Requirement of the Escherichia coli dnaA gene function for ori-2-dependent mini-F plasmid replication.

The mini-F plasmids pSC138, pKP1013, and pKV513 were unable to transform Escherichia coli cells with a dnaA-defective mutation under nonpermissive conditions. The dnaA defect was suppressed for host chromosome replication either by the simultaneous presence of the rnh-199 (amber) mutation or by prophage P2 sig5 integrated at the attP2II locus on the chromosome, both providing new origins for replication independent of dnaA function. The dnaA mutations tested were dnaA17, dnaA5, and dnaA46. dnaA5 and dnaA46 are missense mutations. dnaA17 is an amber mutation whose activity is controlled by the temperature-sensitive amber suppressor supF6. Under permissive conditions in which active DnaA protein was available, the mini-F plasmids efficiently transformed the cells. However, the transformants lost the plasmid as the cells multiplied under conditions in which DnaA protein was inactivated or its synthesis was arrested. As controls, plasmids pSC101 and pBR322 were examined along with mini-F; pSC101 behaved in the same manner as mini-F, showing complete dependence on dnaA for stable maintenance, whereas pBR322 was indifferent to the dnaA defect. Thus, ori-2-dependent mini-F plasmid replication seems to require active dnaA gene function. This notion was strengthened by the results of deletion analysis which revealed that integrity of at least one of the two DnaA boxes present as a tandem repeat in ori-2 was required for the origin activity of mini-F replication.     

DnaA Protein of Escherichia coli: oligomerization at the E. coli chromosomal origin is required for initiation and involves specific N-terminal amino acids

Iterated DnaA box sequences within the replication origins of bacteria and prokaryotic plasmids are recognized by the replication initiator, DnaA protein. At the E. coli chromosomal origin, oriC, DnaA is speculated to oligomerize to initiate DNA replication. We developed an assay of oligomer formation at oriC that relies on complementation between two dnaA alleles that are inactive by themselves. One allele is dnaA46; its inactivity at the non-permissive temperature is due to a specific defect in ATP binding. The second allele, T435K, does not support DNA replication because of its inability to bind to DnaA box sequences within oriC. We show that the T435K allele can complement the dnaA46(Ts) allele. The results support a model of oligomer formation in which DnaA box sequences of oriC are bound by DnaA46 to which T435K then binds to form an active complex. Relying on this assay, leucine 5, tryptophan 6 and cysteine 9 in a predicted alpha helix were identified that, when altered, interfere with oligomer formation. Glutamine 8 is additionally needed for oligomer formation on an oriC-containing plasmid, suggesting that the structure of the DnaA-oriC complex at the chromosomal oriC locus is similar but not identical to that assembled on a plasmid. Other evidence suggests that proline 28 of DnaA is involved in the recruitment of DnaB to oriC. These results provide direct evidence that DnaA oligomerization at oriC is required for initiation to occur.     

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.     

Overexpression of the dnaA gene in Escherichia coli B/r: chromosome and minichromosome replication in the presence of rifampin.

The replication of chromosomes and minichromosomes in Escherichia coli B/r was examined under conditions in which the dnaA gene product was overproduced. Increased levels of the DnaA protein were achieved by thermoinduction of the dnaA gene, under the control of the lambda pL promoter, or by cellular maintenance of multicopy plasmids carrying the dnaA gene under the control of its own promoters. Previous work has shown that overproduction of DnaA protein stimulates replication of the chromosomal origin, oriC, but that the newly initiated forks do not progress along the length of the chromosome (T. Atlung, K. V. Rasmussen, E. Clausen, and F. G. Hansen, p. 282-297, in M. Schaechter, F. C. Neidhardt, J. L. Ingraham, and N. O. Kjeldgaard, ed., The Molecular Biology of Bacterial Growth, 1985). In the present study, it was found that overproduction of DnaA protein caused both a two- to threefold increase in the amount of residual chromosome replication and an extended synthesis of minichromosome DNA in the presence of rifampin. The amount of residual chromosome replication was consistent with the appearance of functional replication forks on the majority of the chromosomes. Since the rate of DNA accumulation and the cellular DNA/mass ratios were not increased significantly by overexpression of the dnaA gene, we concluded that the addition of rifampin either enabled stalled replication forks to proceed beyond oriC or enabled new forks to initiate on both chromosomes and minichromosomes, or both.     

Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein.

Plasmids carrying the intact Bacillus subtilis dnaA-like gene and two reciprocal hybrids between the B. subtilis and Escherichia coli dnaA genes were constructed. None of the plasmids could transform wild-type E. coli cells unless the cells contained surplus E. coli DnaA protein (DnaAEc). A dnaA (Ts) strain integratively suppressed by the plasmid R1 origin could be transformed by plasmids carrying either the B. subtilis gene (dnaABs) or a hybrid gene containing the amino terminus of the E. coli gene and the carboxyl terminus of the B. subtilis gene (dnaAEc/Bs). In cells with surplus E. coli DnaA protein, expression of the E. coli dnaA gene was derepressed by the B. subtilis DnaA protein and by the hybrid DnaAEc/Bs protein, whereas it was strongly repressed by the reciprocal hybrid protein DnaABs/Ec. The plasmids carrying the different dnaA genes probably all interfere with initiation of chromosome replication in E. coli by decreasing the E. coli DnaA protein concentration to a limiting level. The DnaABs and the DnaAEc/Bs proteins effect this decrease possibly by forming inactive oligomeric proteins, while the DnaABs/Ec protein may decrease dnaAEc gene expression.     

Escherichia coli dnaA initiation function is required for replication of plasmids derived from coliphage lambda

The dnaA gene function, indispensable for the initiation of Escherichia coli replication from oriC is not essential for the growth of phage lambda. The in-vitro replication of plasmids derived from phage lambda does not seem to require DnaA protein either. However, we present evidence that in vivo the normal replication of lambda plasmids is dnaA-dependent. After inactivating the dnaA gene function, half of the plasmid molecules may enter a single round of replication. Rifampicin sensitivity of this abortive, as well as normal, replication indicates involvement of RNA polymerase. The rifampicin resistance of the normal replication of lambda plasmids in E. coli carrying the dnaAts46 or dnaAts5, but not the dnaAts204 allele at 30 degrees C implies the interaction of DnaA protein and RNA polymerase in this process. We propose that DnaA protein co-operates with RNA polymerase in the initiation of replication at ori lambda. The dispensability of DnaA in the growth of phage lambda and in lambda plasmid replication in vitro is discussed.     

Low-Temperature-Induced DnaA Protein Synthesis Does Not Change Initiation Mass in Escherichia coli K-12

Expression of the dnaA gene continues in the lag phase following a temperature downshift, indicating that DnaA is a cold shock protein. Steady-state DnaA protein concentration increases at low temperatures, being twofold higher at 14 degrees C than at 37 degrees C. DnaA protein was found to be stable at both low and high temperatures. Despite the higher DnaA concentration at low temperatures, the mass per origin, which is proportional to the initiation mass, was the same at all temperatures. Cell size and cellular DNA content decreased moderately below 30 degrees C due to a decrease in the time from termination to division relative to generation time at the lower temperatures. Analysis of dnaA gene expression and initiation of chromosome replication in temperature shifts suggests that a fraction of newly synthesized DnaA protein at low temperatures is irreversibly inactive for initiation and for autorepression or that all DnaA protein synthesized at low temperatures has an irreversible low-activity conformation.     

Reversibility of DnaA protein activity in the'irreversible'dnaA204 mutant of Escherichia coli

The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature downshift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 mutant at 42δC. The data indicate that the dnaA204 mutant after a shift to 42δC still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, initiation occurs very fast both after a shift to 30δC, and after induction of DnaA protein synthesis at 42 C. A model describing the processing of DnaA protein in mutants and in the wild type Is presented.     

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.     

Three distinct chromosome replication states are induced by increasing concentrations of DnaA protein in Escherichia coli.

The DnaA protein concentration in Escherichia coli was increased above the wild-type level by inducing a lacP-controlled dnaA gene located on a plasmid. In these cells with different DnaA protein levels, we measured several parameters: dnaA gene expression; cell size, amount of DNA per cell, and number of origins per cell by flow cytometry; and origin-to-terminus ratio and the frequencies of five other markers on the chromosome by Southern hybridization. The response of the cells to higher levels of DnaA protein could be divided into three states. From the normal level to a level 1.5-fold higher, DnaA protein had little effect on dnaA gene expression and the rate of DNA replication but led to nearly proportional increases in DNA and origin concentrations. Between 1.5- and 3-fold, the normal DnaA protein concentration, dnaA gene expression was gradually decreased. In this interval, the origin concentration increased significantly; however, the replication rate was severely affected, becoming slower--especially near the origin--the higher the DnaA protein concentration, and as a result, the DNA concentration was constant. Further increases in the DnaA protein concentration did not lead to an increased origin concentration. Thus, the initiation mass was set by the DnaA protein from the normal level to an at least twofold-increased level, but the increased initiation did not lead to a large increase in the amount of DNA per unit of mass because of the inhibition of replication fork velocity.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.     

Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12.

The product of the dnaA gene is essential for the initiation of chromosomal DNA replication in Escherichia coli K-12. A cold-sensitive mutation, dnaA(Cs), was originally isolated as a putative intragenic suppressor of the temperature sensitivity of a dnaA46 mutant (G. Kellenberger-Gujer, A. J. Podhajska, and L. Caro, Mol. Gen. Genet. 162:9-16, 1978). The cold sensitivity of the dnaA(Cs) mutant was attributed to a loss of replication control resulting in overinitiation of DNA replication. We cloned and sequenced the dnaA gene from the dnaA(Cs) mutant and showed that it contains three point mutations in addition to the original dnaA46(Ts) mutation. The dnaA(Cs) mutation was dominant to the wild-type allele. Overproduction of the DnaA(Cs) protein blocked cell growth. In contrast, overproduction of wild-type DnaA protein reduced the growth rate of cells but did not stop cell growth. Thus, the effect of elevated levels of the DnaA(Cs) protein was quite different from that of the wild-type protein under the same conditions.     

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.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

DnaA protein is not essential for replication of IncFII plasmid NR1.

By transformation of dnaA null mutant host cells that are suppressed either by an rnh mutation or by chromosomal integration of a mini-R1 plasmid, it was shown that replication of miniplasmids composed of the NR1 minimal replicon had no absolute dependence upon DnaA protein. In addition, the suppression of the dnaA null mutation by the integrated mini-R1, which is an IncFII relative of NR1, was found to be sensitive to the expression of IncFII-specific plasmid incompatibility. This suggests that the integrative suppression by mini-R1 is under the control of the normal IncFII plasmid replication circuitry. Although NR1 replication had no absolute requirement for DnaA, the copy numbers of NR1-derived miniplasmids were lower in dnaA null mutants, and the plasmids exhibited a much reduced stability of inheritance during subculture in the absence of selection. This suggests that DnaA protein may participate in IncFII plasmid replication in some auxiliary way, such as by increasing the efficiency of formation of an open initiation complex at the plasmid replication origin. Such an auxiliary role for DnaA in IncFII replication would be different from that for replication of most other plasmids examined, for which DnaA has been found to be either essential or unimportant.     

Evidence for positive regulation of plasmid prophage P1 replication: integrative suppression by copy mutants.

Like low-copy-number plasmids including P1 wild type, multicopy P1 mutants (P1 cop, maintained at five to eight copies per chromosome) can suppress the thermosensitive phenotype of an Escherichia coli dnaA host by forming a cointegrate. At 40 degrees C in a dnaA host suppressed by P1 cop, the only copy of P1 is the one in the host chromosome. Trivial explanations of the lack of extrachromosomal copies of P1 cop have been eliminated: (i) during integrative suppression, the P1 cop plasmid does not revert to cop+; (ii) the dnaA+ function of the host is not required to maintain P1 cop at a high copy number; and (iii) integrative recombination does not occur within the region of the plasmid involved in regulation of copy number. Since there are no more copies of the chromosomal origin (now located within the integrated P1 plasmid) than in a P1 cop+-suppressed strain, the extra initiation potential of the P1 cop is not used to provide multiple initiations of the chromosome. When a P1 cop-suppressed dnaA strain was grown at 30 degrees C so that replication could initiate from the chromosomal origin as well as from the P1 origin, multicopy supercoiled P1 DNA was found in the cells. This plasmid DNA was lost again when the temperature was shifted back to 40 degrees C.     

DnaA boxes are important elements in setting the initiation mass of Escherichia coli

The binding of DnaA protein to its DNA binding sites-DnaA boxes-in the chromosomal oriC region is essential for initiation of chromosome replication. In this report, we show that additional DnaA boxes affect chromosome initiation control, i.e., increase the initiation mass. The cellular DnaA box concentration was increased by introducing pBR322-derived plasmids carrying DnaA boxes from the oriC region into Escherichia coli and by growing the strains at different generation times to obtain different plasmid copy numbers. In fast-growing cells, where the DnaA box plasmid copy number per oriC locus was low, the presence of extra DnaA boxes caused only a moderate increase in the initiation mass. In slowly growing cells, where the DnaA box plasmid copy number per oriC locus was higher, we observed more pronounced increases in the initiation mass. Our data clearly show that the presence of extra DnaA boxes increases the initiation mass, supporting the idea that the initiation mass is determined by the normal complement of DnaA protein binding sites in E. coli cells.