Chromosome Replication In Bacteria Research Articles

Establishing a biochemical understanding of the initiation of chromosome replication in bacteria

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC. This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria.

Coordination of Polyploid Chromosome Replication with Cell Size and Growth in a Cyanobacterium.

Homologous chromosome number (ploidy) has diversified among bacteria, archaea, and eukaryotes over evolution. In bacteria, model organisms such as Escherichia coli possess a single chromosome encoding the entire genome during slow growth. In contrast, other bacteria, including cyanobacteria, maintain multiple copies of individual chromosomes (polyploid). Although a correlation between ploidy level and cell size has been observed in bacteria and eukaryotes, it is poorly understood how replication of multicopy chromosomes is regulated and how ploidy level is adjusted to cell size. In addition, the advantages conferred by polyploidy are largely unknown. Here we show that only one or a few multicopy chromosomes are replicated at once in the cyanobacterium Synechococcus elongatus and that this restriction depends on regulation of DnaA activity. Inhibiting the DnaA intrinsic ATPase activity in S. elongatus increased the number of replicating chromosomes and chromosome number per cell but did not affect cell growth. In contrast, when cell growth rate was increased or decreased, DnaA level, DnaA activity, and the number of replicating chromosomes also increased or decreased in parallel, resulting in nearly constant chromosome copy number per unit of cell volume at constant temperature. When chromosome copy number was increased by inhibition of DnaA ATPase activity or reduced culture temperature, cells exhibited greater resistance to UV light. Thus, it is suggested that the stepwise replication of the genome enables cyanobacteria to maintain nearly constant gene copy number per unit of cell volume and that multicopy chromosomes function as backup genetic information to compensate for genomic damage.IMPORTANCE Polyploidy has evolved many times across the kingdom of life. The relationship between cell growth and chromosome replication in bacteria has been studied extensively in monoploid model organisms such as Escherichiacoli but not in polyploid organisms. Our study of the polyploid cyanobacterium Synechococcus elongatus demonstrates that replicating chromosome number is restricted and regulated by DnaA to maintain a relatively stable gene copy number/cell volume ratio during cell growth. In addition, our results suggest that polyploidy confers resistance to UV, which damages DNA. This compensatory polyploidy is likely necessitated by photosynthesis, which requires sunlight and generates damaging reactive oxygen species, and may also explain how polyploid bacteria can adapt to extreme environments with high risk of DNA damage.

Lon recognition of the replication initiator DnaA requires a bipartite degron.

DnaA initiates chromosome replication in bacteria. In Caulobacter crescentus, the Lon protease degrades DnaA to coordinate replication with nutrient availability and to halt the cell cycle during acute stress. Here, we characterize the mechanism of DnaA recognition by Lon. We find that the folded state of DnaA appears crucial for its degradation, in contrast to the well-known role of Lon in degrading misfolded proteins. We fail to identify a single degradation motif (degron) sufficient for DnaA degradation, rather we show that both the ATPase domain and a species-specific N-terminal motif are important for productive Lon degradation of full-length DnaA. Mutations in either of these determinants disrupt DnaA degradation in vitro and in vivo. However, analysis of truncation products reveals that appending other extensions to the ATPase domain is sufficient to trigger degradation, suggesting plasticity in Lon recognition. Our final working model is that Lon engages DnaA through at least two elements, one of which anchors DnaA to Lon and the other acting as an initiation site for degradation.

Low Affinity DnaA-ATP Recognition Sites in E. coli oriC Make Non-equivalent and Growth Rate-Dependent Contributions to the Regulated Timing of Chromosome Replication

Although the mechanisms that precisely time initiation of chromosome replication in bacteria remain unclear, most clock models are based on accumulation of the active initiator protein, DnaA-ATP. During each cell division cycle, sufficient DnaA-ATP must become available to interact with a distinct set of low affinity recognition sites in the unique chromosomal replication origin, oriC, and assemble the pre-replicative complex (orisome) that unwinds origin DNA and helps load the replicative helicase. The low affinity oriC-DnaA-ATP interactions are required for the orisome’s mechanical functions, and may also play a role in timing of new rounds of DNA synthesis. To further examine this possibility, we constructed chromosomal oriCs with equal preference for DnaA-ADP or DnaA-ATP at one or more low affinity recognition sites, thereby lowering the DnaA-ATP requirement for orisome assembly, and measured the effect of the mutations on cell cycle timing of DNA synthesis. Under slow growth conditions, mutation of any one of the six low affinity DnaA-ATP sites in chromosomal oriC resulted in initiation earlier in the cell cycle, but the shift was not equivalent for every recognition site. Mutation of τ2 caused a greater change in initiation age, suggesting its occupation by DnaA-ATP is a temporal bottleneck during orisome assembly. In contrast, during rapid growth, all origins with a single mutated site displayed wild-type initiation timing. Based on these observations, we propose that E. coli uses two different, DnaA-ATP-dependent initiation timing mechanisms; a slow growth timer that is directly coupled to individual site occupation, and a fast growth timer comprising DnaA-ATP and additional factors that regulate DnaA access to oriC. Analysis of origins with paired mutated sites suggests that Fis is an important component of the fast growth timing mechanism.

Chromosome 1 licenses chromosome 2 replication in Vibrio cholerae by doubling the crtS gene dosage.

Initiation of chromosome replication in bacteria is precisely timed in the cell cycle. Bacteria that harbor multiple chromosomes face the additional challenge of orchestrating replication initiation of different chromosomes. In Vibrio cholerae, the smaller of its two chromosomes, Chr2, initiates replication after Chr1 such that both chromosomes terminate replication synchronously. The delay is due to the dependence of Chr2 initiation on the replication of a site, crtS, on Chr1. The mechanism by which replication of crtS allows Chr2 replication remains unclear. Here, we show that blocking Chr1 replication indeed blocks Chr2 replication, but providing an extra crtS copy in replication-blocked Chr1 permitted Chr2 replication. This demonstrates that unreplicated crtS copies have significant activity, and suggests that a role of replication is to double the copy number of the site that sufficiently increases its activity for licensing Chr2 replication. We further show that crtS activity promotes the Chr2-specific initiator function and that this activity is required in every cell cycle, as would be expected of a cell-cycle regulator. This study reveals how increase of gene dosage through replication can be utilized in a critical regulatory switch.

Simultaneous regulation of cell size and chromosome replication in bacteria.

Bacteria are able to maintain a narrow distribution of cell sizes by regulating the timing of cell divisions. In rich nutrient conditions, cells divide much faster than their chromosomes replicate. This implies that cells maintain multiple rounds of chromosome replication per cell division by regulating the timing of chromosome replications. Here, we show that both cell size and chromosome replication may be simultaneously regulated by the long-standing initiator accumulation strategy. The strategy proposes that initiators are produced in proportion to the volume increase and is accumulated at each origin of replication, and chromosome replication is initiated when a critical amount per origin has accumulated. We show that this model maps to the incremental model of size control, which was previously shown to reproduce experimentally observed correlations between various events in the cell cycle and explains the exponential dependence of cell size on the growth rate of the cell. Furthermore, we show that this model also leads to the efficient regulation of the timing of initiation and the number of origins consistent with existing experimental results.

Regulating DNA Replication in Bacteria

The replication origin and the initiator protein DnaA are the main targets for regulation of chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an active initiation complex can be formed at the origin resulting in strand opening and recruitment of the replicative helicase. In Escherichia coli, oriC activity is directly regulated by DNA methylation and specific oriC-binding proteins. DnaA activity is regulated by proteins that stimulate ATP-DnaA hydrolysis, yielding inactive ADP-DnaA in a replication-coupled negative-feedback manner, and by DnaA-binding DNA elements that control the subcellular localization of DnaA or stimulate the ADP-to-ATP exchange of the DnaA-bound nucleotide. Regulation of dnaA gene expression is also important for initiation. The principle of replication-coupled negative regulation of DnaA found in E. coli is conserved in eukaryotes as well as in bacteria. Regulations by oriC-binding proteins and dnaA gene expression are also conserved in bacteria.

Invisible Binding: DnaA, the Initiator of Chromosome Replication in Bacteria, Associates with the Escherichia Coli Inner Membrane In Vivo

DnaA initiates chromosome replication in most known bacteria and its activity is controlled to execute only once every cell division cycle. ATP in the active ATP-DnaA is hydrolyzed after initiation and ADP is replaced back to ATP on the verge of next initiation. Thus DnaA acts as a molecular switch, in which the nucleotide recycling couples key processes in the cell. Two putative recycling mechanisms presume binding of DnaA either to the membrane or to specific chromosomal sites, promoting nucleotide dissociation.

ATP negatively regulates the initiator protein of Vibrio cholerae chromosome II replication

Vibrio cholerae, the agent of cholera, has two circular chromosomes. In bacteria that contain a single chromosome, initiation of chromosome DNA replication is mediated by DnaA, a AAA+ ATPase that unwinds the origin of replication. There is little knowledge regarding initiation of chromosome replication in bacteria with more than one chromosome. Here, we purified V. cholerae DnaA and RctB, which have been implicated in the replication of V. cholerae chromosome II, and characterized their activities in vitro. We found that RctB has origin-specific unwinding activity and can melt the origin of chromosome II (oriCIIvc) but not the origin of chromosome I (oriCIvc); conversely, DnaA promoted the unwinding of oriCIvc and not oriCIIvc. The activity of DnaA and several plasmid initiator proteins is stimulated by ATP binding. We found that RctB bound and hydrolyzed ATP even though RctB lacks any apparent ATP-binding motifs. However, we unexpectedly found that ATP inhibited the oriCIIvc binding activity of RctB, suggesting that the ATP-bound form of RctB cannot initiate replication of chromosome II. Supporting this idea, we identified an RctB mutant that does not bind ATP and found that expression of this ATP-blind RctB mutant in V. cholerae leads to significant overinitiation of chromosome II and marked inhibition of V. cholerae growth. These observations suggest that the rules that license the replication of the two V. cholerae chromosomes differ.

Initiation of chromosome replication in bacteria: analysis of an inhibitor control model.

This article contains an analysis of a version of the well-known inhibitor-dilution model for the control of initiation of chromosome replication in bacteria. According to this model, an unstable inhibitor interacts with an initiation primer in a hit-and-destroy fashion to prevent successful initiation; both constituents are presumed to be RNA species that are synthesized constitutively. The model further postulates that the inhibitor interacts cooperatively with the primer, that the inhibitor gene is removed some distance from the origin of replication, and that an eclipse period exists during which the chromosome origin is not able to reinitiate. This unstable-inhibitor version is characterized by four parameters: the inhibitor half-life, the cooperativity index, the location of the inhibitor gene, and the eclipse period; computer simulations are used to study the effect of each of these on the DNA and interdivision time distributions in exponentially growing steady-state cultures. In neither case was any combination of parameter values found that could provide even moderately satisfactory agreement between the simulation results and experimental data. From the examples furnished and the associated discussion, it appears that there are none--that no combination of parameter values exists that can reasonably be expected to produce a significantly better fit than those tested. We conclude that the model in its present form cannot be a valid description of chromosome replication control in bacteria. It is pointed out that this does not necessarily apply to negative initiation control models in general, or even to all inhibitor-dilution systems, merely to the particular ColE1-like mechanism considered here. Nevertheless, recent experimental results, which can only be understood in terms of a very high degree of initiation synchrony within individual cells, offer strong evidence against stochastic models of this kind for the control of chromosome replication.

Control of chromosome replication in bacteria.

The exciting progress that has recently been made in unravelling the replication control mechanisms that determine the copy numbers of several plasmids has been reviewed and updated by several contributors to this volume. In contrast, the control of chromosome replication in bacteria remains largely a closed book.KeywordsChromosome ReplicationdnaA StrainExciting ProgressInitiation ControloriC PlasmidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Initiation of DNA replication in bacteria: Analysis of an autorepressor control model

The precise mechanism by which the initiation of chromosome replication in bacteria is controlled has not yet been established, and several theoretical models have been proposed in an attempt to provide a conceptual framework for the accumulated experimental evidence. The present article contains a detailed quantitative analysis, using computer simulation, of the control model first put forward schematically by Sompayrac & Maaløe in 1973, in which a single operon codes for both the initiator protein and an autorepressor. By comparing the predictions of the model with what is known about the physiology and molecular biology of Escherichia coli under different growth conditions, we are able to delineate the characteristics that such a control system would need to possess in order to be capable of regulating chromosome replication: the control operon has to lie fairly near the origin of replication and contain a moderate to strong promoter and an operator that competes for its repressor with other equally specific binding sites along the chromosome in an interaction that is somewhat weaker than usual; in addition, the messenger molecules encoded for by the repressor gene must have a relatively ineffective ribosome binding site and not too long a halflife.

Growth rate-dependent control of chromosome replication initiation in Escherichia coli.

The initiation mass, defined as cell mass per origin of deoxyribonucleic acid replication (optical density units at 460 nm of culture/origins per milliliter of culture), reflects the intracellular concentration or activity of a hypothetical factor that controls initiation of chromosome replication in bacteria. In Escherichia coli B/r, the initiation mass was found to increase about twofold with increasing growth rate between 0.6 and 1.6 doublings per h; at higher growth rates it remained essentially constant (measured up to 2.4 doublings per h). A low-thymine-requiring (thyA deoB) derivative of E. coli B/r, strain TJK16, was found to have a 60 to 80% greater initiation mass than B/r which was independent of the replication velocity and not related to the thyA and deoB mutations. It is suggested that TJK16 had acquired, during its isolation, a mutation in a gene affecting the initiation of deoxyribonucleic acid replication. The initiation age was not altered by this mutation, but other parameters, including deoxyribonucleic acid concentration and cell size, were changed in comparison with the B/r parent, as expected from theoretical considerations.

Relation between growth and replication in bacteria

The control of chromosome replication in bacteria depends on the cell growth; a round of replication is initiated when the cell mass (or protein) per replication origin has reached a certain critical level. Relative values of this “initiation mass” have previously been estimated from the evaluation of experiments involving age fractionation of radioactively labelled bacterial cultures. Here a theoretical analysis is presented which permits a simple and accurate method to determine this initiation mass from a few parameters easily observable with exponential cultures, without the need for age fractionation.

Replication of the bacterial chromosome.

In this paper I shall confine myself to only one aspect of chromosome replication in bacteria: its control and co-ordination with growth and cell division. The nature of the problem to be considered is made clear by two features of chromosome replication in Escherichia coli , First, under conditions of rapid growth, involving generation times of up to about one hour, DNA synthesis is essentially continuous; there is no detectable resting period corresponding to the G period typically found in higher organisms. Secondly, in glucose minimal media, as the data of Cairns (1963) and others have shown, a single replication point, or growth point, traverses the length of the chromosome during each cycle of replication. It follows that, although the rate of replication in E. coli might be determined by the supply of DNA precursors, the maintenance of the proper sequence of events cannot be controlled in this way since in a system in which DNA synthesis is continuous these precursors must be present at all times. Under the conditions mentioned above in E. coli , for example, the cell must have some means of ensuring that a new cycle of replication is not initiated until the previous one is complete. Consequently the important point of control of replication in bacteria must be over the initiation of replication rather than replication itself.