recA Gene dependence of replication of the Escherichia coli chromosome initiated by plasmid pUC13 integrated at predetermined sites

Summary Plasmid pUC13 was used to clone DNA fragments of known sites from the chromosome of Escherichia coli. Each chimeric plasmid was introduced individually into the same dnaA46 mutant strain LC381 and suppressive integration (Sin) strains were selected. By means of cotransduction the null mutation recA56 was then introduced into each Sin strain and growth of each recA56 derivative at 42° C was scored. Strains that failed to grow at 42° C depended upon the recA gene for replication. Three factors were shown to limit the viability of LC381 harboring different chimeric plasmids and affect the degree of recA gene dependence of chromosome replication in the Sin strains at 42° C. It is suggested that these three constraints are the consequence of the organization of the E. coli chromosome, particularly the unique ability of terC to retard the progression of replication forks. Two classes of hypotheses concerning the function of the recA gene are considered.     

OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12

Summary The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [¹³C]glucose and ¹⁵NH₄Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.     

Bidirectional termination of chromosome replication in Escherichia coli

Summary Autoradiography was used to study the termination of replication of the circular chromosome of Escherichia coli. The experiments were conducted with cells in which termination occurred with a moderate amount of synchrony. Grain tracks were observed that demonstrated the approach at the replication terminus of the two replication forks involved in bidirectional replication. Other grain tracks were formed by replication forks that had met at the replication terminus. The frequency at which these patterns were observed indicates that most, if not all, terminations occur with both replication forks reaching the terminus at approximately the same time.     

Escherichia coli and its chromosome

The Escherichia coli chromosome is a circular DNA molecule that is approximately 1000 times compacted in the living cell, where it occupies approximately 15% of the cellular volume. The genome is organized in a way that facilitates chromosome maintenance and processing. Despite huge efforts, until recently little has been known about how the chromosome is organized within cells, where replication takes place, and how DNA is segregated before cell division. New techniques for labeling genetic loci and molecular machines are allowing the simultaneous tracking of genetic loci and such machines in living cells over time. These studies reveal remarkable organization, yet a highly dynamic flux of genetic loci and macromolecules. It seems likely that the cellular positioning of chromosomal loci is the outcome of the formation of two chromosome arms (replichores) by replication, followed by sequential chromosome segregation, rather than from the presence of cellular positioning markers.     

Curing of Escherichia coli K12 plasmids by coumermycin

Summary Low concentrations of the antibiotic coumermycin A₁, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of replication.     

Map position of the replication terminus on the Escherichia coli chromosome

Summary The 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; rev 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.     

Analysis of the physiological control of replication of ColE1-type plasmids

The physiology of ColE1-type plasmid replication in a growing host has been examined both theoretically, using computer simulation, and experimentally, by observing replication of the plasmid pBR322 after a nutritional shift-up from glycerol minimal medium (doubling time 71 min) to LB medium (doubling time 24 min). The theory was based on a negative control model and uses three rate equations: for the accumulation of cell mass, for the accumulation of the replication inhibitor, and for the rate of plasmid synthesis. The implications of the theory were explored by simulating the effects of changes in the expression of replication control genes. The nutritional shift-up experiment showed that plasmid replication was blocked immediately after the shift for about half a mass doubling time; after that time, replication rapidly increased until plasmid numbers per unit volume of culture parallelled the increase in culture mass. After the establishment of steady-state growth in the post-shift medium, the plasmid concentration (plasmids per cell mass) was reduced in comparison to pre-shift growth in the same proportion as the culture doubling time. The results showed that plasmid replication factors are under metabolic control and that the changes in the control of these factors compensate one another during steady-state growth, but not immediately after the medium shift.     

Curing effect of clorobiocin on Escherichia coli plasmids

Summary Clorobiocin, an inhibitor of the gyrB subunit of DNA gyrase, was used for the curing of some Escherichia coli plasmids. Of the plasmids studied, ampicillin resistant R28K and a miniplasmid derived from R1 drd-19 were effectively eliminated. We also succeeded in eliminating the ColA factor from E. coli strain B834 (pBS103), which was resistant to the effect of currently used curing agents. Although a derivative of ColE1-pBR322 was effectively cured by clorobiocin, the ColE1-plasmid was resistant to its effect. The ColV plasmid determining virulence was effectively eliminated.     

Initiation of DNA replication in Escherichia coli

Summary When E. coli F⁺ cells carrying the dna-167 or dnaC2 mutation, which causes the temperature-sensitive initiation of DNA replication, are exposed to a non-permissive temperature to stop the replication of chromosome and F factor, and then transferred back to a permissive temperature with the addition of chloramphenicol, one round of the chromosomal replication occurs, but further replication is inhibited. Under these conditions, F DNA replicates coincidentally with the initiation of the chromosomal replication in both strains. When rifampicin is added to the cells upon lowering of the temperature, the chromosome can not replicate in the F⁺ dna-167 strain, but can do so in the F⁺ dnaC2 strain. F DNA can replicate in both of the mutant strains under these conditions.     

Maintenance of plasmids in HU and 1HF mutants of Escherichia coli

Summary Complementation and sequencing analyses revealed that the hopD mutants, which could not support stable maintenance of mini-F plasmids (Niki et al. 1988), had mutations in the hupB gene, and that the hopD410 mutation was an ochre mutation at the 5th Gln position of HU-1. Maintenance and stability of various plasmids, mini-P1 plasmids, mini-F plasmids, and oriC plasmids, were studied in the hupA and hupB mutants (HU mutants), and himA and hip mutants (IHF mutants). Mini-P1 plasmids and mini-F plasmids could not be introduced into the hupA-hupB double deletion mutant. Replication of mini-F plasmids was partially inhibited in the hupB mutants, including the hupB and hopD(hupB) mutants, whereas replication of oriC plasmids was not significantly affected even in the hupA-hupB double deletion mutant. The mini-P1 plasmid was slightly unstable in the himA-hip mutant, whereas the mini-F plasmid was stable.     

Bacillussubtilis holo-cytochrome c-550 can be synthesised in aerobic Escherichia coli

Bacillus subtilis membrane-bound holo-cytochrome c-550 was found to be expressed from the structural gene cloned on a plasmid vector in aerobically grown Escherichia coli and exhibited normal biochemical properties. This occurs despite the lack of endogenous eytochrome c and suggests that eytochrome c-heme lyase activity is also present in aerobic E. coli. The membrane topology of B. subtilis eytochrome c-550 was studied using fusions to alkaline phosphatase (PhoA). The results show that the heme domain (at least when fused to PhoA) can be translocated as apo-cytochrome and confirm that the N-terminal part of the cytochrome functions as both export signal and membrane anchor for the C-tenninal heme domain. A model for the organisation of B. subtilis cytochrome c-550 in the cytoplasmic membrane is presented.     

pUB307 mobilizes resistance plasmids from Escherichia coli into Neisseria gonorrhoeae

Summary The plasmid pUB307, a derivative of RP1, is a conjugative, broad-host-range plasmid. We have shown that this element mobilizes gonococcal resistance plasmids from Escherichia coli to Neisseria gonorrhoeae, thus providing evidence that extrachromosomal elements can efficiently enter gonococci by conjugation. Furthermore, pUB307 can also be used as a helper element to mobilize the cloning vector pLES2 into N. gonorrhoeae. This finding significantly increases the usefulness of pLES2 as a shuttle vector between E. coli and gonococcus.     

ColE plasmid replication in DNA polymerase I-deficient strains ofEscherichia coli

Summary Replication of the non-conjugative plasmids ColE1, ColE2 and ColE3 has been examined in a number of DNA polymerase I-deficient strains, two of which contain the amber mutationpolA1 along with either of two temperature-sensitivesupF amber suppressors. These latter two strains produce reduced amounts of DNA polymerase I polymerizing activity of similar, if not identical properties to that produced bypolA⁺ strains. Our results indicate that the ColE plasmids require different amounts of DNA polymerase I for stable plasmid maintenance. Moreover whereas all three plasmids are maintained in a strain defective in the 53 exonuclease activity of DNA polymerase I, ColE2 and ColE3 are not stably maintained between 30° and 43° in a number of DNA polymerase I-deficient strains that are temperature-sensitive for ColE1 replication.     

Requirement of DNA Gyrase for the initiation of chromosome replication in Escherichia coli K-12

Summary It has been found that strains carrying mutations in the dnaA gene are unusually sensitive to COU, NAL or NOV, which are known to inhibit DNA gyrase activities. The delay in the initiation of chromosome replication after COU treatment has been observed in cells with chromosomes synchronized by amino acid starvation or by temperature shift-up (dnaA46). The unusual sensitivity of growth to COU of the initiation mutant runs parallel to a higher sensitivity to the drug of the initiation of chromosome replication.The double mutant, dnaA46 cou-110 has been isolated and mutation cou-110 conferring resistance of growth, initiation and elongation of chromosome replication to COU was mapped in the gene coding for the subunit of DNA gyrase. The reduced frequency of appearance of the mutants resistant to COU, NAL or NOV in the initiation mutant suggests that some mutations in genes coding for DNA gyrase subunits cannot coexist with the dnaA46 mutation. The possible mechanisms of the requirement of DNA gyrase for dnaA-dependent initiation of E. coli chromosome are discussed.Abbreviations used COU coumermycin A₁ - NAL nalidixic acid - NOV novobiocin     

Maintenance of some ColE1-type plasmids in chemostat culture

Summary When cells carrying the plasmids RP1, pDS4101 (a ColK derivative) or pDS1109 (a ColE1 derivative) were maintained in chemostat culture in the absence of antibiotic selection, plasmid-free segregants were not detected after 120 generations of nutrient-limited growth. By contrast, plasmid-free segregants of pMB9- and pBR322-containing cells arose after approximately 30 generations, irrespective of the host genetic background. However, even though pDS1109 was maintained its copy-number fell five-fold during 80 generations of limited growth. It is suggested that loss of pBR322 occurs following a similar copy-number decrease which results in defective segregation of the plasmid to daughter host cells. This defective segregation was not complemented in trans by either RP1 or pDS4101.     

Intramolecular homologous recombination of linearized plasmids in Escherichia coli K12

Summary The efficacy of linear DNA as a substrate for general homologous recombination was demonstrated using BamHI-linearized pKLC8.5, a plasmid that carries internal direct repeats flanking the unique BamHI site. An analogous plasmid, pKLC2.31, was used in a parallel and comparative study of intramolecular homologous recombination in circular DNA substrates. When the rec ⁺ wild-type strain, AB1157, and its isogenic rec ^– derivatives were transformed with linear pKLC8.5 DNA, intramolecular homologous recombination was independent of recA, recB, recN, recO and exonuclease III (xth-1) functions. Although the recBCsbcA and recBCsbcBC cells were both very recombination proficient, only linear but not circular DNA was used as substrate for intramolecular homologous recombination in the recBCsbcA cells. In both the recBCsbcA and recBCsbcBC genetic backgrounds, the recombination frequencies for linearized pKLC8.5 DNA were 100%. A notable difference between the two strains was that none of the recBCsbcA transformants obtained with circular pKLC8.5 DNA were Tc^s recombinants, whereas 11% of the corresponding recBCsbcBC transformants were Tc^s recombinants. The sbcB mutation was responsible for the recombination proficiency of the recBCsbcBC cells. Unlike the case in recBCsbcA cells, intramolecular homologous recombination of linear DNA in the recBCsbcBC cells was dependent on recA and recF as well as recN and recO gene functions, but was independent of recJ and reeL gene functions.