Multiple links connect central carbon metabolism to DNA replication initiation and elongation in Bacillus subtilis.

DNA replication is coupled to growth by an unknown mechanism. Here, we investigated this coupling by analyzing growth and replication in 15 mutants of central carbon metabolism (CCM) cultivated in three rich media. In about one-fourth of the condition tested, defects in replication resulting from changes in initiation or elongation were detected. This uncovered 11 CCM genes important for replication and showed that some of these genes have an effect in one, two or three media. Additional results presented here and elsewhere (Jannière, L., Canceill, D., Suski, C., et al. (2007), PLoS One, 2, e447.) showed that, in the LB medium, the CCM genes important for DNA elongation (gapA and ackA) are genetically linked to the lagging strand polymerase DnaE while those important for initiation (pgk and pykA) are genetically linked to the replication enzymes DnaC (helicase), DnaG (primase) and DnaE. Our work thus shows that the coupling between growth and replication involves multiple, medium-dependent links between CCM and replication. They also suggest that changes in CCM may affect initiation by altering the functional recruitment of DnaC, DnaG and DnaE at the chromosomal origin, and may affect elongation by altering the activity of DnaE at the replication fork. The underlying mechanism is discussed.

SeqA structures behind Escherichia coli replication forks affect replication elongation and restart mechanisms.

The SeqA protein binds hemi-methylated GATC sites and forms structures that sequester newly replicated origins and trail the replication forks. Cells that lack SeqA display signs of replication fork disintegration. The broken forks could arise because of over-initiation (the launching of too many forks) or lack of dynamic SeqA structures trailing the forks. To confirm one or both of these possible mechanisms, we compared two seqA mutants with the oriCm3 mutant. The oriCm3 mutant over-initiates because of a lack of origin sequestration but has wild-type SeqA protein. Cells with nonfunctional SeqA, but not oriCm3 mutant cells, had problems with replication elongation, were highly dependent on homologous recombination, and exhibited extensive chromosome fragmentation. The results indicate that replication forks frequently break in the absence of SeqA function and that the broken forks are rescued by homologous recombination. We suggest that SeqA may act in two ways to stabilize replication forks: (i) by enabling vital replication fork repair and restarting reactions and (ii) by preventing replication fork rear-end collisions.

Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli.

UNLABELLED: The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein. In H-NS mutant cells, foci of SeqA, replisomes, and origins were irregularly positioned in the cell. Further analysis showed that the average distance between the SeqA structures and the replisome was increased by ∼100 nm compared to that in wild-type cells, whereas the colocalization of SeqA-bound sister DNA behind replication forks was not affected. This result may suggest that H-NS contributes to the folding of DNA along adjacent segments. H-NS mutant cells were found to be incapable of adopting the distinct and condensed nucleoid structures characteristic of E. coli cells growing rapidly in rich medium. It appears as if H-NS mutant cells adopt a "slow-growth" type of chromosome organization under nutrient-rich conditions, which leads to a decreased cellular DNA content. IMPORTANCE: It is not fully understood how and to what extent nucleoid-associated proteins contribute to chromosome folding and organization during replication and segregation in Escherichia coli. In this work, we find in vivo indications that cells lacking the nucleoid-associated protein H-NS have a lower degree of DNA condensation than wild-type cells. Our work suggests that H-NS is involved in condensing the DNA along adjacent segments on the chromosome and is not likely to tether newly replicated strands of sister DNA. We also find indications that H-NS is required for rapid growth with high DNA content and for the formation of a highly condensed nucleoid structure under such conditions.

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli.

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated. In this work we have performed cell cycle analysis of cells that contain a surplus of ATP-DnaA and asked whether initiation then occurs earlier. It does not. Cells with more than a 50% increase in the concentration of ATP-DnaA showed no changes in timing of replication. We suggest that although ATP-DnaA is the main actor in initiation of replication, its accumulation does not control the time of initiation. ATP-DnaA is the motor that drives the initiation process, but other factors will be required for the exact timing of initiation in response to the cell's environment. We also investigated the in vivo roles of datA dependent DnaA inactivation (DDAH) and the DnaA-binding protein DiaA. Loss of DDAH affected the cell cycle machinery only during slow growth and made it sensitive to the concentration of DiaA protein. The result indicates that compromised cell cycle machines perform in a less robust manner.

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome.

The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm). The results support the idea that newly replicated sister molecules are kept together behind the fork and suggest the existence of a stretch of DNA between the replisome and SeqA which enjoys added stabilization. This could be important in facilitating DNA transactions such as recombination, mismatch repair and topoisomerase activity. In slowly growing cells without ongoing replication forks the SeqA protein was found to reside at the fully methylated origins prior to initiation of replication.

Escherichia coli SeqA structures relocalize abruptly upon termination of origin sequestration during multifork DNA replication.

The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration. Then, two abrupt relocalization events occurred within 2-6 minutes and resulted in SeqA foci localized at each of the cell's quarter positions. Imaging of cells containing an additional fluorescent tag in the origin region showed that SeqA colocalizes with the origin region during sequestration. This indicates that the newly replicated DNA of first one chromosome, and then the other, is moved from midcell to the quarter positions. At the same time, origins are released from sequestration. Our results illustrate that newly replicated sister DNA is segregated pairwise to the new locations. This mode of segregation is in principle different from that of slowly growing bacteria where the newly replicated sister DNA is partitioned to separate cell halves and the decatenation of sisters a prerequisite for, and possibly a mechanistic part of, segregation.

Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli.

In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication. We observe here that when acidic phospholipids were depleted, replication was inhibited with a concomitant reduction of chromosomal content and cell mass prior to growth arrest. This global shutdown of biosynthetic activity was independent of the stringent response. Restoration of acidic phospholipid synthesis resulted in a resumption of DNA replication prior to restored growth, indicating a possible cell-cycle-specific growth arrest had occurred with the earlier loss of acidic phospholipids. Flow cytometry, thymidine uptake, and quantitative polymerase chain reaction data suggest that a deficiency in acidic phospholipids prolonged the time required to replicate the chromosome. We also observed that regardless of the cellular content of acidic phospholipids, expression of mutant DnaA(L366K) altered the DNA content-to-cell mass ratio.