Biomedical subjects

Kurt Nordström

Publications and source records attributed to Kurt Nordström.

11 recordsLinked to original sources

PubMed2006

Copy-number control of the Escherichia coli chromosome: a plasmidologist's view.

The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number.

Cell Cycle↗

PubMed2005

Plasmid R1--replication and its control.

Plasmid R1 is a low-copy-number plasmid belonging to the IncFII group. The genetics, biochemistry, molecular biology, and physiology of R1 replication and its control are summarised and discussed in the present communication. Replication of R1 starts at a unique origin, oriR1, and proceeds unidirectionally according to the Theta mode. Plasmid R1 replicates during the entire cell cycle and the R1 copies in the cell are members of a pool from which a plasmid copy at random is selected for replication. However, there is an eclipse period during which a newly replicated copy does not belong to this pool. Replication of R1 is controlled by an antisense RNA, CopA, that is unstable and formed constitutively; hence, its concentration is a measure of the concentration of the plasmid. CopA-RNA interacts with its complementary target, CopT-RNA, that is located upstream of the RepA message on the repA-mRNA. CopA-RNA post-transcriptionally inhibits translation of the repA-mRNA. CopA- and CopT-RNA interact in a bimolecular reaction which results in an inverse proportionality between the relative rate of replication (replications per plasmid copy and cell cycle) and the copy number; the number of replications per cell and cell cycle, n, is independent of the actual copy number in the individual cells, the so-called +n mode of control. Single base-pair substitutions in the copA/copT region of the plasmid genome may result in mutants that are compatible with the wild type. Loss of CopA activity results in (uncontrolled) so-called runaway replication, which is lethal to the host but useful for the production of proteins from cloned genes. Plasmid R1 also has an ancillary control system, CopB, that derepresses the synthesis of repA-mRNA in cells that happen to contain lower than normal number of copies. Plasmid R1, as other plasmids, form clusters in the cell and plasmid replication is assumed to take place in the centre of the cells; this requires traffic from the cluster to the replication factories and back to the clusters. The clusters are plasmid-specific and presumably based on sequence homology.

Animals↗

PubMed2004

Bacterial mitotic machineries.

Here, we review recent progress that yields fundamental new insight into the molecular mechanisms behind plasmid and chromosome segregation in prokaryotic cells. In particular, we describe how prokaryotic actin homologs form mitotic machineries that segregate DNA before cell division. Thus, the ParM protein of plasmid R1 forms F actin-like filaments that separate and move plasmid DNA from mid-cell to the cell poles. Evidence from three different laboratories indicate that the morphogenetic MreB protein may be involved in segregation of the bacterial chromosome.

Actin Cytoskeleton↗

PubMed2004

Effect of the CopB auxiliary replication control system on stability of maintenance of Par(+) plasmid R1.

Plasmid R1 is a low-copy-number plasmid that is present at a level of about four or five copies per average cell. The copy number is controlled posttranscriptionally at the level of synthesis of the rate-limiting initiator protein RepA. In addition to this, R1 has an auxiliary system that derepresses a second promoter at low copy numbers, leading to increased repA mRNA synthesis. This promoter is normally switched off by a constitutively synthesized plasmid-encoded repressor protein, CopB; in cells with low copy numbers, the concentration of CopB is low and the promoter is derepressed. Here we show that the rate of loss of a Par(+) derivative of the basic replicon of R1 increased about sevenfold when the cells contained a high concentration of the CopB protein formed from a compatible plasmid.

DNA Helicases↗

PubMed2003

Eclipse-synchrony relationship in Escherichia coli strains with mutations affecting sequestration, initiation of replication and superhelicity of the bacterial chromosome.

Initiation of replication from oriC on the Escherichia coli chromosomes occurs once and only once per generation at the same cell mass per origin. During rapid growth there are overlapping replication cycles, and initiation occurs synchronously at two or more copies of oriC. Since the bacterial growth can vary over a wide range (from three divisions per hour to 2.5 hours or more per division) the frequency of initiation should change in coordination with bacterial growth. Prevention of reinitiation from a newly replicated origin by temporary sequestration of the hemi-methylated GATC-sites in the origin region provides the molecular/genetic basis for the maintenance of the eclipse period between two successive rounds of replication. Sequestration is also believed to be responsible for initiation synchrony, since inactivation of either the seqA or the dam gene abolishes synchrony while drastically reducing the eclipse. In this work, we attempted to examine the functional relationship(s) between the eclipse period and the synchrony of initiation in E.coli strains by direct measurements of these parameters by density-shift centrifugation and flow-cytometric analyses, respectively. The eclipse period, measured as a fraction of DNA-duplication times, varied continuously from 0.6 for the wild-type E.coli K12 to 0.1 for strains with mutations in seqA, dam, dnaA, topA and gyr genes (all of which have been shown to cause asynchrony) and their various combinations. The asynchrony index, a quantitative indicator for the loss of synchrony of initiation, changed from low (synchronous) to high (asynchronous) values in a step-function-like relationship with the eclipse. An eclipse period of approximately 0.5 generation time appeared to be the critical value for the switch from synchronous to asynchronous initiation.

Chromosomes, Bacterial↗

PubMed2003

The replicon theory 40 years: an EMBO workshop held in Villefranche sur Mer, France, January 18-23, 2003.

It is now 40 years since Jacob, Brenner, and Cuzin presented their Replicon Theory at a Cold Spring Harbor Symposium. The theory was based on their fundamental studies of the sexual system of Escherichia coli which led to the realisation that only specific sequences are able to replicate. They introduced the concept of a replicon consisting of a replicator (a DNA sequence) and a structural gene for an initiator protein. They also proposed a model for how replication of the bacterial chromosome might fit into the bacterial cell cycle. To commemorate the anniversary, an EMBO Workshop was organised in Villefranche on the Riviera of France. During the Workshop, the state of the art of cell-cycle studies of prokaryotic and eukaryotic organisms was presented and discussed in the presence of two of the fathers of the Replicon Theory, Jacob and Cuzin.

Animals↗

PubMed2003

Clustering versus random segregation of plasmids lacking a partitioning function: a plasmid paradox?

Plasmids lacking a functional partition system are randomly distributed to the daughter cells; plasmid-free daughter cells are formed with a frequency of (1/2)2n per cell and cell generation where 2n is the (average) copy number at cell division. Hence, the unit of segregation is one plasmid copy. However, plasmids form clusters in the cells. A putative solution to this potential paradox is presented: one plasmid copy at a time is recruited from the plasmid clusters to the replication factories that are located in the cell centres. Hence, replication offers the means of declustering that is necessary in a growing host population. The daughter copies diffuse freely and each copy may with equal probability end up in either of the two cell halves. In this way, the random segregation of the plasmids is coupled to replication and occurs continuously during the cell cycle, and is not linked to cell division. The unit of segregation is the plasmid copy and not the plasmid clusters. In contrast, the two daughters of a Par+ plasmid are directed in opposite directions by the plasmid-encoded partition system, thereby assuring that each daughter cell receives the plasmid.

Bacterial Proteins↗

PubMed2003

Coupling the cell cycle to cell growth.

In order to multiply, both prokaryotic and eukaryotic cells go through a series of events that are collectively called the cell cycle. However, DNA replication, mitosis and cell division may also be viewed as having their own, in principle independent, cycles, which are tied together by mechanisms extrinsic to the cell cycle--the checkpoints--that maintain the order of events. We propose that our understanding of cell-cycle regulation is enhanced by viewing each event individually, as an independently regulated process. The nature of the parameters that regulate cell-cycle events is discussed and, in particular, we argue that cell mass is not such a parameter.

Cell Culture Techniques↗

PubMed2003

Eclipse period during replication of plasmid R1: contributions from structural events and from the copy-number control system.

The eclipse period (the time period during which a newly replicated plasmid copy is not available for a new replication) of plasmid R1 in Escherichia coli was determined with the classic Meselson-Stahl density-shift experiment. A mini-plasmid with the wild-type R1 replicon and a mutant with a thermo-inducible runaway-replication phenotype were used in this work. The eclipses of the chromosome and of the wild-type plasmid were 0.6 and 0.2 generation times, respectively, at temperatures ranging from 30 degrees C to 42 degrees C. The mutant plasmid had a similar eclipse at temperatures up to 38 degrees C. At 42 degrees C, the plasmid copy number increased rapidly because of the absence of replication control and replication reached a rate of 350-400 plasmid replications per cell and cell generation. During uncontrolled replication, the eclipse was about 3 min compared with 10 min at controlled replication (the wild-type plasmid at 42 degrees C). Hence, the copy-number control system contributed significantly to the eclipse. The eclipse in the absence of copy-number control (3 min) presumably is caused by structural requirements: the covalently closed circular plasmid DNA has to regain the right degree of superhelicity needed for initiation of replication and it takes time to assemble the initiation factors.

Centrifugation, Density Gradient↗

PubMed2002

Eclipse period without sequestration in Escherichia coli.

The classical Meselson-Stahl density shift experiment was used to determine the length of the eclipse period in Escherichia coli, the minimum time period during which no new initiation is allowed from a newly replicated origin of chromosome replication, oriC. Populations of bacteria growing exponentially in heavy ((15)NH(4)+ and (13)C(6)-glucose) medium were shifted to light ((14)NH(4)+ and (12)C(6)-glucose) medium. The HH-, HL- and LL-DNA were separated by CsCl density gradient centrifugation, and their relative amounts were determined using radioactive gene-specific probes. The eclipse period, estimated from the kinetics of conversion of HH-DNA to HL- and LL-DNA, turned out to be 0.60 generation times for the wild-type strain. This was invariable for widely varying doubling times (35, 68 and 112 min) and was independent of the chromosome locus at which the eclipse period was measured. For strains with seqA, dam and damseqA mutants, the length of the eclipse period was 0.16, 0.40 and 0.32 generation times respectively. Thus, initiations from oriC were repressed for a considerable proportion of the generation time even when the sequestration function seemed to be severely compromised. The causal relationship between the length of the eclipse period and the synchrony of initiations from oriC is discussed.

Bacterial Outer Membrane Proteins↗

PubMed2002

Effects of the Min system on nucleoid segregation in Escherichia coli.

The Min system of Escherichia coli directs cell division to the mid-cell by a mechanism that involves the dynamic localization of all of its three constituent proteins, MinC, MinD and MinE. Both the Min system and the nucleoid regulate cell division negatively and strains of E. coli lacking a functional Min system can divide at nucleoid-free cell poles in addition to the nucleoid-free region between newly segregated nucleoids. Interestingly, E. coli strains with a defective Min system have disturbed nucleoid segregation and the cause for this disturbance is not known. It is reported here that growth conditions promoting a higher frequency of polar divisions also lead to a more pronounced disturbance in nucleoid segregation. In strains with an intact Min system, expression of MinE, but not of MinD, from an inducible promoter was followed by impaired nucleoid segregation. These results suggest that the disturbed nucleoid segregation in min mutants is not caused by polar divisions per se, nor by impaired resolution of chromosome dimers in min mutants, leaving open the possibility that the Min system has a direct effect on nucleoid segregation. It is also shown how the disturbed nucleoid segregation can explain in part the unexpected finding that the clear majority of cells in min mutant populations contain 2(n) (n=0, 1, 2.) origins of replication.

Adenosine Triphosphatases↗