Rifampicin-resistant initiation of chromosome replication from oriC in ihf mutants.

IHF (integration host factor) mutants exhibit asynchronous initiation of chromosome replication from oriC as determined from flow cytometric analysis of cultures where RNA synthesis was inhibited with rifampicin. However, the run-out kinetics of chromosome replication in ihf mutants shows that they continue to produce oriCs for some time in the absence of RNA synthesis resulting in a twofold increase in the oriC per mass ratio. An ihf dnaA double mutant did not exhibit this continued increase of the oriC per mass ratio. This indicates that ihf mutants can initiate replication from oriC in a rifampicin-resistant initiation mode but requires fully functional DnaA protein. The origin per mass ratio, determined by a quantitative Southern blotting technique, showed that the ihf mutants had an origin per mass ratio that was 60% of the wild type although it had a normal DnaA protein concentration. This shows that the initiation mass was substantially higher in the ihf mutants. The oriC per terminus ratio, which was also determined by Southern blotting, was very low in the ihf mutant, although it grew with the same doubling times as the wild-type strain. This indicates that cells lacking IHF replicate their chromosome(s) very fast.

Suggestions as to quantitative measurements of plasmid loss.

Suggestions are made for the estimation of plasmid loss rates in bacterial populations that multiply by binary fission. The plasmid loss rate is defined as the probability of a division of a plasmid-carrying individual giving birth to one plasmid-free and one plasmid-carrying daughter cell. The unit of time is consequently the interdivision time for plasmid-carrying individuals. The cautions that have to be taken when the population is cultivated in serial transfer (the usual stability experiment) are discussed.

SeqA limits DnaA activity in replication from oriC in Escherichia coli.

A mutant Escherichia coli that transforms minichromosomes with high efficiency in the absence of Dam methylation has been isolated and the mutation mapped to 16.25 min on the E. coli map. The mutant strain containing seqA2 is defective for growth in rich medium but not in minimal medium. A similar mutation in this gene, named seqA1, has also been isolated. Here we show that the product of the seqA gene, SeqA, normally acts as an inhibitor of chromosomal initiation. In the seqA2-containing mutant, the frequency of initiation increases by a factor of three. Introduction of the wild-type seqA gene on a low-copy plasmid suppresses the cold sensitivity of a dnaAcos mutant known to overinitiate at temperatures below 39 degrees C. In addition, the seqA2 mutation is a suppressor of several dnaA (Ts) alleles. The seqA2 mutant overinitiates replication from oriC and displays the asynchronous initiation phenotype. Also the seqA2 mutant has an elevated level of DnaA protein (twofold). The introduction of minichromosomes or a low-copy-number plasmid carrying five DnaA-boxes from the oriC region increases the growth rate of the seqA2 mutant in rich medium to the wild-type level, reduces overinitiation but does not restore synchrony. We propose that the role of SeqA is to limit the activity level of the E. coli regulator of chromosome initiation, DnaA.

The initiation cascade for chromosome replication in wild-type and Dam methyltransferase deficient Escherichia coli cells.

'Newborn' Escherichia coli B/r cells, obtained by membrane elution, were used to study the cell cycles of wild-type and Dam methyltransferase mutants. In wild-type cells, initiation of chromosome replication was synchronous and tightly controlled. In dam mutants, initiation was altered, but not random. We propose that this is due to the absence of an initiation cascade caused by liberated DnaA molecules, and that this cascade normally synchronizes initiation. The dam- cells contained mainly two, three or four replication origins, and this affected nucleoid partitioning as well as cell division. In cultures growing with a 50 min doubling time, a variety of cell cycles were present and half the origins were used every 25 min. Some cells had a 25 min interdivision time, whereas others had an interdivision time longer than the generation time. Partitioning of nucleoids containing unequal numbers of replication origins could also be readily observed by fluorescence microscopy in the dam mutant. Based upon these observations we propose that the dam mutant is also an initiation cascade mutant.

The level of supercoiling affects the regulation of DNA replication in Escherichia coli.

The chromosome of Escherichia coli is negatively supercoiled. This favours processes that unwind the two DNA strands, such as DNA replication. In this paper, we have investigated the effect of changed levels of overall chromosomal supercoiling on the initiation of DNA replication. Specifically, we have used flow cytometry to reveal effects on the synchrony of initiations of DNA replication in single cells. An increase in the level of supercoiling moderately reduced initiation synchrony. In contrast, decreased supercoiling led to pronounced asynchrony. We have excluded the possibility that this asynchrony is caused by changes in the level of the Dam methyltransferase or the DnaA protein. We suggest that the global level of supercoiling influences the topology of oriC and thereby the sequence of events leading to initiation of DNA replication in E. coli.

DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry.

We have investigated the initiation of DNA replication in Escherichia coli gyrB (Ts) mutants and find that initiations in single cells, even those that occur at non-permissive temperature, are synchronous. Furthermore, our results indicate that the gradual arrest of DNA replication at non-permissive temperature reflects a general decrease in transcription activity and not an initiation-specific function of the DNA gyrase. At an intermediate temperature, the only effect observed was a lack of segregation (decatenation) of replicated chromosomes in a sizeable fraction of the cell population.

Escherichia coli minichromosomes: random segregation and absence of copy number control.

Minichromosomes, i.e. plasmids that can replicate from an integrated oriC, have been puzzling because of their high copy numbers compared to that of the chromosomal oriC, their lack of incompatibility with the chromosome and their high loss frequencies. Using single cell resistance to tetracycline or ampicillin as an indicator of copy number we followed the development of minichromosome distributions in Escherichia coli cells transformed with minichromosomes and then allowed to grow towards the steady state. The final copy number distribution was not reached within 15 to 20 generations. If the minichromosome carried the sop (partitioning) genes from plasmid F, the development of the copy number distribution was further drastically delayed. We conclude that E. coli cells have no function that directly controls minichromosomal copy numbers, hence the absence of incompatibility in the sense of shared copy number control. We suggest that minichromosomes are subject to the same replication control as the chromosome but segregate randomly in the absence of integrated partitioning genes. This, combined with evidence that the lowest copy number classes are normally present despite high average copy numbers, can account for the high loss frequencies.

Stability and replication control of Escherichia coli minichromosomes.

A stabilized minichromosome--a plasmid replicating from the chromosomal origin oriC--was constructed by cloning the sopA,B,C, genes from plasmid F. This minichromosome had a loss frequency of less than 10(-3), while that of the nonstabilized parental plasmid was 2 X 10(-2) to 4 X 10(-2). Both minichromosomes had the same average copy number per chromosomal origin, and the copy numbers were constant over an eightfold range of growth rates. Different mutations in the mioC gene and promoter, from which transcription enters oriC, were constructed, and their effects on minichromosome copy number and stability were tested. The results indicated that normal replication control at oriC was independent of the MioC protein and most of the sequences between the promoter and oriC, but required both transcription from the mioC promoter and probably also the presence of the DnaA box (DnaA protein-binding site) just upstream of the mioC promoter. Transcription from the mioC promoter was shown to be efficiently repressed in vivo after overproduction of DnaA protein and to be derepressed at the nonpermissive temperature in six different dnaA(Ts) mutants.

Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli.

A temperature-sensitive mutant of Escherichia coli with a temperature-dependent change in the control of initiation of DNA replication was isolated. The phenotype of the mutant was dependent on a mutation in the RNA polymerase gene rpoC. In vitro RNA polymerase activity was temperature sensitive. The mutant grew and synthesized DNA at 30 degrees C as did the wild type. After a shift to 39 degrees C, a temperature still permissive for growth, the mutant increased its origin concentration more than twofold. After a shift from 39 to 30 degrees C, initiation of DNA replication was inhibited until the normal origin concentration was reestablished.

Regulation of the dnaA product in Escherichia coli.

When an E. coli mutant (CRT46, dnaA46), thermosensitive in the initiation of DNA replication, grows at intermediate temperatures its DNA/mass ratio is somewhat lower than normal, but the cells possess an excess of initiation capacity, which can be expressed in the absence of protein synthesis and lead to the accumulation of anomalously high amounts of DNA. A shift-up in temperature causes inhibition of initiation, and at the same time the production of initiation capacity is accelerated. After a shift-down in temperature initiation is released but the production of capacity is inhibited. The initiation capacity is thermolabile. The simplest explanation of these observations is that the dnaA product has a dual role: a positive function as an initiator of replication and a negative control function in its own synthesis.