Jacqueline Louarn

Origin and sequence of chromosome replication in Escherichia coli

Two methods have been used to determine the origin and direction of chromosome replication in Escherichia coli: gradient of marker frequency and sequence of replication in synchronized cultures. In both cases, DNA-DNA hybridization was used to assay for gene dosage. A series of isogenic strains were made lysogenic for phage λ and for phage Mu-1, with phage Mu-1 in a different chromosomal location in each strain. In a first group of experiments, DNA from exponential cultures of the various strains was extracted, denatured, immobilized on filters and hybridized against a mixture of differentially labeled phage λ and phage Mu-1 DNA. This was done for several culture conditions. The ratio of hybridization Mu-1/λ gives a measurement of the dosage of the chromosome region where phage Mu-1 is integrated. A plot of this ratio versus map position reflects the marker frequency distribution. In another group of experiments, several of the strains were synchronized by amino-acid starvation, then restitution of essential amino acids. The cultures thus synchronized were density labeled with bromouracil and their DNA extracted at various times, separated by density on a CsCl gradient, and the amount of phage Mu-1 specific DNA present in each band was measured by DNA-DNA hybridization. The following conclusions have been reached. 1. (1) The origin of replication in near ilv, at 74 minutes on the standard genetic map of Escherichia coli. 2. (2) Replication proceeds simultaneously in the two opposite directions, at approximately the same velocity in both directions. 3. (3) This velocity is influenced, in a thymineless mutant, by the concentration of exogenous thymine. 4. (4) The two growing forks meet at a point diametrically opposed to the origin, near trp (25 min).

Characterization and properties of very large inversions of the E. coli chromosome along the origin-to-terminus axis

SummarySuppression of a dnaA46 mutation by integration of plasmid R100.1 derivatives in the termination region of chromosome replication in E. coli results in medium dependence, the suppressed bacteria being sensitive to rich medium at 42° C. Derivatives of such bacteria have been selected for growth at 42° C in rich medium and we have analyzed representatives of the most frequently observed type: bacteria displaying, once cured of the suppressor plasmid, both rich-medium sensitivity and temperature sensitivity. We found, in all cases, that the chromosome had undergone a major inversion event between two inverted IS5s. One is located at 29.2 min on the chromosome map and the other at either one of two positions between 69 and 80 min. The consequences of such inversions for cell growth are discussed. Some of them result from the fact that the replication terminator T2 is located, in inverted chromosomes, close to oriC in the orientation which allows its functioning as a terminus (de Massy et al. in press). Our observations allow an estimation of the frequency of inversions arising from recombination between pairs of inverted chromosomal IS, which could be as high as 10-2 per cell per generation. We also found that inversion reversal occurs frequently after Hfr conjugational transfer of one of the IS5s, in its wild-type location. This led us to propose a new mechanisms of recombination, in which the incoming DNA strands serve as guides to favor recombination between the resident sequences.

Evidence for a fixed termination site of chromosome replication in Escherichia coli K12

We have investigated the possibility of a fixed terminus for bidirectional replication in Escherichia coli by determining whether a displacement of the chromosome replication origin results in an inversion of the direction of replication for markers located in the region where termination normally occurs. Three prophages have been used to mark four chromosomal sites: Mu-1, integrated in either malA (74 min) or malB (90 min); P2 in location H (43 min) and φ80 (27 min). Integrative suppression, promoted by a resistance transfer factor, resulted in origin displacements greater than 20 minutes in each direction. In the parental strains and in their integratively suppressed derivatives we have established, for each prophage: (a) the direction of replication (by hybridizing labelled Okazaki fragments to separated phage strands); (b) the relative frequency, in the exponential phase of growth (by DNA-DNA hybridization of long-term labelled DNA to denatured phage DNA). The following conclusions have been reached. (1) In conditions of integrative suppression, chromosome replication is bidirectional, starting from the inserted episome. (2) The direction of replication of each of the two prophages, P2 and φ80, is invariant in the termination region. (3) Marker frequency analysis has revealed that P2 prophage and φ80 prophage are on two different replication units. These results suggest that replication forks, travelling in either direction, must stop at a site located between 27 and 43 minutes on the genetic map, presumably the terminus of replication (tre).

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli

SummaryA strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30° C and 42° C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the Δ(topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.

Map position of the replication terminus on the Escherichia coli chromosome

SummaryThe directions of replication of several prophages integrated with a known orientation in the vicinity of the terminus (tre) of chromosome replication (trp::Mu, min 27; λrev integrated within rac, min 31, man::Mu, min 35), have been established by determining the molecular polarity of Okazaki pieces specific to these prophages. The results obtained strongly suggest that the site tre is located between rac and man, an otherwise genetically silent region.

Constraints in chromosomal inversions in Escherichia coli are not explained by replication pausing at inverted terminator-like sequences.

Regions close to the replication terminus of the Escherichia coli chromosome are strongly refractory to genomic inversions (Rebollo et al., 1988). Since these regions also harbour polar replication terminator‐like sequences or pause sites (François et al., 1989), we have investigated the possibility that slowing of replication as a result of pausing at inverted pause sites is responsible for inability to isolate stable inversions affecting these regions. A mutation in the tus gene is known to abolish replication pausing at terminators (Hill et al., 1988). We show here that the distribution of invertible and noninvertible segments along the chromosome is not affected by tus mutations. This observation eliminates replication pausing as a cause for the reduced fitness of bacteria harbouring certain chromosomal inversions.

Chromosome replication pattern in dam mutants of Escherichia coli

SummaryThe replication cycle of Escherichia coli dam mutants was analysed and compared with that of isogenic Dam+ strains. Marker frequency analyses indicated no gross difference between the strains. In the Dam− as well as in the Dam+ bacteria, initiation most likely occurs at oriC, replication forks move at a constant and invariant velocity, and termination takes place in the terC region. An analysis of replication terminator activity indicated that this activity is unaffected by the methylation status. Taken together with previous results, our data are compatible with Dam methylation controlling initiation timing but no subsequent step of the replication process.

The Terminus of Chromosome Replication of E. coli Phenotypic Suppression of a dnaA Mutation by Plasmid Integration near terC

The existence of a fixed termination region for chromosome replication in E. coli has been proposed1,2,3,4. In particular, we have shown that when a dnaAts mutation is phenotypically suppressed by an integrated plasmid (Integrative Suppression5), the replication forks initiated from the plasmid always meet in the rac (min 30)-man (min 35.5) region, irrespective of the plasmid insertion site on the chromosome. The terminus of replication, terC, was thus described primarily as a locus inhibiting replication fork movement in either direction. In addition, the termination step might be involved in regulatory operations of the cell cycle, as previously proposed6,7, but this possibility remains poorly documented. In the course of our previous analyses, as well as in other studies on integrative suppression by plasmid R100 derivatives8, integrative suppression by plasmid integration in a large region surrounding terC (grossly between 15 min and 45 min on the genetic map of Bachmann et al9 was never observed. If the restriction in the distribution of integration sites along the chromosome is related to terC functions, its analysis could constitute a way to investigate the role of the terminus.