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2 Abstract: Two contrasting experimental descriptions of P1 plasmid replication during the cell cycle of Escherichia coli have been described. One set of results led to the proposal that replication of P1 plasmid occurs at a specific time during the cell cycle over a wide range of growth rates and follows rules similar to that governing bacterial chromosome replication. Experiments supporting this proposal utilized membrane-elution experiments, radioactive double-labeling of DNA and scintillation counting of purified plasmids. An alternative experimental description of P1 plasmid replication during the cell cycle, also based on membrane-elut ion methodology but measuring radioactivity incorporated into plasmid DNA by autoradiograp hy and scanning of films, proposed that P1 plasmid replicates at all stages of the cell cycle in rapidly growing cells, but with a slight periodicity or increase in P1 replication probability within the cell cycle of slower growing cells. These discordant experimental results are analyzed. It is concluded that the direct double-label counting approach is to be preferred, as the results are consistent with a large number of experiments, are supported by theoretical considerations and yield a unified view of plasmid replication over a wide range of growth rates. Theoretical ramifications of each view of P1 plasmid replication-cycle-dependent and cycle-independent are compared. An analysis of P1 plasmid segregation is also presented.

For a number of years now, we have argued that current models for the control of initiation of DNA synthesis, chromosomal partitioning and septum formation in Escherichia coli are unsatisfactory. Indeed, we could argue that despite considerable efforts, with the possible exception of dnaA and ftsZ, no genes specifically implicated in these control processes have been identified. In the cases of DnaA and FtsZ, no evidence has appeared to indicate how such molecules might be regulated to act once per cycle. In 1988, we formulated a specific proposal that the timing of cell cycle events in E. coli might be determined by a Ca++ flux, mediated by calcium-binding proteins and protein kinases and culminating, in the case of chromosome segregation and division, in the action of force-generating proteins such as myosin (Norris et al., 1988). In formulating this proposal, we took the view that the fundamental elements of cell cycle regulation are likely to be highly conserved across all species including prokaryotes. In this presentation, we shall describe the approaches we have been taking in order to test this hypothesis and to summarize the data obtained, in particular in relation to new genes identified which may play a role in the E. coli cell cycle. We shall also briefly indicate recent data from other laboratories consistent with our general hypothesis.

RstB/RstA is an uncharacterized Escherichia coli two-component system, the regulatory effects of which on the E. coli cell cycle remain unclear. We found that the doubling time and average number of replication origins per cell in an ΔrstB mutant were the same as the wild-type, and the average number of replication origins in an ΔrstA mutant was 18.2% lower than in wild-type cells. The doubling times were 34 min, 35 min, and 40 min for the wild-type, ΔrstB, and ΔrstA strains, respectively. Ectopic expression of RstA from plasmid pACYC-rstA partly reversed the ΔrstA mutant phenotypes. The amount of initiator protein DnaA per cell was reduced by 40% in the ΔrstA mutant compared with the wild-type, but the concentration of DnaA did not change as the total amount of cellular protein was also reduced in these cells. Deletion or overproduction of RstA does not change the temperature sensitivity of dnaA46, dnaB252 and dnaC2. The expression of hupA was decreased by 0.53-fold in ΔrstA. RstA interacted with Topoisomerase I weakly in vivo and increased its activity of relaxing the negative supercoiled plasmid. Our data suggest that deletion of RstA leads to delayed initiation of DNA replication, and RstA may affect initiation of replication by controlling expression of dnaA or hupA. Furthermore, the delayed initiation may by caused by the decreased activity of topoisomerase I in RstA mutant.

Initiation of E. coli replication starts with formation of a DnaA protein filament on specific DnaA boxes within the oriC locus of bacterial chromosome. That binding forces proximal DNA to melt, forming an open replication bubble, which allows loading of replication machinery onto the DNA. We have explored how targeting a nuclease-deficient CRISPR system to the DnaA boxes inhibits the initiation of replication. Cells simultaneously expressing dCas9 protein and the CRISPR guide RNA that bind to the one of DnaA boxes, are not able to initiate chromosome replication and divide leading to cell cycle arrest phenotype as shown by both microscopy and FACS assays, thus resulting in inability to form colonies on the plate. We conclude that the dCas9 competes with DnaA proteins for binding to the same sequence resulting in inhibition of the earliest step of DNA replication. Preliminary data suggest that the arrest is reversible and new rounds of replication can be established after removing the CRISPR block. We suggest that this new method of E. coli cell cycle control may be used in several different areas, such as chromosome structure studies or genetic circuits design.

The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division.

The cellular response to DNA damage that has been most extensively studied is the SOS response of Escherichia coli. Analyses of the SOS response have led to new insights into the transcriptional and posttranslational regulation of processes that increase cell survival after DNA damage as well as insights into DNA-damage-induced mutagenesis, i.e., SOS mutagenesis. SOS mutagenesis requires the recA and umuDC gene products and has as its mechanistic basis the alteration of DNA polymerase III such that it becomes capable of replicating DNA containing miscoding and noncoding lesions. Ongoing investigations of the mechanisms underlying SOS mutagenesis, as well as recent observations suggesting that the umuDC operon may have a role in the regulation of the E. coli cell cycle after DNA damage has occurred, are discussed.

Over 80% of the genes in the E. coli chromosome express fewer than a hundred copies each of their protein products per cell. It is argued here that transcription of these genes is neither constitutive nor regulated by protein factors, but rather, induced by the act of replication. The utility of such replication-induced (RI) transcription to the temporal regulation of synthesis of determinate quantities of low copy number (LCN) proteins is described. It is suggested that RI transcription may be necessitated, as well as facilitated, by the folding of the bacterial chromosome into a compact nucleoid. Mechanistic aspects of the induction of transcription by replication are discussed with respect to the modulation of transcriptional initiation by negative supercoiling effects, promoter methylation status and derepression. It is shown that RI transcription offers plausible explanations for the constancy of the C period of the E. coli cell cycle and the remarkable conservation of gene order in the chromosomes of enteric bacteria. Some experimental tests of the hypothesis are proposed.

Efficient partitioning of chromosomes into dividing cells is important for cell survival. In Escherichia coli and Bacillus subtilis, chromosomes partition to daughter cells with high fidelity (Hiraga et al. 1989; Ireton et al. 1994). Although proteins, sites, and mechanisms involved in physical separation (decatenation) of bacterial chromosomes have been characterized, much less is known about the mechanisms governing efficient partitioning to daughter cells (for review, see Hiraga 1992; Wake and Errington 1995). Recent work has shown that the chromosomal region around the origin of replication (oriC) is in a defined orientation for most of the bacterial (B. subtilis and E. coli) cell cycle and that newly replicated oriC regions are rapidly separated from each other (Glaser et al. 1997; Gordon et al. 1997; Lin et al. 1997; Webb et al. 1997). The origin regions are found toward the poles of the highly condensed nucleoid body, oriented toward the ends of the cell. The rapid separation and localization of oriC regions indicate the function of a mitotic-like apparatus in prokaryotes (Glaser et al. 1997; Gordon et al. 1997; Lin et al. 1997; Webb et al. 1997). Cellular proteins contributing to efficient chromosome partitioning have recently been characterized. Spo0J from B. subtilis and ParB from Caulobacter crescentus are required for efficient chromosome partitioning and are similar to a family of plasmid-encoded proteins required for plasmid partitioning in E. coli (e.g., ParB for P1 and SopB for F). ParB of C. crescentus is essential for growth, and overexpression causes a defect in chromosome partitioning (Mohl and Gober 1997). Deletion of spo0J in B. subtilis causes an ∼100-fold increase in the number of anucleate cells, resulting in accumulation of 1%–2% anucleate cells in a growing culture (Ireton et al. 1994). Spo0J binds to at least eight sites located in the origin proximal 20% of the chromosome (Lin and Grossman 1998). Spo0J is found in the cell in single discrete foci located near the poles of the nucleoid body (Glaser et al. 1997; Lin et al. 1997), in a pattern similar to that observed for the region around the origin of replication (Lewis and Errington 1997; Webb et al. 1997). Visualization of the foci of Spo0J by immunofluorescence microscopy or by use of a Spo0J–green fluorescent protein (GFP) fusion indicates the assembly of a large nucleoprotein complex containing Spo0J. The function of Spo0J and other proteins of this family is still unknown, though they are thought to be involved in pairing and/or positioning sister chromosomes (Nordstrom and Austin 1989; Niki and Hiraga 1997; Lin and Grossman 1998). The muk genes of E. coli were identified in an elegant screen for mutants that produce anucleate cells (Hiraga et al. 1989). The mukB gene product has features of a myosin-like motor protein and is involved in chromosome condensation and/or movement [(Niki et al. 1991; Hiraga 1992; Wake and Errington 1995; Hu et al. 1996), and references therein]. mukE and mukF, which are in an operon with mukB, are also required for efficient partitioning and their products are thought to interact with MukB (Yamanaka et al. 1996). Of the ∼12 bacterial genomes that have been sequenced, mukB, mukE, and mukF are found only in E. coli and Haemophilus influenzae. B. subtilis, along with many other bacterial species (but not E. coli or H. influenzae), contains a homolog of the eukaryotic Smc (structural maintenance of chromosomes) proteins (Oguro et al. 1996). Several eukaryotes have multiple smc genes, and eukaryotic Smc proteins play a role in chromosome condensation, pairing, and/or segregation (for review, see Hirano et al. 1995; Koshland and Strunnikov 1996; Heck 1997). For example, mutations in the SMC genes of Saccharomyces cerevisiae cause defects in chromosome condensation, segregation, and sister chromatid cohesion (Guacci et al. 1997; Michaelis et al. 1997). DNA condensation by the 13S condensin of Xenopus laevis requires two Smc proteins, XCAP-C and XCAP-E (Hirano et al. 1997). Dosage compensation in Caenorhabditis elegans involves specific interaction of an Smc homolog, Dpy-27, and other proteins, with the X chromosome (Chuang et al. 1994, 1996). Although the precise biochemical function of the Smc proteins is not known, recent work has shown that Smc proteins, or complexes containing Smc proteins, can affect DNA topology in vitro (Kimura and Hirano 1997; Sutani and Yanagida 1997). The smc gene of B. subtilis encodes a 135-kD protein that is homologous to eukaryotic Smc proteins (Oguro et al. 1996). B. subtilis Smc is ∼24% identical and ∼46% similar to SMC1 and SMC2 (S. cerevisiae), XCAP-C and XCAP-E (X. laevis), Dpy-27 (C. elegans), and Cut3 and Cut14 (Schizosaccaromyces pombe). It contains all of the domains associated with the Smc family; an amino-terminal NTP-binding domain, two internal coiled–coil regions separated by a hinge, and the carboxy-terminal signature “DA-box” motif (Hirano et al. 1995; Koshland and Strunnikov 1996). Whereas the existence of Smc proteins in eukaryotes is well documented, their prevalence in bacteria and archaebacteria is only beginning to be appreciated. A search of GenBank and individual sequence databases (both completed and in progress) revealed that at least 11 bacteria and 2 archaebacteria contain genes encoding homologs of Smc. In addition to B. subtilis, the list includes Streptococcus pyogenes, Streptococcus pneumoniae, Mycobacterium tuberculosis, several Mycoplasma species (M. genitalium, M. pneumoniae, and M. hyorhinis), Borrelia burgdorferi, Treponema pallidum, Synechocystis sp., Neisseria gonorrhoeae, and the archaebacteria Methanococcus jannaschii and Archaeoglobus fulgidus. We report the characterization of the B. subtilis smc gene. Null mutations in smc caused a conditional lethal phenotype, alterations in nucleoid appearance, a defect in chromosome partitioning, and a synthetic phenotype with a null mutation in spo0J. These findings indicate that the function of Smc proteins is highly conserved.

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minB deletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minB mutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleoid conformation and distribution were considerably disturbed. The results obtained call for a re-evaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle.

As an alternative approach in the study of the Escherichia coli cell cycle, we have constructed strains in which chromosome replication is under the control of various plasmid R1 derivatives, IntR1 strains. The physiological properties of such strains are described. In intR1 strains, chromosome replication can be manipulated independently of cell-cycle-related control mechanisms, and the effects on cell division can be analysed. Using this approach, we have found that the timing of replication during the cell cycle is random in intR1 strains, that overreplication of the chromosome is lethal, and that chromosome replication does not trigger cell division. Current investigations include the study of the E. coli cell cycle in the absence of the DnaA protein, the effect on cell division of a specific inhibition of the initiation of chromosome replication, and the molecular basis of uni- and bidirectional replication.

The promoter/regulatory region of the dnaA gene, whose gene product is required for the initiation of DNA replication in Escherichia coli K-12, contains an unusually large number of Dam methylation sites. In this paper we report that the expression of the dnaA gene is decreased in Dam- strains of E. coli. The decrease in the expression of dnaA was measured in vivo using a dnaA-lacZ gene fusion. In vivo S1 nuclease mapping demonstrated that the decrease was due to a differential decrease in expression from the more proximal of the two dnaA promoters, dnaA2P. Comparison of the strengths of the two dnaA promoters in an in vitro transcription system using methylated and unmethylated DNA templates suggests that the effect of methylation on dnaA2P is probably at the level of RNA polymerase/DNA interaction. We suggest that this effect of methylation may be important in controlling the expression of dnaA during the E. coli cell cycle.

In synchronous cultures of Escherichia coli, the pool sizes of ATP and ADP increase continuously, probably exponentially, such that the ATP/ADP ratio is invariant during the cell cycle. Resolution of cells from an exponentially growing culture into size, and thus age, classes reveals no significant variation in succinate dehydrogenase activity during the cell cycle. These data reinforce earlier findings that respiratory metabolism in the E. coli cell cycle is not manifestly periodic.