Chromosome replication and cell division in plasmid-containing Escherichia coli B/r.

The kinetics of chromosome replication and cell division have been examined in recA mutants of Escherichia coli B/r containing F' plasmids of various sizes. Plasmid-mediated alterations in growth properties were detected only with the presence of the larger F' plasmids, and were reflected in decreased mean cell sizes and growth rates. The lengths of C and D in all plasmid-containing strains were in accord with the values for plasmid-free parental strains growing with similar generations times. The findings were consistent with an absence of competition between the chromosomal and extrachromosomal replicons for rate-limiting components involved in the initiation of deoxyribonucleic acid synthesis or in the elongation of deoxyribonucleic acid chains.     

Length growth of two Escherichia coli B/r substrains.

Length growth of synchronized Escherichia coli B/r substrain A (ATCC 12407) and B/r substrain F26 (Thy his) was followed with an electron microscope. Cells were grown with doubling times (tau) of 60 min (B/rA) and of 82 and 165 min (B/rF26). Different length growth patterns were found for the two substrains. In B/rF, the length growth rate increased about midway in the cell cycle. For tau = 165 min, the rate increase was preceded by a short period of slow growth. For B/r A (r = 60 min), this period seemed to occur at the beginning of the cell cycle. The possibility is raised that the different length growth patterns are related to different deoxyribonucleic acid replication patterns of the respective strains.     

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.     

Chromosome Replication and the Division Cycle of Escherichia coli B/r

The average amount of deoxyribonucleic acid (DNA) per cell was measured in steady-state cultures of Escherichia coli B/r grown at 37 C in glucose-limited chemostats or in batch cultures in the exponential growth phase as maintained with one of several carbon sources. Within experimental errors, DNA content was dependent only on growth rate and independent of the type of culture, the carbon source, or the addition of growth factors. The amount of DNA per cell increased continuously with growth rate over the range of 0.02 to 3 divisions per hour. The data over the entire range of growth rates are in agreement with a constant time for a single replication point to traverse the entire genome, 47 min, and with cell division following 25 min after termination of replication. The measured amount of DNA per genome was 4.2 x 10(-15) g (or 2.5 x 10(9) daltons).     

Initiation and Velocity of Chromosome Replication in Escherichia coli B/r and K-12

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common Escherichia coli strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (C period) were determined. For these two strains, the initiation mass approached values of 8 × 10⁻¹⁰ and 10 × 10⁻¹⁰ units of optical density (at 460 nm) of culture mass per oriC, respectively, at growth rates above 1 doubling/h (at 37°C). The amount of protein per oriC decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The C period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and C period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.     

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.     

Accumulation of carbohydrate by Escherichia coli B/r/1 during growth at low water activity

The carbohydrate content of Escherichia coli B/r/1, grown in a glucose or arabinose-limited salts medium in a chemostat, increased by a factor of 2–4 when the water activity (a_w) of the medium was reduced to 0.986 by addition of NaCl, KCl or sucrose. The biomass decreased by 30–45%. The sucrose system resulted in the lowest biomass and carbohydrate content. The monosaccharide part of the accumulated carbohydrate consisted of glucose or glucose and arabinose in the cultures fed glucose and arabinose, respectively, and accounted for 50% or more of the total carbohydrate in the NaCl and KCl systems and 16.79% in the sucrose system. In addition, the K⁺ content depended on the solute and related inversely to the monosaccharide content, being highest in the sucrose system. The combined molarity of the monosaccharide and K⁺ was deduced to be far in excess of that required for osmotic equilibration of the cultures, especially in the sucrose system. These observations are discussed in the context of osmoregulation, the effects of solutes on glucose metabolism and the morphological changes that occur in cultures at low a_w.     

Polypeptide-chain-elongation rate in Escherichia coli B/r as a function of growth rate.

By evaluating the kinetics of radioactive labelling of nascent and finished polypeptides, the peptide-chain elongation rate for Escherichia coli B/r at three different growth rates (mu) was determined to be 17 amino acids/s for the fast-growing cells (mu equals 1.3 and 2.0 doublings/h) and 12 amino acids/s for slow-growing cells (mu equals 0.67 doublings/h). The results agree with the growth-rate-dependence of the rate of peptide-chain elongation found for the translation of newly induced beta-galactosidase messenger in this strain and under these conditions of growth [Dalbow & Young (1975) Biochem. J. 150, 13-20]. Together with the previously observed ribosome efficiency at these growth rates [Dennis & Bremer (1974) J. Mol. Biol. 84, 407-422] the results indicate that the fraction of ribosomes engaged in protein synthesis is about 0.8 at all three growth rates.     

Kinetics of minichromosome replication in Escherichia coli B/r.

Replication control of the minichromosome pAL2 was found to differ from that of the chromosome in synchronously dividing populations of Escherichia coli B/r. Initiation of minichromosome replication took place at an increasing rate throughout synchronous growth. No coupling to initiation of chromosome replication was detected. Minichromosome replication was further examined in a dnaA5(Ts) temperature-sensitive initiation mutant. When cultures held at nonpermissive temperature (41 degrees C) for 60 min were shifted to permissive temperature (25 degrees C), initiation of both pAL2 and chromosome replication ensued in two waves spaced 25 to 35 min apart. Evidence is presented that minichromosomes terminate replication by passing slowly through a series of dimeric intermediate forms before reaching the closed circular monomeric form. The consequence of this slow passage as a rate-limiting step in the initiation reaction is discussed.     

The cell cycle in Escherichia coli B/r

Cell division and DNA synthesis were measured in synchronous cultures of E. coll B/r growing in glucose minimal medium at 37 °. The kinetic curves were analysed in order to find the variability of replication initiation, termination, and cell division events during the cell cycle. It is inferred that under the conditions used, cells begin to divide 17 min (D₀ = minimum D-period) after each termination of chromosome replication with a constant probability per unit of time (half-life = 4·5–6 min). This randomness produces an asymmetric frequency distribution of D-periods, similar but mirror-symmetric frequency distributions of initiation and termination periods, a symmetric, non-Gaussian distribution of interdivision intervals, and complex kinetic changes in the rate of DNA synthesis as a function of cell age. The results suggest that replication and division are precisely controlled with respect to mass accumulation, and the apparent variability of cell cycle events would only result from the use of the time of cell separation as a reference point for the definition of cell age rather than initiation or termination of replication.     

Sensitivity of Escherichia coli to cephaloridine at different growth rates.

Steady-state populations of Escherichia coli B/r were treated with cephaloridine at minimal inhibitory concentrations. The antibiotic sensitivity of the cells and the localization of spheroplast emergence along the cell surface were examined as a function of cell length and growth rate. In fast-growing populations (greater than 1 division per h) the sites of cephaloridine interaction occurred preferentially at the cell pole in the smaller cells and at the cell center in dividing cells. At decreasing growth rates the cells became more resistant to cephaloridine, and a gradual shift from the cell pole toward the cell center was observed for the sphere position. A similar growth rate-dependent change in localization was found for sucrose-induced plasmolysis vacuoles.     

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.     

Isolation of rel mutants of Escherichia coli B/r.

A method that relies on the biological effect of near-UV (340-nm) irradiation is described by which large numbers of independent rel mutants of Escherichia coli B/r may be rapidly isolated.     

Murein segregation in Escherichia coli.

Peptidoglycan (murein) segregation has been studied by means of a new labeling method. The method relies on the ability of Escherichia coli cells to incorporate D-Cys into macromolecular murein. The incorporation depends on a periplasmic amino acid exchange reaction. At low concentrations, D-Cys is innocuous to the cell. The distribution of modified murein in purified sacculi can be traced and visualized by immunodetection of the -SH groups by fluorescence and electron microscopy techniques. Analysis of murein segregation in wild-type and cell division mutant strains revealed that murein in polar caps is metabolically inert and is segregated in a conservative fashion. Elongation of the sacculus apparently occurs by diffuse insertion of precursors over the cylindrical part of the cell surface. At the initiation of cell division, there is a FtsZ-dependent localized activation of murein synthesis at the potential division sites. Penicillin-binding protein 3 and the products of the division genes ftsA and ftsQ are dispensable for the activation of division sites. As a consequence, under restrictive conditions ftsA,ftsI,or ftsQ mutants generate filamentous sacculi with rings of all-new murein at the positions where septa would otherwise develop.     

Studies on the biosynthesis of acylphosphatidylglycerol in Escherichia coli B and B/r.

The present study has demonstrated that one molecule of acylphosphatidylglycerol was synthesized from two molecules of phosphatidylgycerol by the transacylation reaction in which phosphatidylglycerol acted both as an acyl donor and an acceptor. Phosphatidylethanolamine was identified as an another acyl donor, participating in acylphosphatidylglycerol formation. These results are discussed in terms of a new pathway for the turnover of phosphatidylglycerol in Escherichia coli.     

Evolution of the D-ribose operon on Escherichia coli B/r.

The D-ribose operon (rbs) of Escherichia coli K-12 maps at 83 min and is inducible. The rbs operon of E. coli B/r maps at 2 min and is constitutive. Evidence is presented showing that a second inducible copy of the rbs operons is present in E. coli B/r mapping at 83 min. The data indicated that the duplication of the rbs operon represented a transposition of the 83-min region to 2 min. The identification of a second copy of the rbs operon in B/r and the determination of its inducibility were based on the reactivation, through mutagenesis, of inducible rbs expression, mapping by P1 transduction of the mutation site to 83 min, and merodiploid complementation analysis of the D-ribokinase expression in E. coli B/r. We also show that the rbs transposition to 2-min continued to generate transposable elements coding for the 1- to 2-min region of the chromosome and transposing onto extrachromosomal DNA target molecules such as pBR322.