New Rounds Of DNA Replication Research Articles

Link Between Antibiotic Persistence and Antibiotic Resistance in Bacterial Pathogens.

Both, antibiotic persistence and antibiotic resistance characterize phenotypes of survival in which a bacterial cell becomes insensitive to one (or even) more antibiotic(s). However, the molecular basis for these two antibiotic-tolerant phenotypes is fundamentally different. Whereas antibiotic resistance is genetically determined and hence represents a rather stable phenotype, antibiotic persistence marks a transient physiological state triggered by various stress-inducing conditions that switches back to the original antibiotic sensitive state once the environmental situation improves. The molecular basics of antibiotic resistance are in principle well understood. This is not the case for antibiotic persistence. Under all culture conditions, there is a stochastically formed, subpopulation of persister cells in bacterial populations, the size of which depends on the culture conditions. The proportion of persisters in a bacterial population increases under different stress conditions, including treatment with bactericidal antibiotics (BCAs). Various models have been proposed to explain the formation of persistence in bacteria. We recently hypothesized that all physiological culture conditions leading to persistence converge in the inability of the bacteria to re-initiate a new round of DNA replication caused by an insufficient level of the initiator complex ATP-DnaA and hence by the lack of formation of a functional orisome. Here, we extend this hypothesis by proposing that in this persistence state the bacteria become more susceptible to mutation-based antibiotic resistance provided they are equipped with error-prone DNA repair functions. This is - in our opinion - in particular the case when such bacterial populations are exposed to BCAs.

Energy Starvation Induces a Cell Cycle Arrest in Escherichia coli by Triggering Degradation of the DnaA Initiator Protein.

During steady-state Escherichia coli growth, the amount and activity of the initiator protein, DnaA, controls chromosome replication tightly so that initiation only takes place once per origin in each cell cycle, regardless of growth conditions. However, little is known about the mechanisms involved during transitions from one environmental condition to another or during starvation stress. ATP depletion is one of the consequences of long-term carbon starvation. Here we show that DnaA is degraded in ATP-depleted cells. A chromosome replication initiation block is apparent in such cells as no new rounds of DNA replication are initiated while replication events that have already started proceed to completion.

The Stringent Response Inhibits DNA Replication Initiation in E. coli by Modulating Supercoiling of oriC.

The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA, but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA. Instead, we provide evidence that a global reduction of transcription by ppGpp prevents replication initiation by modulating the supercoiling state of the origin of replication, oriC Active transcription normally introduces negative supercoils into oriC to help promote replication initiation, so the accumulation of ppGpp reduces initiation potential at oriC by reducing transcription. We find that maintaining transcription near oriC, either by expressing a ppGpp-blind RNA polymerase mutant or by inducing transcription from a ppGpp-insensitive promoter, can strongly bypass the inhibition of replication by ppGpp. Additionally, we show that increasing global negative supercoiling by inhibiting topoisomerase I or by deleting the nucleoid-associated protein gene seqA also relieves inhibition. We propose a model, potentially conserved across proteobacteria, in which ppGpp indirectly creates an unfavorable energy landscape for initiation by limiting the introduction of negative supercoils into oriCIMPORTANCE To survive bouts of starvation, cells must inhibit DNA replication. In bacteria, starvation triggers production of a signaling molecule called ppGpp (guanosine tetraphosphate) that helps reprogram cellular physiology, including inhibiting new rounds of DNA replication. While ppGpp has been known to block replication initiation in Escherichia coli for decades, the mechanism responsible was unknown. Early work suggested that ppGpp drives a decrease in levels of the replication initiator protein DnaA. However, we found that this decrease is not necessary to block replication initiation. Instead, we demonstrate that ppGpp leads to a change in DNA topology that prevents initiation. ppGpp is known to inhibit bulk transcription, which normally introduces negative supercoils into the chromosome, and negative supercoils near the origin of replication help drive its unwinding, leading to replication initiation. Thus, the accumulation of ppGpp prevents replication initiation by blocking the introduction of initiation-promoting negative supercoils. This mechanism is likely conserved throughout proteobacteria.

The nucleoid occlusion factor Noc controls DNA replication initiation in Staphylococcus aureus.

Successive division events in the spherically shaped bacterium Staphylococcus aureus are oriented in three alternating perpendicular planes. The mechanisms that underlie this relatively unique pattern of division and coordinate it with chromosome segregation remain largely unknown. Thus far, the only known spatial regulator of division in this organism is the nucleoid occlusion protein Noc that inhibits assembly of the cytokinetic ring over the chromosome. However, Noc is not essential in S. aureus, indicating that additional regulators are likely to exist. To search for these factors, we screened for mutants that are synthetic lethal with Noc inactivation. Our characterization of these mutants led to the discovery that S. aureus Noc also controls the initiation of DNA replication. We show that cells lacking Noc over-initiate and mutations in the initiator gene dnaA suppress this defect. Importantly, these dnaA mutations also partially suppress the division problems associated with Δnoc. Reciprocally, we show that over-expression of DnaA enhances the over-initiation and cell division phenotypes of the Δnoc mutant. Thus, a single factor both blocks cell division over chromosomes and helps to ensure that new rounds of DNA replication are not initiated prematurely. This degree of economy in coordinating key cell biological processes has not been observed in rod-shaped bacteria and may reflect the challenges posed by the reduced cell volume and complicated division pattern of this spherical pathogen.

Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy.

ABSTRACTBacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum.

The DnaA inhibitor SirA acts in the same pathway as Soj (ParA) to facilitate oriC segregation during Bacillus subtilis sporulation.

DNA replication and chromosome segregation must be carefully regulated to ensure reproductive success. During Bacillus subtilis sporulation, chromosome copy number is reduced to two, and cells divide asymmetrically to produce the future spore (forespore) compartment. For successful sporulation, oriC must be captured in the forespore. New rounds of DNA replication are prevented in part by SirA, a protein that utilizes residues in its N-terminus to directly target Domain I of the bacterial initiator, DnaA. Using a quantitative forespore chromosome organization assay, we show that SirA also acts in the same pathway as another DnaA regulator, Soj, to promote oriC capture in the forespore. By analyzing loss-of-function variants of both SirA and DnaA, we observe that SirA's ability to inhibit DNA replication can be genetically separated from its role in oriC capture. In addition, we identify substitutions near the C-terminus of SirA and in DnaA Domain III that enhance interaction between the two proteins. One such variant, SirAP141T , remained functional in regard to inhibiting replication, but was unable to support oriC capture. Collectively, our results support a model in which SirA targets DnaA Domain I to inhibit DNA replication, and DnaA Domain III to facilitate Soj-dependent oriC capture in the forespore.

Failsafe Mechanisms Couple Division and DNA Replication in Bacteria

The past 20 years have seen tremendous advances in our understanding of the mechanisms underlying bacterial cytokinesis, particularly the composition of the division machinery and the factors controlling its assembly [1]. At the same time, we understand very little about the relationship between cell division and other cell-cycle events in bacteria. Here we report that inhibiting division in Bacillus subtilis and Staphylococcus aureus quickly leads to an arrest in the initiation of new rounds of DNA replication, followed by a complete arrest in cell growth. Arrested cells are metabolically active but are unable to initiate new rounds of either DNA replication or division when shifted to permissive conditions. Inhibiting DNA replication results in entry into a similar quiescent state in which cells are unable to resume growth or division when returned to permissive conditions. Our data suggest the presence of two failsafe mechanisms: one linking division to the initiation of DNA replication and another linking the initiation of DNA replication to division. These findings contradict the prevailing view of the bacterial cell cycle as a series of coordinated but uncoupled events. Importantly, the terminal nature of the cell-cycle arrest validates the bacterial cell-cycle machinery as an effective target for antimicrobial development.

Visualizing spatiotemporal dynamics of multicellular cell-cycle progressions with fucci technology.

The visualization of cell-cycle behavior of individual cells within complex tissues presents an irresistible challenge to biologists studying multicellular structures. However, the transition from G1 to S in the cell cycle is difficult to monitor despite the fact that the process involves the critical decision to initiate a new round of DNA replication. Here, we use ubiquitination oscillators that control cell-cycle transitions to develop genetically encoded fluorescent probes for cell-cycle progression. Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes exploit the regulation of cell-cycle-dependent ubiquitination to effectively label individual nuclei in G1 phase red, and those in S/G2/M phases green. Cultured cells and transgenic mice constitutively expressing the probes have been generated, such that every cell nucleus shows either red or green fluorescence. This protocol details two experiments that use biological samples expressing Fucci probes. One experiment involves time-lapse imaging of cells stably expressing a Fucci derivative (Fucci2), which allows for the exploration of the spatiotemporal patterns of cell-cycle dynamics during structural and behavioral changes of cultured cells. The other experiment involves large-field, high-resolution imaging of fixed sections of Fucci transgenic mouse embryos, which provides maps that illustrate cell proliferation versus differentiation in various developing organs.

The deoxynucleotide triphosphohydrolase SAMHD1 is a major regulator of DNA precursor pools in mammalian cells

Sterile alpha motif and HD-domain containing protein 1 (SAMHD1) is a triphosphohydrolase converting deoxynucleoside triphosphates (dNTPs) to deoxynucleosides. The enzyme was recently identified as a component of the human innate immune system that restricts HIV-1 infection by removing dNTPs required for viral DNA synthesis. SAMHD1 has deep evolutionary roots and is ubiquitous in human organs. Here we identify a general function of SAMHD1 in the regulation of dNTP pools in cultured human cells. The protein was nuclear and variably expressed during the cell cycle, maximally during quiescence and minimally during S-phase. Treatment of lung or skin fibroblasts with specific siRNAs resulted in the disappearence of SAMHD1 accompanied by loss of the cell-cycle regulation of dNTP pool sizes and dNTP imbalance. Cells accumulated in G1 phase with oversized pools and stopped growing. Following removal of the siRNA, the pools were normalized and cell growth restarted, but only after SAMHD1 had reappeared. In quiescent cultures SAMHD1 down-regulation leads to a marked expansion of dNTP pools. In all cases the largest effect was on dGTP, the preferred substrate of SAMHD1. Ribonucleotide reductase, responsible for the de novo synthesis of dNTPs, is a cytosolic enzyme maximally induced in S-phase cells. Thus, in mammalian cells the cell cycle regulation of the two main enzymes controlling dNTP pool sizes is adjusted to the requirements of DNA replication. Synthesis by the reductase peaks during S-phase, and catabolism by SAMHD1 is maximal during G1 phase when large dNTP pools would prevent cells from preparing for a new round of DNA replication.

Quantitative analysis of the mechanism of DNA binding by Bacillus DnaA protein

DnaA protein has the sole responsibility of initiating a new round of DNA replication in prokaryotic organisms. It recognizes the origin of DNA replication, and initiates chromosomal DNA replication in the bacterial genome. In Gram-negative Escherichia coli, a large number of DnaA molecules bind to specific DNA sequences (known as DnaA boxes) in the origin of DNA replication, oriC, leading to the activation of the origin. We have cloned, expressed, and purified full-length DnaA protein in large quantity from Gram-positive pathogen Bacillus anthracis (DnaA(BA)). DnaA(BA) was a highly soluble monomeric protein making it amenable to quantitative analysis of its origin recognition mechanisms. DnaA(BA) bound DnaA boxes with widely divergent affinities in sequence and ATP-dependent manner. In the presence of ATP, the K(D) ranged from 3.8 × 10(-8) M for a specific DnaA box sequence to 4.1 × 10(-7) M for a non-specific DNA sequence and decreased significantly in the presence of ADP. Thermodynamic analyses of temperature and salt dependence of DNA binding indicated that hydrophobic (entropic) and ionic bonds contributed to the DnaA(BA)·DNA complex formation. DnaA(BA) had a DNA-dependent ATPase activity. DNA sequences acted as positive effectors and modulated the rate (V(max)) of ATP hydrolysis without any significant change in ATP binding affinity.

CULLIN 4-RING FINGER-LIGASE plays a key role in the control of endoreplication cycles in Arabidopsis trichomes

One of the predominant cell-cycle programs found in mature tissues is endoreplication, also known as endoreduplication, that leads to cellular polyploidy. A key question for the understanding of endoreplication cycles is how oscillating levels of cyclin-dependent kinase activity are generated that control repeated rounds of DNA replication. The APC/C performs a pivotal function in the mitotic cell cycle by promoting anaphase and paving the road for a new round of DNA replication. However, using marker lines and plants in which APC/C components are knocked down, we show here that outgrowing and endoreplicating Arabidopsis leaf hairs display no or very little APC/C activity. Instead we find that RBX1-containing Cullin-RING E3 ubiquitin-Ligases (CRLs) are of central importance for the progression through endoreplication cycles; in particular, we have identified CULLIN4 as a major regulator of endoreplication in Arabidopsis trichomes. We have incorporated our findings into a bio-mathematical simulation presenting a robust two-step model of endoreplication control with one type of cyclin-dependent kinase inhibitor function for entry and a CRL-dependent oscillation of cyclin-dependent kinase activity via degradation of a second type of CDK inhibitor during endoreplication cycles.

The Myxococcus xanthus Developmental Program Can Be Delayed by Inhibition of DNA Replication

Under conditions of nutrient deprivation, Myxococcus xanthus undergoes a developmental process that results in the formation of a fruiting body containing environmentally resistant myxospores. We have shown that myxospores contain two copies of the genome, suggesting that cells must replicate the genome prior to or during development. To further investigate the role of DNA replication in development, a temperature-sensitive dnaB mutant, DnaB(A116V), was isolated from M. xanthus. Unlike what happens in Escherichia coli dnaB mutants, where DNA replication immediately halts upon a shift to a nonpermissive temperature, growth and DNA replication of the M. xanthus mutant ceased after one cell doubling at a nonpermissive temperature, 37 degrees C. We demonstrated that at the nonpermissive temperature the DnaB(A116V) mutant arrested as a population of 1n cells, implying that these cells could complete one round of the cell cycle but did not initiate new rounds of DNA replication. In developmental assays, the DnaB(A116V) mutant was unable to develop into fruiting bodies and produced fewer myxospores than the wild type at the nonpermissive temperature. However, the mutant was able to undergo development when it was shifted to a permissive temperature, suggesting that cells had the capacity to undergo DNA replication during development and to allow the formation of myxospores.

Cyclin Degradation: Don’t Mes(s) with Meiosis

Cyclin degradation is required for exit from mitosis and enables a new round of DNA replication in the subsequent S phase. A recent study in fission yeast shows that, during exit from meiosis I, the Mes1 protein partially inhibits cyclin degradation and thereby allows entry into meiosis II without an intervening S phase.

E.coli cell-cycle regulation by bacteriophage lambda.

We re-examined the old but surprising claim of Kourilsky and Knapp that transient expression of genes located downstream of the p(L) promoter of bacteriophage lambda can induce cell-cycle synchrony in a population of Escherichia coli cells. Although we were unable to reproduce a lasting synchrony, a cessation of division, followed by one or two fairly synchronous cell divisions was observed. This line up of the cell cycle was found to be due to two genetically separable events: a temporary block of cell division and, at the same time, a block to the initiation of new rounds of DNA replication. These blocks then release after about one mass doubling so that chromosome replication and cell division occur during a short time interval in all the cells in the population. The cell division block is a result of the transient expression of the lambda kil gene. The block to initiation of DNA replication requires a region that we term bin (blocks initiation) immediately upstream of the xis gene. The region consists of ea22 and ea8.5 and two small open reading frames (ORFs) that flank them. Deletion-substitution mutagenesis suggests that all four ORFs may be required for the initiation block. The ability of the phage to modify two aspects of the host cell cycle presumably reflects a stratagem that provides the phage with an advantage for lysogeny or lytic growth.

Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae.

Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.

The extrusion-capture model for chromosome partitioning in bacteria.

Successful cellular reproduction requires accurate duplication and partitioning (segregation) of the genome. Failure to correctly partition the sister genomes results in aneuploidy. The consequences of these errors range from loss of normal cellular function (e.g., the loss of normal growth controls in tumor cells) to cell death. In prokaryotes with a single chromosome, partitioning failures are fatal for at least one of the two daughter cells; a so-called anucleate cell forms when one daughter receives no chromosome and the other daughter receives two chromosomes. Several findings in recent years have fundamentally altered our view of chromosome partitioning in prokaryotes (for review, see Gerdes et al. 2000; Gordon and Wright 2000; Hiraga 2000; Moller-Jensen et al. 2000; Donachie 2001; Sawitzke and Austin 2001). The flurry of new observations was ignited by adaptation of cell biological techniques used in eukaryotes (immunofluorescence, GFP, and fluorescent in situ hybridization) for use in prokaryotes (Harry et al. 1995; Pogliano et al. 1995; Webb et al. 1995; Niki and Hiraga 1998). This review focuses on chromosome partitioning in bacteria that have a single circular chromosome, specifically, the gram-positive organism, Bacillus subtilis, and the gramnegative organisms, Escherichia coli and Caulobacter crescentus. In these bacteria, DNA replication initiates once per cell division cycle from a specific chromosomal locus, oriC, and proceeds bidirectionally to terminate in a defined region opposite the origin, terC (Fig. 1). The basic components of the DNA replication machinery are highly conserved in bacteria (Kornberg and Baker 1992); in fact, these basic components are functionally conserved from bacteria to mammals (Baker and Bell 1998). Fundamental differences exist between chromosome partitioning in eukaryotes and prokaryotes. In eukaryotes, chromosomes are duplicated in S phase, and sister chromosomes remain together during G2. Chromosome partitioning occurs during M phase, when sister chromosomes are lined up on a metaphase plate, separated from each other, and finally segregated in opposite directions by the combined action of the microtubular spindle and mitotic motors. In contrast to the temporal separation of chromosome replication and partitioning in eukaryotes, regions of the bacterial chromosome, starting with the origin, appear to be partitioned soon after duplication, whereas the remainder of the chromosome awaits replication (Glaser et al. 1997; Gordon et al. 1997; Lewis and Errington 1997; Lin et al. 1997; Mohl and Gober 1997; Webb et al. 1997, 1998; Niki and Hiraga 1998; Sharpe and Errington 1998; Teleman et al. 1998; Jensen and Shapiro 1999; Niki et al. 2000). Thus, in bacteria, DNA replication, chromosome refolding, and chromosome partitioning are concurrent. Given these differences, it is not surprising that there is no evidence that bacteria contain eukaryotic-like mitotic spindles or mitotic motors. As discussed below, it appears that bacteria with circular chromosomes probably power chromosome partitioning differently from eukaryotes. This may be possible because of the much smaller distances that chromosomes move in bacteria. Many bacteria, including B. subtilis and E. coli, are capable of dividing in one-half to one-third of the time it takes to duplicate the genome. To accomplish this, new rounds of DNA replication are initiated before a previous round is completed, giving the replication cycle a head start on the division cycle. This results in cells with multiple bidirectional DNA replication forks (so-called multifork replication), multiple copies of oriC (2, 4, or 8), but only a single, unduplicated terminus region (Fig. 2). Soon after duplication, sister origins are each partitioned in opposite directions. Thus, during multifork replication, bacteria contain positional information regarding not only the current medial division site, but also the future division planes (the cell quarters and cell eighths). Other bacteria, for exampleC. crescentus, are not known to be capable of multifork replication and do not have more than two copies of the origin per cell. Recent studies have revealed common mechanisms involved in chromosome partitioning, along with differences in some of the details between organisms. This review summarizes the following recent findings that have contributed new insights into the mechanism by which bacteria accomplish accurate chromosome partitioning. First, the bacterial chromosome has a defined orientation within cells, and individual regions move 3Corresponding author. E-MAIL adg@mit.edu; FAX (617) 253-2643. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.913301.

Cell cycle-dependent transcriptional and proteolytic regulation of FtsZ in Caulobacter.

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.

Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning.

The Bacillus subtilis spo0J gene is required for accurate chromosome partitioning during growth and sporulation. We have characterized the subcellular localization of Spo0J protein by immunofluorescence and, in living cells, by use of a spo0J-gfp fusion. We show that the Spo0J protein forms discrete stable foci usually located close to the cell poles. The foci replicate in concert with the initiation of new rounds of DNA replication, after which the daughter foci migrate apart inside the cell. This migration is independent of cell length extension, and presumably serves to direct the daughter chromosomes toward opposite poles of the cell, ready for division. During sporulation, the foci move to the extreme poles of the cell, where they function to position the oriC region of the chromosome ready for polar septation. These observations provide strong evidence for the existence of a dynamic, mitotic-like apparatus responsible for chromosome partitioning in bacteria.

Pds1p is required for faithful execution of anaphase in the yeast, Saccharomyces cerevisiae

To identify mutations that cause defects in mitosis, a collection of mutants in Saccharomyces cerevisiae was screened by a rapid visual assay for abnormal chromosome segregation. From this screen we identified one mutation, pds1-1 that was independently identified in an alternative screen for mutants that exhibit inviability after transient exposure to nocodazole and precocious disassociation of sister chromatids (Guacci, V., A. Yamamoto, A. Strunnikov, J. Kingsbury, E. Hogan, P. Meluh, and D. Koshland. 1993. CSH Symp. Quant. Biol. 58:677-685; Yamamoto, T.J., G. Li, B. Schaar, I. Szilak, and D.W. Cleveland. 1992. Nature (Lond.). 359:536-539). At 23 degrees C pds1-1 mutants exhibit frequent cell death and a 300-fold increase in chromosome loss compared to wild type. At 37 degrees C pds1-1 cells fail to elongate their spindles during anaphase. This spindle defect of pds1 mutants results from a temperature-sensitive step that occurs around the G1/S boundary about the time of spindle assembly. In the absence of spindle elongation pds1 mutants undergo cytokinesis, leading to the missegregation of both chromosomes and spindle pole bodies. After abnormal cell division pds1-1 mutants also initiate new rounds of DNA replication, spindle pole body duplication, and bud formation. Thus, in the pds1-1 mutant at 37 degrees C, cell cycle progression is uncoupled from the completion of anaphase. A pds1 deletion allele has similar phenotypes to the original allele. Taken together these results suggest that Pds1 protein plays an important role in chromosome segregation at 23 degrees C and an essential role for this process at 37 degrees C. The PDS1 gene encodes a novel 42-kD nuclear protein that has both basic and acidic domains. The level of PDS1 mRNA varies with the cell cycle with maximal accumulation around the G1/S boundary. The stability of Pds1 protein also appears to change during the cell cycle as overproduced Pds1p is stable in S and M but degraded in early G1. Therefore, expression of Pds1p is regulated apparently both transcriptionally and postranslationally during the cell cycle. The phenotypes of pds1 mutants and expression pattern of Pds1p are discussed in the context of other spindle-defective mutants and the knowledge that Pds1 protein is an inhibitor of anaphase (Yamamoto, T.J., G. Li, B. Schaar, I. Szilak, and D.W. Cleveland. 1992. Nature (Lond.). 359:536-539).

Use of Flow cytometry to identify a Caulobacter4.5 S RNA Temperature-sensitive Mutant Defective in the Cell Cycle

Flow cytometry was used to screen a collection of temperature-sensitive mutants for those blocked at discrete points in the cell cycle with respect to the replicative status of the chromosome. At the non-permissive temperature, one such mutant, LS439, could not initiate new rounds of DNA replication and arrested primarily as cells with two completed chromosomes. Extended incubation at the restrictive temeprature resulted in filament formation. following the shift to the restrictive temperature protein synthesis continued, but at a reduced rate. A 0.2 kb fragment of DNA located immediately upstream of the Caulobacterhomolog of the Escherichia coil dnaX gene was able to completely rescue the temperature-sensitive phenotype of LS439. The 0.2 kb fragment contained a homolog of the bacterial gene encoding 4.5 S RNA. The original point mutation is predicted to disrupt the stem structure in the 4.5 S RNA thus providing a rationale for the genetic basis of the LS439 phenotype.