The bacteriophage lambda DNA replication protein P inhibits the oriC DNA- and ATP-binding functions of the DNA replication initiator protein DnaA of Escherichia coli

Under the condition of expression of lambda P protein at lethal level, the oriC DNA-binding activity is significantly affected in wild-type E. coli but not in the rpl mutant. In purified system, the lambda P protein inhibits the binding of both oriC DNA and ATP to the wild-type DnaA protein but not to the rpl DnaA protein. We conclude that the lambda P protein inhibits the binding of oriC DNA and ATP to the wild-type DnaA protein, which causes the inhibition of host DNA synthesis initiation that ultimately leads to bacterial death. A possible beneficial effect of this interaction of lambda P protein with E. coli DNA initiator protein DnaA for phage DNA replication has been proposed.     

Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.     

Modulation of pPS10 host range by DnaA

Narrow-host-range plasmid pPS10, originally found in Pseudomonas savastanoi, is unable to replicate in other strains such as Escherichia coli. Here, we report that the establishment of pPS10 in E. coli can be achieved by a triple mutation in the dnaA gene of E. coli (dnaA403), leading to Q14amber, P297S and A412V changes in the DnaA host replication protein (DnaA403 mutant). As the E. coli strain used contained double amber suppressor mutations (supE, supF), the amber codon in dnaA403 can be translated into glutamine or tyrosine. Genetic analysis of DnaA proteins containing either the individual changes or their different combinations suggests that the P297S mutation is crucial for the establishment of the pPS10 replicon in E. coli. The data also indicate that the P297S change is toxic to the cell and that the additional mutations in DnaA403 could contribute to neutralize this toxicity. To our knowledge, this work reports the first chromosome mutant described in the literature that allows the host range broadening of a plasmid, highlights the essential role played by DnaA in the establishment of pPS10 replicon in E. coli and provides support for the hypothesis that interactions between RepA and DnaA modulate the establishment of pPS10 in that bacteria and probably in other species.     

Conditionally lethal genes in the N pilus region of plasmid pCU1.

Plasmid genes or regions that are conditionally lethal to Escherichia coli have been called kil and those lethal to Klebsiella but not to E. coli have been called kik. Both classes of genes are found in or close to the N pilus region of the plasmid pCU1 and the closely related plasmid pKM101. Here we describe two new and overlapping lethal genes that are located between kikA and traA of the plasmid pCU1 and display host specificity. KilC is lethal in E. coli and Klebsiella while kikC is lethal only in Klebsiella. The previously identified korA gene is sufficient to override the lethality of kilC in trans or in cis but is insufficient to override kikC. kilC expression in E. coli leads to cell death accompanied by an increase in average cell length without affecting septum formation.     

Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication.

Bacteriophage T4 rnh encodes an RNase H that removes ribopentamer primers from nascent DNA chains during synthesis by the T4 multienzyme replication system in vitro (H. C. Hollingsworth and N. G. Nossal, J. Biol. Chem. 266:1888-1897, 1991). This paper demonstrates that either T4 RNase HI or Escherichia coli DNA polymerase I (Pol I) is essential for phage replication. Wild-type T4 phage production was not diminished by the polA12 mutation, which disrupts coordination between the polymerase and the 5'-to-3' nuclease activities of E. coli DNA Pol I, or by an interruption in the gene for E. coli RNase HI. Deleting the C-terminal amino acids 118 to 305 from T4 RNase H reduced phage production to 47% of that of wild-type T4 on a wild-type E. coli host, 10% on an isogenic host defective in RNase H, and less than 0.1% on a polA12 host. The T4 rnh(delta118-305) mutant synthesized DNA at about half the rate of wild-type T4 in the polA12 host. More than 50% of pulse-labelled mutant DNA was in short chains characteristic of Okazaki fragments. Phage production was restored in the nonpermissive host by providing the T4 rnh gene on a plasmid. Thus, T4 RNase H was sufficient to sustain the high rate of T4 DNA synthesis, but E. coli RNase HI and the 5'-to-3' exonuclease of Pol I could substitute to some extent for the T4 enzyme. However, replication was less accurate in the absence of the T4 RNase H, as judged by the increased frequency of acriflavine-resistant mutations after infection of a wild-type host with the T4 rnh (delta118-305) mutant.     

Physiological properties of cold-sensitive suppressor mutations of a temperature-sensitive dnaZ mutant of Escherichia coli

Suppressors of a temperature-sensitive dnaZ polymerization mutant of Escherichia coli have been identified by selecting temperature-insensitive revertants. Those suppressed strains which concomitantly became cold sensitive were chosen for further study. Intragenic suppressor mutations, which caused cold-sensitive defects in DNA polymerization, were located in dnaZ by transduction with lambda dnaZ+ phages. Extragenic suppressor mutations were mapped within the initiation gene dnaA. These suppressor-containing strains were defective in initiation at low temperature as determined by measurements of DNA synthesis in vivo and in toluene-treated cells. The occurrence of suppressor mutations of dnaZ(Ts) within the dnaA gene is considered evidence that the dnaA and dnaZ products interact in vivo. A second indication of a dnaA-dnaZ protein-protein interaction was provided by the observation that the introduction of additional copies of the dnaZ+ gene into a strain carrying the dnaA suppressor mutation was lethal [whether the strain was dnaZ+ or dnaZ(Ts)].     

Construction and characterization of mutations in hupB, the gene encoding HU-beta (HU-1) in Escherichia coli K-12.

Plasmid pJMC21 contains Escherichia coli chromosomal DNA encoding Lon protease, HU-beta (HU-1), and an unidentified 67,000-dalton protein. A kanamycin resistance cassette was used in the construction of insertion and deletion mutations in hupB, the gene encoding HU-beta on plasmid pJMC21. The reconstructed plasmids were linearized and used to introduce hupB chromosomal mutations into JC7623 (recBC sbcBC). These mutations, as expected, mapped in the 9.8-min region of the E. coli chromosome by P1 transduction (16% linkage to proC+). Southern blot hybridization of chromosomal fragments verified that hupB+ was replaced by the mutant allele, with no indication of gene duplication. All the mutant strains had growth rates identical to that of wild-type E. coli, were resistant to UV irradiation and nitrofurantoin, and supported the in vivo transposition-replication of bacteriophage Mu, Mu lysogenization, Tn10 transposition from lambda 1098, and lambda replication-lysogenization. The only observable phenotypic variation was a reduced Mu plaque size on the hupB mutant strains; however, the yield of bacteriophage Mu in liquid lysates prepared from the mutant strains was indistinguishable from the yield for the wild type.     

Essential amino acid residues in the single-stranded DNA-binding protein of bacteriophage T7. Identification of the dimer interface

Gene 2.5 of bacteriophage T7 is an essential gene that encodes a single-stranded DNA-binding protein. T7 phage with gene 2.5 deleted can grow only on Escherichia coli cells that express gene 2.5 from a plasmid. This complementation assay was used to screen for lethal mutations in gene 2.5. By screening a library of randomly mutated plasmids encoding gene 2.5, we identified 20 different single amino acid alterations in gene 2.5 protein that are lethal in vivo. The location of these essential residues within the three-dimensional structure of gene 2.5 protein assists in the identification of motifs in the protein. In this study we show that a subset of these alterations defines the dimer interface of gene 2.5 protein predicted by the crystal structure. Recombinantly expressed and purified gene 2.5 protein-P22L, gene 2.5 protein-F31S, and gene 2.5 protein-G36S do not form dimers at salt concentrations where the wild-type gene 2.5 protein exists as a dimer. The basis of the lethality of these mutations in vivo is not known because altered proteins retain the ability to bind single-stranded DNA, anneal complementary strands of DNA, and interact with T7 DNA polymerase.     

Repair promoted by plasmid pKM101 is different from SOS repair.

In E. coli K12 bacteria carrying plasmid pKM101, prophage lambda was induced at UV doses higher than in plasmid-less parental bacteria. UV-induced reactivation per se was less effective. Bacteria with pKM101 showed no alteration in their division cycle. Plasmid pKM101 coded for a constitutive error-prone repair different from the inducible error-prone repair called SOS repair. Plasmid pKM101 protected E. coli bacteria from UV damage but slightly sensitized them to X-ray lesions. Protection against UV damage was effective in mutant bacteria deficient in DNA excision-repair provided that the recA, lexA and uvrE genes were functional. Survival of phages lambda and S13 after UV irradiation was enhanced in bacteria carrying plasmid pKM101; phage lambda mutagenesis was also increased. Plasmid pKM101 repaired potentially lethal DNA lesions, although wild-type DNA sequences may not necessarily be restored; hence the mutations observed are the traces of the original DNA lesions.     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Conditionally lethal amber mutations in the dnaA region of the Escherichia coli chromosome that affect chromosome replication.

Three amber mutations, dna-801, dna-803, and dna-806, were isolated by localized mutagenesis of the dnaA-oriC region of the chromosome from an Escherichia coli strain carrying temperature-sensitive amber suppressors. When the mutations were not suppressed at 42 degrees C, the cells did not grow and DNA synthesis was arrested. They were very closely linked to each other and to the dnaA46 mutation. The mutant phenotype of each strain was converted to the wild type by infecting the mutants with specialized transducing phase lambda i21 dnaA-2 but not with lambda i21 tna. Derivatives of lambda i21 dnaA-2, each of which carried the amber mutation dna-801 dna-803, or dna-806, converted the dnaA mutant phenotype to Dna+ but did not convert rhe amber mutants to the wild-type phenotype. E. coli uvrB cells were irradiated with ultraviolet light and infected with each of these phage strains. An analysis of proteins synthesized in the cells revealed that two proteins with molecular weights of 50,000 and 43,000 were specified by lambda i21 dnaA-2 but not by lambda i21 tna. When the ultraviolet-irradiated cells did not carry an amber suppressor, the derivative phage with the amber mutation invariably failed to produce the 43,000-dalton protein, but when the host cell carried supF (tyrT), the protein was produced. The 50,000-dalton protein was unaffected.     

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.     

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.     

ClpP/ClpX-mediated degradation of the bacteriophage λ O protein and regulation of λ phage and λ plasmid replication

The O protein is a replication initiator that binds to the orilambda region and promotes assembly of the bacteriophage lambda replication complex. This protein, although protected from proteases by other elements of the replication complex, in a free form is rapidly degraded in the host, Escherichia coli, by the ClpP/ClpX protease. Nevertheless, the physiological role of this rapid degradation remains unclear. Here we demonstrate that the copy number of plasmids derived from bacteriophage lambda is significantly higher in wild-type cells growing in rich media than in slowly growing bacteria. However, lambda plasmid copy number in bacteria devoid of the ClpP/ClpX protease was not dependent on the bacterial growth rate and in all minimal media tested was comparable to that observed in wildtype cells growing in a rich medium. Contrary to lambda plasmid replication, the efficiency of lytic growth of bacteriophage lambda was found to be dependent on the host growth rate in both wild-type bacteria and clpP and clpX mutants. The activities of two major lambda promoters operating during the lytic development, p(R) and p(L), were found to be slightly dependent on the host growth rate. However, when p(R) activity was significantly decreased in the dnaA mutant, production of phage progeny was completely abolished at low growth rates. These results indicate that the O protein (whose level in E. coli cells depends on the activity of ClpP/ClpX protease) is a major limiting factor in the regulation of lambda plasmid replication at low bacterial growth rates. However, this protein seems to be only one of the limiting factors in the bacteriophage lambda lytic development under poor growth conditions of host cells. Therefore, it seems that the role of the rapid ClpP/ClpX-mediated proteolysis of the O protein is to decrease the efficiency of early DNA replication of the phage in slowly growing host cells.     

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.     

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.