Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures.

We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.     

Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K.

The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol. It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA. An Hfr strain designated as Hfr(R6K) no. 1 was thus obtained and characterized. It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C. The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol. These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.     

Suppression of Escherichia coli dnaA46 mutations by integration of plasmid R100.1. derivatives: constraints imposed by the replication terminus.

We have studies the phenotypic suppression of a dnaA46 mutation by plasmid integration at preselected chromosomal sites after introducing homologous sequences (Mu prophages) onto both the chromosomes and the suppressive plasmid. The plasmids used were all derived from plasmid R100.1. We found that the conditions required to get viable suppressive integration varied as the plasmid integration site moved from the origin to the terminus of chromosome replication. Two constraints were observed. Both appeared to be linked to the new characteristics acquired by chromosome replication from the integrated plasmid. One constraint was that strains with integrative suppression near the terminus terC were viable only in minimal medium. The rich medium sensitivity of these strains was correlated with a loss of regulation of initiation. The other constraint was a requirement for a specific orientation in certain regions of the chromosome. The two branches defined by normally initiated replication, between oriC and terC, were also symmetrical with respect to these plasmid orientation constraints. In studying the possible reasons for a plasmid orientation constraint, we found that, of the two forks initiated in bidirectional replication from the integrated plasmid, one was capable of moving across the terC region with a higher movability than the other.     

Suppression of an Escherichia coli dnaA mutation by the integrated R factor R100.1: generation of small plasmids after integration.

We have observed that integration of the R plasmid R100.1 into the chromosome of Escherichia coli is associated with the formation of small, covalently closed circular elements. Contour length measurements, partial denaturation mapping, and analysis of the deoxyribonucleic acid fragments produced by digestion of one of these, pLC1, with the restriction endonuclease EcoRI indicate that it is the r-determinant element of R100.1.     

Two types of cold sensitivity associated with the A184-->V change in the DnaA protein

Multicopy dnaA(Ts) strains carrying the dnaA5 or dnaA46 allele are high-temperature resistant but are cold sensitive for colony formation. The DnaA5 and DnaA46 proteins both have an A184-->V change in the ATP binding motif of the protein, but they also have one additional mutation. The mutations were separated, and it was found that a plasmid carrying exclusively the A184-->V mutation conferred a phenotype virtually identical to that of the dnaA5 plasmid. Strains carrying plasmids with either of the additional mutations behaved like a strain carrying the dnaA+ plasmid. In temperature downshifts from 42 degrees C to 30 degrees C, chromosome replication was stimulated in the multicopy dnaA46 strain. The DNA per mass ratio increased threefold, and exponential growth was maintained for more than four mass doublings. Strains carrying plasmids with the dnaA(A184-->V) or the dnaA5 gene behaved differently. The temperature downshift resulted in run out of DNA synthesis and the strains eventually ceased growth. The arrest of DNA synthesis was not due to the inability to initiate chromosome replication because marker frequency analysis showed high initiation activity after temperature downshift. However, the marker frequencies indicated that most, if not all, of the newly initiated replication forks were stalled soon after the onset of chromosome replication. Thus, it appears that the multicopy dnaA(A184-->V) strains are cold sensitive because of an inability to elongate replication at low temperature. The multicopy dnaA46 strains, on the contrary, exhibit productive initiation and normal fork movement. In this case, the cold-sensitive phenotype may be due to DNA overproduction.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Replication of ColE1 plasmid deoxyribonucleic acid in a thermosensitive dnaA mutant of Escherichia coli.

The replication of ColE1 deoxyribonucleic acid (DNA) took place at the restrictive temperature in a dnaA mutant, dnaA167(Ts). It proceeded at a constant rate at 42 degrees C for at least 3 h. The replication was insensitive to rifampin, which blocked replication at the permissive temperature or in the presence of chloramphenicol, even at the restrictive temperature. A linear DNA strand of ColE1 longer than unit genome size was synthesized. The structure of the replicating molecules observed by electron microscopy was mostly sigma shaped, composed of a circle of a unit genome length with a double-stranded tail. These observations suggest that the replication of ColE1 DNA proceeds via a rolling-circle type of structure in the absence of dnaA function.     

Suppression of temperature-sensitive chromosome replication of an Escherichia coli dnaX(Ts) mutant by reduction of initiation efficiency

Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40 degrees C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20 degrees C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the tau and gamma subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40 degrees C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34 degrees C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39 degrees C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39 degrees C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38 degrees C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.     

Formation of Escherichia coli Hfr strains by integrative suppression with the P group plasmid RP1.

Hfr strains of Escherichia coli were obtained by integrative suppression of a dnaA(Ts) mutation by the Inc P-1 plasmid RP1 without prior creation of an unnatural homology between the plasmid and the E. coli chromosome. Unmodified RP1 mobilized the polarized transfer of the chromosome in a counterclock-wise direction from a distinct origin between 81 min (pyrE) and 82 min (dnaA) with pyrE as a leading marker. Inheritance of RP1-Hfr chromosomal and antibiotic resistance genes was due to recombination with the recipient chromosome, as shown by the need for a functional recA system. The acquisition of temperature resistance and donor ability was accompanied by the disappearance of free plasmid when the selection pressure for integration was maintained (growth at 41 degrees C); the loss of temperature resistance and donor ability was accompanied by the reappearance of autonomous RP1 when the selection pressure was removed (growth at 30 degrees C).     

Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids.

Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX+ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20 degrees C (J.R. Walker, J.A. Ramsey, and W.G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA+ allele restored growth to dnaA (Cs, Sx) strains at 20 degrees C independently of which allele was present on the plasmid. The dnaA (Cs, Sx) alleles provided initiator function at 39 degrees C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA (Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.     

Chromosome-plasmid interaction in Escherichia coli K-12 carrying a thermosensitive plasmid, Rts1, in autonomous and in integrated states.

An Hfr strain of Escherichia coli K-12 was obtained by integrative suppression with a thermosensitive plasmid, Rts1. The R plasmid was integrated into the chromosome between rif and thr, and transfer of the chromosome occurred counterclockwise. The thermosensitivity of host cell growth due to the dnaA mutation was markedly but not completely reduced in this integratively suppressed Hfr strain. When the dnaA mutation was removed by transducing the dnaA+ genome to this Hfr, the thermosensitivity of cell growth due to existence of Rts1 was suppressed in contrast to strains carrying it autonomously. Thermosensitivity of cell growth appeared again when the plasmid was detached from the chromosome to exist autonomously. Contrary to the effect on cell growth, the transfer of the chromosome and the plasmid itself and the ability to "restrict" T-even phages were still thermosensitive in all of these strains carrying Rts1, irrespective of its state of existence. The detached plasmid as well as the original Rts1 were segregated upon growth at 42 C. These data are discussed in relation to chromosome-plasmid interaction. One of the most important conculusions is that some plasmid genes, related to their replication, are phenotypically suppressed by the chromosome when it is integrated.     

Host cell variations resulting from F plasmid-controlled replication of the Escherichia coli chromosome.

Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA (Ts) mutation that has been suppressed by the insertion of the F plasmid into the chromosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39 degrees C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30 degrees C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39 degrees C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39 degrees C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle number ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.     

Requirement of the Escherichia coli dnaA gene product for plasmid F maintenance.

There are DnaA protein-binding sites in at least one F origin of replication, and only potentially leaky dnaA(Ts) mutations had ever been used in previous studies indicating that F replication was independent of the dnaA gene product. Here we show that an Escherichia coli dnaA::Tn10 host which does not make a dnaA gene product cannot sustain autonomous or integrated F plasmid maintenance.     

Transposition of a deoxyribonucleic acid sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons.

R483, a plasmid of the Ialpha incompatibility group, contained a deoxyribonucleic acid (DNA) sequence encoding resistance to trimethoprim (TpR) and streptomycin (SmR) that could be transposed to other replicons, i.e., to the Escherichia coli chromosome and to related and unrelated plasmids. Each transposition resulted in the acquisition by the recipient replicon of a segment of DNA of about 9 X 10(6) daltons, both resistance genes, but never the colicin Ia or pilus genes of R483. Transposition took place at a single chromosomal site between dnaA and ilv and did not suppress the DnaA phenotype, in contrast to integration of the whole R483 plasmid. The chromosome, having received the transposition, could secondarily act as a transposition donor to another plasmid. Such a plasmid was indistinguishable from one having received a direct transposition from R483. TpR SmR transposition was very site specific and did not require a functional recA+ gene. We postulate that the TpR SmR segment of R483 is a transposon (TnC) with specific boundary sequences.     

Integrative suppression of dnaA46 by broad host-range plasmid R1162

Summary Replication of plasmid R1162 DNA does not require the product of the dnaA gene. An integrated copy of the plasmid can suppress the temperature-sensitive dnaA46 allele when (1) additional plasmid copies are present in the cytoplasm and (2) an inactive replication origin, generated by deletion, is also present in the chromosome. We propose that the inactive origin sets the rate of initiation of chromosome replication at a level compatible with cell viability, possibly by providing additional binding sites for an R1162-encoded protein that is rate-limiting for plasmid replication.     

Mutations of temperature sensitivity in R plasmid pSC101.

Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.     

Host-dependent, thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae.

The thermosensitive replication of an R plasmid, pJY5, isolated from Enterobacter cloacae, was studied. pJY5 consisted of 61 million daltons of covalently closed circular (CCC) deoxyribonucleic acid (DNA) with a buoyant density of 1.714 g/cm3 (55 mol % guanine plus cytosine). In Escherichia coli, this plasmid replicated stringently at 32 degrees C, but ceased its CCC DNA replication after a short incubation at 42 degrees C, resulting in production of R- segregants. The thermosensitive replication of pJY5 was not overcome by the coexistence of non-thermosensitive R plasmids. The plasmid manifested an inhibitory effect on host bacterial cell growth at 42 degrees C, although the effect was less prominent than that of R plasmids belonging to the T-incompatibility group, Rts1, R401, and R402. When the pJY5 plasmid was transferred into E. cloacae, however, no R- segregants were detected at any culture temperature, even 42 degrees C. Alkaline sucrose gradient analysis revealed that a significant amount of pJY5 CCC DNA was synthesized in E. cloacae at the high temperature but not in E. coli. Furthermore, the growth-inhibitory effect of pJY5 on hosts at 42 degrees C was not observed in E. cloacae. On the other hand, Rts1 and R401 were found to be thermosensitive in E. cloacae as well as in E. coli.     

Overinitiation of chromosome and plasmid replication in a dna Acos mutant of Escherichia coli K12. Evidence for dnaA-dnaB interactions

The dnaAcos mutations are phenotypic suppressors of dnaAts46 that are co-transduced with dnaA, render the cell cold sensitive, and cause an excess of chromosome replication relative to cell mass when the cells are shifted from 42 degrees C to 32 degrees C. We have used pulse labelling and DNA-DNA hybridization to follow the effect of a temperature shift on the replication of the chromosome and of the plasmids pSC101, RTF-Tc, and lambda dv in such strains. After a shift of a dnaAcos strain from 42 degrees C to 32 degrees C (non-permissive temperature), initiation of the chromosome and replication of the plasmid pSC101 are stimulated, while the dnaA-independent plasmid RTF-Tc is not affected. The presence of pSC101 does not affect the level of overinitiation of the chromosome. The presence of lambda dv suppresses the cold sensitivity of dnaAcos mutants and allows the cells to grow at both 32 degrees C and 42 degrees C. The presence of lambda dv suppresses the overinitiation of chromosome and of pSC101 replication at 32 degrees C. Previous reports had shown that these suppressions involve an interaction between the dnaA product and the lambda P protein, which is also known to interact with dnaB. We show here that the mutant prophage P1 bac-crr, which produces high levels of a dnaB analogue, suppresses the dnaAcos phenotype, while wild type P1 does not. These results suggest that initiation involves interactions between the dnaA and dnaB products.