Biomedical subjects

R Bernander

Publications and source records attributed to R Bernander.

10 recordsLinked to original sources

PubMed1992

Mapping of the in vivo start site for leading strand DNA synthesis in plasmid R1.

We have previously constructed Escherichia coli strains in which an R1 plasmid is integrated into the origin of chromosome replication, oriC. In such intR1 strains, oriC is inactive and initiation of chromosome replication instead takes place at the integrated R1 origin. Due to the large size of the chromosome, replication intermediates generated at the R1 origin in these strains are considerably more long-lived than those in unintegrated R1 plasmids. We have taken advantage of this and performed primer extensions on total DNA isolated from intR1 strains, and mapped the free 5' DNA ends that were generated as replication intermediates during R1 replication in vivo. The sensitivity of the mapping was considerably improved by the use of a repeated primer extension method (RPE). The free DNA ends were assumed to represent normal in vivo start sites for leading strand DNA synthesis in plasmid R1. The ends were mapped to a short region approximately 380 bp away from the R1 minimal origin, and the positions agreed well with previous in vitro mappings. The same start positions were also utilized in the absence of the DnaA protein, indicating that DnaA is not required for determination of the position at which DNA synthesis starts during initiation of replication at the R1 origin.

Bacterial Proteins↗

PubMed1992

Cell division in Escherichia coli minB mutants.

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.

Cell Cycle↗

PubMed1992

Direct visualization of plasmid DNA in bacterial cells.

The direct visualization of plasmid DNA inside Escherichia coli cells is demonstrated using phase-fluorescence microscopy of DAPI (4',6-diamidino-2-phenylindole)-stained bacteria. Small as well as large plasmids could be detected, both in minicells and in cells of larger size. For large plasmids, even single molecules appeared to be within the detection limit. The fluorescence generated from monomers of small plasmids was probably below this limit, and for these plasmids the observed signals may represent aggregates. The distribution of the fluorescence foci might reflect specific plasmid positioning during partition and/or replication.

Cell Division↗

PubMed1991

The E. coli cell cycle and the plasmid R1 replication cycle in the absence of the DnaA protein.

In E. coli strain EC::71CW chromosome replication is under the control of the R1 miniplasmid pOU71. A dnaA850::Tn10 derivative of EC::71CW was viable, which confirmed that R1 can replicate in the absence of the DnaA protein. The frequency of initiation of replication was, however, lowered and cell division was severely disturbed due to underreplication of the chromosome. Both replication and cell division could be restored to normal by increasing the production of RepA, the rate-limiting protein for initiation of replication from the integrated R1 origin. Therefore, the RepA protein seems to compensate for the absence of DnaA in the initiation of replication and assembly of replisomes. The role of the DnaA protein in the initiation of DNA replication, and as an overall regulator of the chromosome replication and cell division cycles of E. coli, is discussed in view of these results.

Bacterial Proteins↗

PubMed1991

The Escherichia coli cell cycle: one cycle or multiple independent processes that are co-ordinated?

In the life cycle of a bacterium there are several key processes: cellular growth, chromosome replication and decatenation, nucleoid partition, septum formation, and cell division. These processes have to be carefully controlled and co-ordinated both with respect to each other and to the growth of the cell, and could be viewed as parts of a single cycle in which each step is dependent upon the previous one. Alternatively, they could be independently controlled and carefully tuned to each other without actually constituting a true cycle. In this review, using Escherichia coli as model system, we discuss these two ways of describing the bacterial life cycle. The evidence supporting independent control of the processes is presented, and some of the key questions in the elucidation of the regulation of the bacterial life cycle are discussed.

Biological Evolution↗

PubMed1990

Chromosome replication does not trigger cell division in E. coli.

An essential part of the chromosome replication origin of E. coli K-12 and B/r was replaced by the plasmid pOU71. The average initiation mass of replication for pOU71 decreases with increasing temperature. The constructed strains were grown exponentially at different temperatures, and cell sizes and DNA content were measured by flow cytometry. The average DNA content increased with increasing temperature, but the cell size distribution was largely unaffected. Furthermore, cells in which DNA replication had not yet initiated (cells in the B period) became less abundant with increasing temperature. The increased DNA content could not be explained by an increase in the length of the C period. It is concluded that chromosome replication does not trigger cell division in E. coli, but that the chromosome replication and cell division cycles of E. coli run in parallel independently of each other.

Cell Cycle↗

PubMed1989

Overinitiation of replication of the Escherichia coli chromosome from an integrated runaway-replication derivative of plasmid R1.

A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFII plasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfold increase in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution was broader in the constructed strains relative to that in the wild type because of asynchrony between the chromosome replication and cell division cycles. This difference was more pronounced for counterclockwise initiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant. Following a temperature shift the cell size distributions became even more broad, showing that changes in the frequency of chromosomal replication affect cell division and emphasizing the interplay between these two processes.

Chromosomes, Bacterial↗

PubMed1987

Direct selection for the exchange of alleles between a plasmid and the Escherichia coli chromosome.

Recombination is extensively used in order to move alleles between replicons. The exchange of wild-type chromosomal and mutant plasmid-borne alleles is a two-step process entailing the formation of a cointegrate between the entire plasmid and the chromosome, followed by resolution of such cointegrates to give a mutant chromosome and a plasmid carrying the wild-type chromosomal sequence. Often the cointegrate and the resolved forms cannot be distinguished phenotypically. To enable the direct isolation of the resolved products we have developed a positive selection technique. Cells containing a cointegrated plasmid R1 were constructed by transduction using a P1 lysate prepared from cells harbouring a plasmid comprising a mutant chromosomal allele and the so-called omega fragment which carries an aad (aminoglycoside adenylyltransferase) gene. P1 transduction from the cointegrate strain into an SmD recipient allowed direct selection for the resolved complex, since transduction of the aad gene is lethal to an SmD strain.

Alleles↗

PubMedDate not supplied

In vivo effect of the tus mutation on cell division in an Escherichia coli strain where chromosome replication is under the control of plasmid R1.

The phenotypic effect of the tus::kan mutation in an Escherichia coli strain, where the chromosome is replicated unidirectionally by an integrated R1 miniplasmid, was examined by flow cytometry and phase fluorescence microscopy. The tus+ cells exhibited perturbed cell division, as indicated by the presence of many elongated cells and filaments. Inactivation of the tus gene led to a reduction in the frequency of such elongated cells, presumably by eliminating Tus-mediated polar arrests of replication forks at ter sites, thereby shortening the time required for completion of chromosome replication.

Bacterial Proteins↗

PubMedDate not supplied

Analysis of the bacterial cell cycle using strains in which chromosome replication is controlled by plasmid R1.

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.

Cell Cycle↗