Changes in Cell Size and Shape Associated with Changes in the Replication Time of the Chromosome of Escherichia coli

Average cell mass is shown to be inversely related to the concentration of thymine in the growth medium of a thy⁻ strain of Escherichia coli. The kinetics of the transition from one steady-state average cell mass to another was followed in an attempt to determine the relationship between the chromosome replication time and the time between completion of a round of chromosome replication and the subsequent cell division. Differences in average cell mass are shown to be associated with similar differences in average cell volume. Changes in volume associated with changes in thymine concentration are shown to be due primarily to differences in the width of cells. It is proposed that extension in length of the cell envelope occurs at a linear rate which is proportional to the growth rate and which doubles at the time of termination of rounds of replication. Changes in volume not associated with a change in growth rate are therefore accommodated by a change in cell width. Conditions are described under which average cell mass can continue to increase in successive generations and no steady-state average cell mass is achieved.     

Elongation of rod-shaped bacteria.

Three models relating cell length to generation time are considered for rod-shaped bacteria growing under steady-state conditions; all three presuppose linear elongation. The first model assumes that the rate of elongation is proportional to the instantaneous number of chromosome replication forks per cell; the others, that it is inversely related to the generation time and doubles a fixed time prior to cell division. One of these (model 2) treats this relationship as continuous, with the doubling occurring during the last division cycle (at chromosome termination), while the other is a discrete model in which the doubling in rate takes place at chromosome initiation. Expressions are derived for mean cell length and length at birth in each case.Comparison with experimental data on E. coli B/r using non-linear least-squares techniques results in an excellent fit for model 2 and unsatisfactory ones for the others, the best estimate for the time at which the rate doubles being 15·3 min prior to cell division and for the minimum length at birth (i.e., as the growth rate of the culture tends to zero), 1·47 μm.The functional relationship between cell radius and generation time implied by model 2 is also presented. This model again produces a good fit to the experimental data and provides, for the first time, a direct estimate of the volume/origin ratio at initiation of chromosome replication 0·35 ± 0·05 μm³ (s.e.).The results obtained here are compared with various qualitative observations reported in the literature and with such numerical data as are available.     

Condensin aids sister chromatid decatenation by topoisomerase II

The condensin complex is a key determinant of mitotic chromosome architecture. In addition, condensin promotes resolution of sister chromatids during anaphase, a function that is conserved from prokaryotes to human. Anaphase bridges observed in cells lacking condensin are reminiscent of chromosome segregation failure after inactivation of topoisomerase II (topo II), the enzyme that removes catenanes persisting between sister chromatids following DNA replication. Circumstantial evidence has linked condensin to sister chromatid decatenation but, because of the difficulty of observing chromosome catenation, this link has remained indirect. Alternative models for how condensin facilitates chromosome resolution have been put forward. Here, we follow the catenation status of circular minichromosomes of three sizes during the Saccharomyeces cerevisiae cell cycle. Catenanes are produced during DNA replication and are for the most part swiftly resolved during and following S-phase, aided by sister chromatid separation. Complete resolution, however, requires the condensin complex, a dependency that becomes more pronounced with increasing chromosome size. Our results provide evidence that condensin prevents deleterious anaphase bridges during chromosome segregation by promoting sister chromatid decatenation.     

Properties of membrane-associated folded chromosomes of E. coli related to initiation and termination of DNA replication

Analysis of folded chromosomes prepared from amino acid-starved E. coli cells or from a dnaC initiation mutant indicates that a unique structure is associated with completion or near completion of rounds of chromosome replication in E. coli. Chromosomes remain associated with portions of the bacterial cell envelope throughout the DNA replication cycle, but become more rapidly sedimenting as replication proceeds in the absence of reinitiation. Before reinitiation of chromosome replication occurs after restoring required amino acids to amino acid-starved cells or after lowering the temperature in a thermosensitive dnaC mutant, sedimentation velocities of the membrane-associated folded chromosomes decrease substantially. The decrease in sedimentation velocity does not depend on renewed DNA synthesis, but does require the activity of at least the dnaC gene product.     

Comparison among patterns of macromolecular synthesis in Escherichia coli B/r at growth rates of less and more than one doubling per hour at 37 degrees C.

In Escherichia coli B/r, the relationship between the patterns of chromosome replication and of synthesis of envelope components differs at various growth rates. At growth rates greater than 1.0 doubling per h at 37 degrees C, the average mass and age at initiation of rounds of chromosome replication are similar to those at increase in incorporation of precursors into a major outer membrane protein and phosphatidylethanolamine. At growth rates less than 1.0 doubling per h at 37 degrees C the average mass and age at increase in the synthesis of these envelope components differ from those at initiation of chromosome replication. The average cell mass per chromosomal origin at initiation of rounds of chromosome replication is not a constant and varies between growth rates greater and less than 1.0 doubling per h.     

FtsK-dependent dimer resolution on multiple chromosomes in the pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.     

Control of membrane protein synthesis in bacillus subtilis

In synchronous cultures of Bacillus subtilis 168/S grown on succinate as a sole carbon source (mean generation time 115 min), chromosome initiation occurs at the beginning of the cell cycle but the rate of membrane protein synthesis doubles in mid-cycle more or less coincident with nuclear segregation. In glucose-grown cultures, the doubling in rate of membrane protein synthesis occurs at about the same time as nuclear segregation and DNA initiation at the beginning of the cycle. Control of the rate of membrane synthesis by the chromosome has been demonstrated by inhibiting DNA synthesis using thymine starvation and showing that membrane protein synthesis continues at a constant rate, whereas the rate of cytoplasmic protein synthesis almost doubles.I suggest that the replication of a region at or close to the chromosome terminus is required to allow the doubling in rate of membrane synthesis.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Stomatal dimensions and resistance to diffusion

In the past, relations of diffusive resistance to stomatal geometry have concerned circular pores or pores that are replaced by equivalent circles of the same area. We calculated the resistance for general shapes that include the realistic slit. The resistance comprises two terms. The first is an outer resistance that depends only on ventilation and leaf geometry and is independent of stomata. The second is an inner resistance and is a function of stomatal interference and of stomatal geometry only. If interstomatal spacing is at least three times stomatal length, interstomatal interference is negligible. The inner resistance can then be calculated by adding the resistance of the two ends and the throat of each stoma. In the case of an elongated stoma, the part of the diffusive resistance per square centimeter determined by stomatal geometry is [Formula: see text] where a, b, d, and n are the semilength, semiwidth, depth, and density of the stomata, and D is the diffusivity. This is the familiar Brown and Escombe result applied to slits.     

Chromosome ends and the nuclear envelope at premeiotic interphase in the male grasshopper Brachystola magna by 3-D,E.M. reconstruction

During premeiotic interphase in the male grasshopper Brachystola magna the nucleus is divided into two nuclear envelope bound compartments, one containing the X chromosome and one the autosomes. — The autosomal compartment is characterized by an invaginated nuclear envelope with nuclear pores distributed throughout the envelope. In a polarized region of the cell the pericentric heterochromatic chromocenters are associated with the inner membrane of the envelope invaginations. In this species the chromosomes are telocentric (acrocentric?) and the pericentric heterochromatin marks the proximal chromosome ends. It is concluded that the chromosome ends are attached to the nuclear envelope at premeiotic interphase. — Comparisons are made between the present observations on chromosome arrangements and the nuclear envelope at premeiotic interphase to earlier observations at early meiotic prophase in the same species (Church, 1976). It is concluded that a rearrangement of both the proximal chromosome ends and the nuclear envelope occurs as cells enter meiotic prophase.     

Excess SeqA leads to replication arrest and a cell division defect in Vibrio cholerae

Although most bacteria contain a single circular chromosome, some have complex genomes, and all Vibrio species studied so far contain both a large and a small chromosome. In recent years, the divided genome of Vibrio cholerae has proven to be an interesting model system with both parallels to and novel features compared with the genome of Escherichia coli. While factors influencing the replication and segregation of both chromosomes have begun to be elucidated, much remains to be learned about the maintenance of this genome and of complex bacterial genomes generally. An important aspect of replicating any genome is the correct timing of initiation, without which organisms risk aneuploidy. During DNA replication in E. coli, newly replicated origins cannot immediately reinitiate because they undergo sequestration by the SeqA protein, which binds hemimethylated origin DNA. This DNA is already methylated by Dam on the template strand and later becomes fully methylated; aberrant amounts of Dam or the deletion of seqA leads to asynchronous replication. In our study, hemimethylated DNA was detected at both origins of V. cholerae, suggesting that these origins are also subject to sequestration. The overproduction of SeqA led to a loss of viability, the condensation of DNA, and a filamentous morphology. Cells with abnormal DNA content arose in the population, and replication was inhibited as determined by a reduced ratio of origin to terminus DNA in SeqA-overexpressing cells. Thus, excessive SeqA negatively affects replication in V. cholerae and prevents correct progression to downstream cell cycle events such as segregation and cell division.     

Segregation of Induced Frameshift Mutations and the Sequence of Gene Replication in ESCHERICHIA COLI K12

We have constructed a strain of E. coli K12 carrying six mutations induced by the acridine half-mustard ICR-191. The mutations are widely spaced on the E. coli linkage map and are all easily reverted by ICR-191. Mapping of ten independent revertants for each of five markers indicated that the reversions induced by ICR-191 occurred near the original mutations. Exponentially and nonsynchronously growing cultures of this strain were exposed to ICR-191 for 0.85 generation, quickly washed free of mutagen, and resuspended in the original medium minus mutagen. Total viable cell number maintained its exponential increase both during and immediately after exposure to mutagen, whereas the number of revertants of any particular type remained constant for a characteristic period after removal of mutagen before finally assuming an exponential increase. Theoretically, the length of such a segregation lag should depend on the position of the particular reverted gene in the sequence of gene replication: the earlier a gene is replicated in the chromosome replication cycle, the longer its segregation lag should be. Our results are consistent with this prediction and fit a unidirectional, clockwise replication scheme with an origin between 55 and 74 min on the E. coli linkage map. The results also fit a very asymmetric bidirectional replication scheme.     

Tracking of Chromosome and Replisome Dynamics in Myxococcus xanthus Reveals a Novel Chromosome Arrangement

Cells closely coordinate cell division with chromosome replication and segregation; however, the mechanisms responsible for this coordination still remain largely unknown. Here, we analyzed the spatial arrangement and temporal dynamics of the 9.1 Mb circular chromosome in the rod-shaped cells of Myxococcus xanthus. For chromosome segregation, M. xanthus uses a parABS system, which is essential, and lack of ParB results in chromosome segregation defects as well as cell divisions over nucleoids and the formation of anucleate cells. From the determination of the dynamic subcellular location of six genetic loci, we conclude that in newborn cells ori, as monitored following the ParB/parS complex, and ter regions are localized in the subpolar regions of the old and new cell pole, respectively and each separated from the nearest pole by approximately 1 µm. The bulk of the chromosome is arranged between the two subpolar regions, thus leaving the two large subpolar regions devoid of DNA. Upon replication, one ori region remains in the original subpolar region while the second copy segregates unidirectionally to the opposite subpolar region followed by the rest of the chromosome. In parallel, the ter region of the mother chromosome relocates, most likely passively, to midcell, where it is replicated. Consequently, after completion of replication and segregation, the two chromosomes show an ori-ter-ter-ori arrangement with mirror symmetry about a transverse axis at midcell. Upon completion of segregation of the ParB/parS complex, ParA localizes in large patches in the DNA-free subpolar regions. Using an Ssb-YFP fusion as a proxy for replisome localization, we observed that the two replisomes track independently of each other from a subpolar region towards ter. We conclude that M. xanthus chromosome arrangement and dynamics combine features from previously described systems with new features leading to a novel spatiotemporal arrangement pattern.     

The effect on chromosome stability of deleting replication origins.

The observed spacing between chromosomal DNA replication origins in Saccharomyces cerevisiae is at least four times shorter than should be necessary to ensure complete replication of chromosomal DNA during the S phase. To test whether all replication origins are required for normal chromosome stability, the loss rates of derivatives of chromosome III from which one or more origins had been deleted were measured. In the case of a 61-kb circular derivative of the chromosome that has two highly active origins and one origin that initiates only 10 to 20% of the time, deletion of either highly active origin increased its rate of loss two- to fourfold. Deletion of both highly active origins caused the ring chromosome to be lost in approximately 20% of cell divisions. This very high rate of loss demonstrates that there are no efficient cryptic origins on the ring chromosome that are capable of ensuring its replication in the absence of the origins that are normally used. Deletion of the same two origins from the full-length chromosome III, which contains more than six replication origins, had no effect on its rate of loss. These results suggest that the increase in the rate of loss of the small circular chromosome from which a single highly active origin was deleted was caused by the failure of the remaining highly active origin to initiate replication in a small fraction (approximately 0.003) of cell cycles.     

The Escherichia coli cell cycle

This review summarizes present knowledge of the bacterial cell cycle with particular emphasis on Escherichia coli. We discuss data coming from three different types of approaches to the study of cell extension and division: The search for discrete events occurring once per division cycle. It is generally agreed that the initiation and termination of DNA replication and cell septation are discrete events; there is less agreement on the sudden doubling in rate of cell surface extension, murein biosynthesis and the synthesis of membrane proteins and phospholipids. We discuss what is known about the temporal relationship amongst the various cyclic events studied. The search for discrete growth zones in the cell envelope layers. We discuss conflicting reports on the existence of murein growth zones and protein insertion sites in the inner and outer membranes. Elucidation of the mechanism regulating the initiation of DNA replication. The concept of "critical initiation mass" is examined. We review data suggesting that the DNA is attached to the envelope and discuss the role of the latter in the initiation of DNA replication.     

Dimensions of Escherichia coli at various growth rates: model for envelope growth.

The duplication of Escherichia coli B/r is described based on two independent sequences, the replication of the genome and the growth of the envelope. It is proposed that (i) new envelope growth zones are activated coincident with the initiation of new rounds of chromosome replication; (ii) each zone is active in envelope synthesis from the time of its inauguration to the division which follows the completion of the round of chromosome replication (that is, for C + D min); and (iii) the rate of envelope synthesis at each site is constant, independent of the growth rate. Measurements of the surface areas of two E. coli B/r substrains growing at a variety of rates and during nutritional transitions are consistent with the predictions of the model.     

Mechanism for chromosome and minichromosome segregation in Escherichia coli

A mechanism for the segregation of chromosomes and minichromosomes into daughter cells during division of Escherichia coli is presented. It is based on the idea that the cell envelope contains a large number of sites capable of binding to the chromosomal replication origin, oriC, and that a polymerizing DNA strand becomes attached to one of the sites at initiation of a round of replication. The attachment sites are distributed throughout the actively growing cell envelope, i.e. lateral envelope and septum, but not in the existing cell poles. This asymmetric distribution of oriC attachment sites accounts for the experimentally observed non-random chromosome and minichromosome segregation, and for the variation in the degree of non-random segregation with cell strain and growth rate. The multi-site attachment concept also accounts for the unstable maintenance of minichromosomes.     

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.     

Termination of Bacillus subtilis chromosome replication as visualized by autoradiography

A mutant of Bacillus subtilis W23 thy his, temperature sensitive for the initiation of rounds of chromosome replication, has been used to investigate the manner in which the two growing points on the replicating circular chromosome approach one another during termination (completion) of the round in progress.The mutant, growing exponentially at 34 °C, was transferred to 45 °C and [³H]thymidine added shortly afterwards. After a further short interval, the specific radioactivity was lowered by a factor of three and rounds of replication were allowed to complete under these conditions. The pattern of heavy and light grain density regions in chromosomal structures made visible by autoradiography was examined, and many with both ends more heavily labelled than their internal region were found. From an analysis of the relative frequency and size of these structures it is concluded that the two growing points in a single chromosome can continue to move at similar rates (within a factor of 2) as they approach one another to within < 10% of the length of the whole circular chromosome in the vicinity of the terminus. The data obtained do not prove that all chromosomes behave in this manner, but are consistent with such an interpretation.