Suppression of ColE1 high-copy-number mutants by mutations in the polA gene of Escherichia coli.

We isolated three Escherichia coli suppressor strains that reduce the copy number of a mutant ColE1 high-copy-number plasmid. These mutations lower the copy number of the mutant plasmid in vivo up to 15-fold; the wild-type plasmid copy number is reduced by two- to threefold. The suppressor strains do not affect the copy numbers of non-ColE1-type plasmids tested, suggesting that their effects are specific for ColE1-type plasmids. Two of the suppressor strains show ColE1 allele-specific suppression; i.e., certain plasmid copy number mutations are suppressed more efficiently than others, suggesting specificity in the interaction between the suppressor gene product and plasmid replication component(s). All of the mutations were genetically mapped to the chromosomal polA gene, which encodes DNA polymerase I. The suppressor mutational changes were identified by DNA sequencing and found to alter single nucleotides in the region encoding the Klenow fragment of DNA polymerase I. Two mutations map in the DNA-binding cleft of the polymerase region and are suggested to affect specific interactions of the enzyme with the replication primer RNA encoded by the plasmid. The third suppressor alters a residue in the 3'-5' exonuclease domain of the enzyme. Implications for the interaction of DNA polymerase I with the ColE1 primer RNA are discussed.     

Plasmid rolling circle replication: identification of the RNA polymerase-directed primer RNA and requirement for DNA polymerase I for lagging strand synthesis.

Plasmid rolling circle replication involves generation of single-stranded DNA (ssDNA) intermediates. ssDNA released after leading strand synthesis is converted to a double-stranded form using solely host proteins. Most plasmids that replicate by the rolling circle mode contain palindromic sequences that act as the single strand origin, sso. We have investigated the host requirements for the functionality of one such sequence, ssoA, from the streptococcal plasmid pLS1. We used a new cell-free replication system from Streptococcus pneumoniae to investigate whether host DNA polymerase I was required for lagging strand synthesis. Extracts from DNA polymerase I-deficient cells failed to replicate, but this was corrected by adding purified DNA polymerase I. Efficient DNA synthesis from the pLS1-ssoA required the entire DNA polymerase I (polymerase and 5'-3' exonuclease activities). ssDNA containing the pLS1-ssoA was a substrate for specific RNA polymerase binding and a template for RNA polymerase-directed synthesis of a 20 nucleotide RNA primer. We constructed mutations in two highly conserved regions within the ssoA: a six nucleotide conserved sequence and the recombination site B. Our results show that the former seemed to function as a terminator for primer RNA synthesis, while the latter may be a binding site for RNA polymerase.     

Analysis of dominant copy number mutants of the plasmid pMB1.

We characterize two dominant copy number mutants of a derivative of plasmid pMB1. One of the two mutations maps in the -35 region of the primer promoter and results in increased promoter activity. The analysis of the secondary structure in the proximity of the mutant sequence suggests a possible mechanism which could be the basis of the promoter-up phenotype. By comparing the properties of the mutant and the wild type plasmid in an in vitro system, we confirm that the primer and not its coding sequence is the target of RNA I inhibition. The second mutation affects the sequence of the primer so that it is less sensitive to inhibition by RNA I. We propose that this mutation stabilizes a secondary structure necessary for primer formation.     

High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II

The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C. The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.     

Copy-number of broad host-range plasmid R1162 is regulated by a small RNA.

We have shown previously [Kim, K. and Meyer, R.J. (1985) J. Mol. Biol. 185,755-767] that copy-number of the broad host-range plasmid R1162 is controlled by the amounts of two proteins, encoded by cotranscribed genes comprising a region of the plasmid called RepI. We have now demonstrated that expression of RepI is negatively regulated by a 75 base RNA that is complementary to a segment of the RepI message. Increased intracellular amounts of RNA molecules that include this segment relieve the inhibition of RepI gene expression, suggesting that the target for regulation is the mRNA itself. A mutation decreasing the amount of the 75 base RNA results in elevated plasmid copy-number. Thus, consistent with our previous observations, regulation of the expression of the RepI genes is a factor in controlling plasmid copy-number.     

High efficiency of site-directed mutagenesis mediated by a single PCR product.

We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which incorporates both the mutations at the selection site and the desired single base substitutions at the mutant site. This primer is annealed to the denatured plasmid and directs the synthesis of the mutant strand. After digestion with selection enzyme, the plasmid DNA is amplified into Escherichia coli strain BMH71-18 and subjected to a second digestion and amplification into the bacterial strain DH5alpha. A mutagenesis efficiency >80% was consistently achieved in the case of two unrelated plasmids.     

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.     

Site-directed mutations in the relaxase operon of RP4.

Mutations were constructed by site-directed mutagenesis in the relaxase operon of the broad-host-range plasmid RP4. The mutations were constructed in smaller plasmids, recombined into the 60-kb RP4 plasmid, and tested for their ability to transfer. The relaxase operon contains the transfer genes traJ, traH, and traI, which are involved in nicking at the transfer origin to generate the single strand destined to be transferred to the recipient cell. In the first mutant, the C terminus of TraI was truncated, leaving TraH intact. This mutant decreased transfer by approximately 500-fold in Escherichia coli, and the traI mutation could be complemented by a wild-type copy of traI in trans in the donor. The traI mutation similarly decreased transfer between a variety of gram-negative bacteria. A site-specific mutation was made by the polymerase chain reaction-based unique-site mutagenesis procedure to alter the start site of traH. This mutation had no effect on intraspecific E. coli transfer but reduced transfer by up to sevenfold for some gram-negative bacteria. The traH mutation had no effect on plasmid stability. Thus, neither TraH nor the C terminus of TraI is required for conjugative transfer, but both increase mating efficiency in some hosts.     

In vitro transcription of Drosophila ribosomal genes using Drosophila RNA polymerases

Drosophila RNA polymerases I &; II were used to transcribe a recombinant bacterial plasmid containing one copy of Drosophila ribosomal DNA. Both supercoiled and relaxed, closed circular plasmids were used. With Mg⁺² as the divalent cation, enzyme I is much more active on both forms of the plasmid; the relaxed form in particular supports almost no RNA synthesis by enzyme II. When Mn⁺² is present, differences in template efficiencies are minimal. The differences observed in the absence of Mn⁺² seem to depend only on different preferences for the physical state of the template and not on recognition of specific promotor sequences, since enzyme I shows no strand selection when transcribing these plasmids.