Replication of Vibrio cholerae Chromosome I in Escherichia coli: Dependence on Dam Methylation

We successfully substituted Escherichia coli''s origin of replication oriC with the origin region of Vibrio cholerae chromosome I (oriCI_Vc). Replication from oriCI_Vc initiated at a similar or slightly reduced cell mass compared to that of normal E. coli oriC. With respect to sequestration-dependent synchrony of initiation and stimulation of initiation by the loss of Hda activity, replication initiation from oriC and oriCI_Vc were similar. Since Hda is involved in the conversion of DnaA^ATP (DnaA bound to ATP) to DnaA^ADP (DnaA bound to ADP), this indicates that DnaA associated with ATP is limiting for V. cholerae chromosome I replication, which similar to what is observed for E. coli. No hda homologue has been identified in V. cholerae yet. In V. cholerae, dam is essential for viability, whereas in E. coli, dam mutants are viable. Replacement of E. coli oriC with oriCI_Vc allowed us to specifically address the role of the Dam methyltransferase and SeqA in replication initiation from oriCI_Vc. We show that when E. coli''s origin of replication is substituted by oriCI_Vc, dam, but not seqA, becomes important for growth, arguing that Dam methylation exerts a critical function at the origin of replication itself. We propose that Dam methylation promotes DnaA-assisted successful duplex opening and replisome assembly at oriCI_Vc in E. coli. In this model, methylation at oriCI_Vc would ease DNA melting. This is supported by the fact that the requirement for dam can be alleviated by increasing negative supercoiling of the chromosome through oversupply of the DNA gyrase or loss of SeqA activity.The genomes of Vibrio cholerae and several related Vibrio spp. are distributed between two circular chromosomes. Characterization of the origins of replication of V. cholerae chromosomes I and II (oriCI_Vc and oriCII_Vc, respectively) has shown that oriCI_Vc is similar to the origin of replication of the Escherichia coli chromosome, oriC, whereas oriCII_Vc is completely different (20). Like oriC, oriCI_Vc has five R-type DnaA boxes (53) as well as boxes conforming to the I and τ types (52, 61), and the DnaA protein is the rate-limiting factor in the initiation of replication in both cases (18). In E. coli, DnaA associates with both ATP and ADP, and the ATP-bound form is absolutely required for initiation to take place (reviewed in reference 60). When reaching a critical level, DnaA^ATP (DnaA bound to ATP) protein is proposed to form a helical filament, anchored at one or more R-boxes (54, 69), in which origin DNA wraps around the outside of the DnaA core (21) or where the DnaA wraps around oriC (61). In both cases, the topology of the DnaA-oriC nucleoprotein complex leads to formation of compensatory negative supercoiling that facilitates unwinding of the adjacent AT-rich region resulting in initiation. In both models, DnaA^ATP is absolutely required for initiation, and in agreement with this, DnaA^ATP was found to be the rate-limiting factor for initiation in vivo (69).The V. cholerae oriCI_Vc also resembles oriC in having many potential sites for methylation by DNA adenine methyltransferase (Dam), although the number and position of the GATC sites differ slightly (see Fig. ​Fig.1).1). The role of Dam in initiation of chromosome replication has been studied mainly in E. coli. After initiation of DNA replication has occurred on a fully methylated oriC, the newly replicated hemimethylated origins are sequestered from the Dam methyltransferase and from reinitiation for approximately one-third of a doubling time. During this time interval, the activity and amount of DnaA available for initiation are reduced to prevent immediate reinitiation (reviewed in references 57 and 83). The sequestration is carried out by the SeqA protein that binds hemimethylated oriC GATC sequences with high affinity (48). In the absence of Dam methylation or SeqA, the same origin can be reinitiated in the same cell cycle, and initiations become asynchronous (9, 48).Open in a separate windowFIG. 1.Alignment of the E. coli minimal oriC with the corresponding region from V. cholerae chromosome I. The AT-rich sequence and the three 13-mer repeats L, M, and R found in E. coli (5) are indicated above the alignment. The 6-mer (A/T)GATCT boxes (80) are underlined. Other DnaA binding sites, i.e., R-boxes (53), I-boxes (52), and τ-boxes (61), are shown as boxed regions. Dam methylation sites (GATC) are shaded gray. The experimentally defined binding sites for integration host factor (IHF) (22) and factor for inversion stimulation (FIS) (65) in E. coli are indicated, and bases that match the consensus sequence are in boldface type. The single base difference between oriCI_Vc and oriCI_Vc* (see Materials and Methods) in the minimal origin region is shown below the two sequences. A gap introduced to maximize alignment of the two sequences is indicated by a dash in the sequence. Nucleotides that are identical in the two sequences are indicated by an asterisk below the two sequences.Genes encoding a Dam homologue and a SeqA homologue are present on Vibrio genomes, but there appear to be some differences between the functions of the proteins in E. coli and V. cholerae. dam has been found to be an essential gene in V. cholerae (33, 15), which is not the case in E. coli (48, 51). Conflicting data exist concerning the essentiality of seqA in V. cholerae (15, 72). The roles of Dam and SeqA in oriCI_Vc replication have been studied using minichromosomes, i.e., plasmids replicating exclusively from a cloned copy of oriCI_Vc (20). oriCI_Vc-based minichromosomes can replicate in wild-type E. coli cells but were unable to replicate in dam, seqA, and seqA dam mutants (20). The extrachromosomal existence of minichromosomes is dependent on their ability to initiate replication in synchrony with the chromosomal origin (46, 75). In E. coli cells mutated in dam or seqA, incompatibility exists between the oriC carried on minichromosomes and that of the chromosome due to origin competition (13), and when minichromosomes are maintained under selective pressure, they integrate into the origin region of the host chromosome (46, 75). Minichromosomes based on oriCI_Vc may also compete with the E. coli oriC for initiations in dam or seqA mutant cells. However, due to limited sequence identity, they may not be able to integrate into the E. coli chromosome. This could provide an explanation for the failure to introduce oriCI_Vc minichromosomes into dam and seqA mutant cells (20). Both dam and seqA genes could therefore be required for viability of V. cholerae for reasons not related to chromosome replication. In addition to its role in DNA replication, roles for Dam methylation in gene regulation and DNA repair have also been demonstrated in a number of bacteria (for reviews, see references 11, 45, 47, and 50). For V. cholerae as well as for Salmonella spp. and Yersinia pseudotuberculosis, Dam plays a role in virulence possibly through regulation of virulence gene expression (33). Less is known about the functions of seqA apart from its role in E. coli replication, but it has been suggested that SeqA functions as a nucleoid-organizing protein (for a review, see reference 83), and the E. coli chromosome has been demonstrated to have increased supercoiling in a seqA strain (85).Here we describe the first in vivo evidence that Dam plays an important role in the initiation of replication by facilitating the replication initiation at oriCI_Vc in E. coli. In addition, we show that SeqA does not carry an essential role in the initiation of replication.     

Importance of Multiple Methylation Sites in Escherichia coli Chemotaxis

Bacteria navigate within inhomogeneous environments by temporally comparing concentrations of chemoeffectors over the course of a few seconds and biasing their rate of reorientations accordingly, thereby drifting towards more favorable conditions. This navigation requires a short-term memory achieved through the sequential methylations and demethylations of several specific glutamate residues on the chemotaxis receptors, which progressively adjusts the receptors’ activity to track the levels of stimulation encountered by the cell with a delay. Such adaptation also tunes the receptors’ sensitivity according to the background ligand concentration, enabling the cells to respond to fractional rather than absolute concentration changes, i.e. to perform logarithmic sensing. Despite the adaptation system being principally well understood, the need for a specific number of methylation sites remains relatively unclear. Here we systematically substituted the four glutamate residues of the Tar receptor of Escherichia coli by non-methylated alanine, creating a set of 16 modified receptors with a varying number of available methylation sites and explored the effect of these substitutions on the performance of the chemotaxis system. Alanine substitutions were found to desensitize the receptors, similarly but to a lesser extent than glutamate methylation, and to affect the methylation and demethylation rates of the remaining sites in a site-specific manner. Each substitution reduces the dynamic range of chemotaxis, by one order of magnitude on average. The substitution of up to two sites could be partly compensated by the adaptation system, but the full set of methylation sites was necessary to achieve efficient logarithmic sensing.     

Partitioning of the Escherichia coli chromosome: superhelicity and condensation

Segregation in Escherichia coli, the process of separating the replicated chromosomes into daughter progeny cells, seems to start long before the duplication of the genome reaches completion. Soon after initiation in mid-cell region, the daughter oriCs rapidly move apart to fixed positions inside the cell (quarter length positions from each pole) and are anchored there by yet unknown mechanism(s). As replication proceeds, the rest of the chromosome is sequentially unwound and then refolded. At termination, the two sister chromosomes are unlinked by decatenation and separated by supercoiling and/or condensation. Muk and Seq proteins are involved in different stages of this replication-cum-partition process and thus can be categorized as important partition proteins along with topoisomerases. E. coli strains, lacking mukB or seqA functions, are defective in segregation and cell division. The nucleoids in these mutant strains exhibit altered condensation and superhelicity as can be demonstrated by sedimentation analysis and by fluorescence microscopy. As the supercoiling of an extrachromosomal element (a plasmid DNA) was also influenced by the mukB and seqA mutations we concluded that the MukB and SeqA proteins are possibly involved in maintaining the general supercoiling activity in the cell. The segregation of E. coli chromosome might therefore be predominantly driven by factors that operate by affecting the superhelicity and condensation of the nucleoid (MukB, SeqA, topoisomerases and additional unknown proteins). A picture thus emerges in which replication and partition are no longer compartmentalized into separable stages with clear gaps (S and M phases in eukaryotes) but are parallel processes that proceed concomitantly through a cell cycle continuum.     

Chromosome segregation by the Escherichia coli Min system

The mechanisms underlying chromosome segregation in prokaryotes remain a subject of debate and no unifying view has yet emerged. Given that the initial disentanglement of duplicated chromosomes could be achieved by purely entropic forces, even the requirement of an active prokaryotic segregation machinery has been questioned. Using computer simulations, we show that entropic forces alone are not sufficient to achieve and maintain full separation of chromosomes. This is, however, possible by assuming repeated binding of chromosomes along a gradient of membrane‐associated tethering sites toward the poles. We propose that, in Escherichia coli, such a gradient of membrane tethering sites may be provided by the oscillatory Min system, otherwise known for its role in selecting the cell division site. Consistent with this hypothesis, we demonstrate that MinD binds to DNA and tethers it to the membrane in an ATP‐dependent manner. Taken together, our combined theoretical and experimental results suggest the existence of a novel mechanism of chromosome segregation based on the Min system, further highlighting the importance of active segregation of chromosomes in prokaryotic cell biology.     

Chromosome structuring limits genome plasticity in Escherichia coli

Chromosome organizations of related bacterial genera are well conserved despite a very long divergence period. We have assessed the forces limiting bacterial genome plasticity in Escherichia coli by measuring the respective effect of altering different parameters, including DNA replication, compositional skew of replichores, coordination of gene expression with DNA replication, replication-associated gene dosage, and chromosome organization into macrodomains. Chromosomes were rearranged by large inversions. Changes in the compositional skew of replichores, in the coordination of gene expression with DNA replication or in the replication-associated gene dosage have only a moderate effect on cell physiology because large rearrangements inverting the orientation of several hundred genes inside a replichore are only slightly detrimental. By contrast, changing the balance between the two replication arms has a more drastic effect, and the recombinational rescue of replication forks is required for cell viability when one of the chromosome arms is less than half than the other one. Macrodomain organization also appears to be a major factor restricting chromosome plasticity, and two types of inverted configurations severely affect the cell cycle. First, the disruption of the Ter macrodomain with replication forks merging far from the normal replichore junction provoked chromosome segregation defects. The second major problematic configurations resulted from inversions between Ori and Right macrodomains, which perturb nucleoid distribution and early steps of cytokinesis. Consequences for the control of the bacterial cell cycle and for the evolution of bacterial chromosome configuration are discussed.     

Absence of oligomeric murein intermediates in Escherichia coli.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.     

Effect of p-Fluorophenylalanine on Chromosome Replication in Escherichia coli

The effect of p-fluorophenylalanine (FPA) on deoxyribonucleic acid (DNA) synthesis and chromosome replication was studied in a thymine-requiring mutant of Escherichia coli. The rate and extent of chromosome replication were followed by labeling the DNA with isotopic thymine and a density marker, bromouracil. The DNA was extracted and analyzed by CsCl gradient centrifugation. The block in chromosome replication caused by high concentrations of FPA occurred at the same point on the chromosome as that caused by amino acid starvation. In a random culture, DNA in cells treated with FPA replicated only slightly slower than the DNA from cells that were not exposed to the analogue. In cultures which had been previously starved for thymine, however, the DNA from the cells treated with FPA showed a marked decrease in the rate and extent of replication. It was concluded that the E. coli cell is most sensitive to FPA when a new cycle of chromosome replication is being initiated at the beginning of the chromosome.     

Chromosome replication in an Hfr strain of Escherichia coli

The pattern of chromosome replication in an exponentially growing culture of an Hfr strain of Escherichia coli has been compared to that obtained with the same Hfr following a procedure which synchronizes rounds of DNA replication. The results indicate that there is significant replication from the integrated plasmid following the synchronization procedure, whereas in the exponentially growing culture replication starts most frequently from the normal origin with little, if any, replication from the sex factor, F.     

Chromosome replication pattern in dam mutants of Escherichia coli

Summary The replication cycle of Escherichia coli dam mutants was analysed and compared with that of isogenic Dam⁺ strains. Marker frequency analyses indicated no gross difference between the strains. In the Dam^– as well as in the Dam⁺ bacteria, initiation most likely occurs at oriC, replication forks move at a constant and invariant velocity, and termination takes place in the terC region. An analysis of replication terminator activity indicated that this activity is unaffected by the methylation status. Taken together with previous results, our data are compatible with Dam methylation controlling initiation timing but no subsequent step of the replication process.     

Methylation of chemotaxis-specific proteins in Escherichia coli cells permeable to S-adenosylmethionine

Using a modification of the EGTA treatment of Oishi and Smith [Oishi, M., & Smith, C. L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3569], Escherichia coli cells have been made permeable to S-adenosylmethionine and other related molecules in order to facilitate the study of methylation in chemotaxis. The permeable cells are nonmotile but respond to chemotactic stimuli by reversible methylation of their methyl-accepting chemotactic proteins (MCP I and MCP II) in a manner similar to that of untreated, motile cells. Addition of S-adenosyl-L-[methyl-3H]methionine to the permeable cells specifically labels two proteins, MCP I and MCP II. Methylation of these MCP's is dependent on the presence of wild-type gene products of flaI, flaA, cheB, cheX, tsr, and tar. The extent of methylation of the MCP's is affected by the presence of attractants or repellents: addition of attractant increases the steady-state level of methylation; addition of repellent causes rapid demethylation to a new steady-state level. Methylation is inhibited by the addition of the transmethylase inhibitors A9145C and Sinefungin, which are S-adenosylmethionine analogues, and by S-adenosylhomocysteine.     

Chromosome replication in Escherichia coli induced by oversupply of DnaA

Summary Overexpression of DnaA protein from a multicopy plasmid accompanied by a shift to 42°C causes initiation of one extra round of replication in a dnaA ⁺ strain grown in glycerol minimal medium. This extra round of replication does not lead to an extra cell division, such that cells contain twice the normal number of chromosomes.     

Effects of Chromosome Underreplication on Cell Division in Escherichia coli

The key processes of the bacterial cell cycle are controlled and coordinated to match cellular mass growth. We have studied the coordination between replication and cell division by using a temperature-controlled Escherichia coli intR1 strain. In this strain, the initiation time for chromosome replication can be displaced to later (underreplication) or earlier (overreplication) times in the cell cycle. We used underreplication conditions to study the response of cell division to a delayed initiation of replication. The bacteria were grown exponentially at 39°C (normal DNA/mass ratio) and shifted to 38 and 37°C. In the last two cases, new, stable, lower DNA/mass ratios were obtained. The rate of replication elongation was not affected under these conditions. At increasing degrees of underreplication, increasing proportions of the cells became elongated. Cell division took place in the middle in cells of normal size, whereas the longer cells divided at twice that size to produce one daughter cell of normal size and one three times as big. The elongated cells often produced one daughter cell lacking a chromosome; this was always the smallest daughter cells, and it was the size of a normal newborn cell. These results favor a model in which cell division takes place at only distinct cell sizes. Furthermore, the elongated cells had a lower probability of dividing than the cells of normal size, and they often contained more than two nucleoids. This suggests that for cell division to occur, not only must replication and nucleoid partitioning be completed, but also the DNA/mass ratio must be above a certain threshold value. Our data support the ideas that cell division has its own control system and that there is a checkpoint at which cell division may be abolished if previous key cell cycle processes have not run to completion.     

Role of the C Terminus of FtsK in Escherichia coli Chromosome Segregation

FtsK is essential for Escherichia coli cell division. We report that cells lacking the C terminus of FtsK are defective in chromosome segregation as well as septation, often exhibiting asymmetrically positioned nucleoids and large anucleate regions. Combining the corresponding truncated ftsK gene with a mukB null mutation resulted in a synthetic lethal phenotype. When the truncated ftsK was combined with a minCDE deletion, chains of minicells were generated, many of which contained DNA. These results suggest that the C terminus of FtsK has an important role in chromosome partitioning.