8, 55). As first characterized in Escherichia coli, the SOS response includes an enhanced capacity for recombinational repair, enhanced capacity for excision repair, enhanced mutagenesis (due to error-prone repair), and inhibition of cell division (i.e., filamentation). Induction of the SOS response is due to the coordinate derepression of a number of SOS or din (for damage-inducible) genes. The SOS response to DNA damage in Bacillus subtilis is similar to that of E. coli (26,56,58), but unlike E. coli, the B. subtilis SOS system is also induced in competent cells in the absence of any DNA-damaging treatment (25, 57, 58). As in E. coli, SOS gene expression in B. subtilis is controlled by two proteins (which are themselves products of SOS genes): the LexA protein (also called DinR) (40,54), which represses the transcription of din genes by binding to the SOS operator (31), and the RecA protein (30), which is activated by single-stranded DNA (29,42) to stimulate the proteolytic autodigestion of LexA (24,31). Thus, an SOS gene is defined by two criteria-RecA-dependent induction by DNA damage and a binding site for LexA overlapping its promoter.By contrast with E. coli, where more than 30 SOS genes have been identified (7,8), only 5 B. subtilis SOS genes have been shown to meet both SOS gene criteria thus far: recA, lexA, uvrB (formerly dinA), dinB, and dinC (also called tagC) (4,9,15,25).

Cell growth comprises changes in both mass and volume-two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell's three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications.biosensor | cell growth | cell size | microfluidics | Saccharomyces

Cell growth is an essential requirement for cell cycle progression. While it is often held that growth is independent of cell cycle position, this relationship has not been closely scrutinized. Here we show that in budding yeast, the ability of cells to grow changes during the cell cycle. We find that cell growth is faster in cells arrested in anaphase and G1 than in other cell cycle stages. We demonstrate that the establishment of a polarized actin cytoskeleton-either as a consequence of normal cell division or through activation of the mating pheromone response-potently attenuates protein synthesis and growth. We furthermore show by population and single-cell analysis that growth varies during an unperturbed cell cycle, slowing at the time of polarized growth. Our study uncovers a fundamental relationship whereby cell cycle position regulates growth.[Keywords: Actin polarization; cdc28; cell cycle; cell growth; cell size; Saccharomyces] Supplemental material is available at http://www.genesdev.org.

No abstract