Bacterial cell cycle classification. Application to DNA synthesis and DNA content at any cell age.

A proposal is presented for classifying bacterial cell cycles into twelve discrete groups. This classification translates the three temporal parameters that define a cell cycle into numbers that facilitate an algorithmic approach to analyse the replication state of a single bacterium and of a bacterial population during steady-state of exponential growth. The classification and its implementation offer easy ways to obtain the rate of DNA synthesis and the amount of DNA per cell at any age in batch cultures.

Chromosome replication status and DNA content at any cell age in a bacterial cell cycle.

An algorithm is presented to determine the chromosome replication status, the rate of DNA synthesis per fork, and the amount of DNA in chromosome equivalents (G) per chromosome, per cell and per age throughout a bacterial cell cycle. This algorithm is the only attempt to study replication and the G value at any cell age since the general model of the bacterial cell cycle by Cooper and Helmstetter (1968, J. Mol.

Organization of ribonucleoside diphosphate reductase during multifork chromosome replication in Escherichia coli.

Ribonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that fork : focus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.

Correlation between ribonucleoside-diphosphate reductase and three replication proteins in Escherichia coli.

Background: There has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm.

Toward a hyperstructure taxonomy.

Bacterial cells contain many large, spatially extended assemblies of ions, molecules, and macromolecules, called hyperstructures, that are implicated in functions that range from DNA replication and cell division to chemotaxis and secretion. Interactions between these hyperstructures would create a level of organization intermediate between macromolecules and the cell itself. To explore this level, a taxonomy is needed.

Double-strand break generation under deoxyribonucleotide starvation in Escherichia coli.

Stalled replication forks produced by three different ways of depleting deoxynucleoside triphosphate showed different capacities to undergo "replication fork reversal." This reaction occurred at the stalled forks generated by hydroxyurea treatment, was impaired under thermal inactivation of ribonucleoside reductase, and did not take place under thymine starvation.

Functional taxonomy of bacterial hyperstructures.

The levels of organization that exist in bacteria extend from macromolecules to populations. Evidence that there is also a level of organization intermediate between the macromolecule and the bacterial cell is accumulating. This is the level of hyperstructures.

Defective ribonucleoside diphosphate reductase impairs replication fork progression in Escherichia coli.

The observed lengthening of the C period in the presence of a defective ribonucleoside diphosphate reductase has been assumed to be due solely to the low deoxyribonucleotide supply in the nrdA101 mutant strain. We show here that the nrdA101 mutation induces DNA double-strand breaks at the permissive temperature in a recB-deficient background, suggesting an increase in the number of stalled replication forks that could account for the slowing of replication fork progression observed in the nrdA101 strain in a Rec(+) context. These DNA double-strand breaks require the presence of the Holliday junction resolvase RuvABC, indicating that they have been generated from stalled replication forks that were processed by the specific reaction named "replication fork reversal.

Differences in the degree of inhibition of NDP reductase by chemical inactivation and by the thermosensitive mutation nrdA101 in Escherichia coli suggest an effect on chromosome segregation.

NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA (ts) mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells.

Initiation of heat-induced replication requires DnaA and the L-13-mer of oriC.

An upshift of 10 degrees C or more in the growth temperature of an Escherichia coli culture causes induction of extra rounds of chromosome replication. This stress replication initiates at oriC but has functional requirements different from those of cyclic replication. We named this phenomenon heat-induced replication (HIR).

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli.

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes.