Discriminatory function of ribonuclease H in the selective initiation of plasmid DNA replication.

The initiation stage of ColE1-type plasmid replication was reconstituted with purified protein fractions from Escherichia coli. The reconstituted system included DNA polymerase I, DNA ligase, RNA polymerase, DNA gyrase, and a discriminating activity copurifying with RNAase H (but free of RNAase III). Initiation of DNA synthesis in the absence of RNAase H did not occur at the normal replication origin and was non-selective with respect to the plasmid template. In the presence of RNAase H the system was selective for ColE1-type plasmids and could not accept the DNA of non-amplifiable plasmids. Electron microscopic analysis of the reaction product formed under discriminatory conditions indicated that origin usage and directionally of ColE1, RSF1030, and CloDF13 replication were consistent with the normal replication pattern of these plasmids. It is proposed that the initiation of ColE1-type replication depends on the formation of an extensive secondary structure in the origin primer RNA that prevents its degradation by RNAase H.     

Inhibition of DNA synthesis at the hemimethylated pBR322 origin of replication by a cell membrane fraction.

The replication of both ColE1-type plasmids and plasmids bearing the origin of replication of the Escherichia coli chromosome (oriC) has been shown to be inhibited by hemimethylation of adenine residues within GATC sequences. In the case of oriC plasmids, this inhibition was previously shown to be mediated by the specific affinity of the hemimethylated origin DNA for an outer cell membrane fraction. Here, we suggest that a similar mechanism is operating in the case of the ColE1-like plasmid pBR322 as (i) a hemimethylated DNA fragment carrying the promoter for the RNA which primes DNA synthesis (RNAII) is specifically bound by the same membrane fraction and, (ii) the addition of the membrane fraction to a soluble assay of pBR322 replication results in preferential inhibition of initiation on the hemimethylated template. We suggest that membrane sequestration of hemimethylated origin DNA and/or associated replication genes following replication may be a common element restricting DNA replication to precise moments in the cell cycle.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells

It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation. Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids. According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II. Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids.     

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.     

Control of ColE1 replication: low affinity specific binding of Rop (Rom) to RNAI and RNAII.

We have studied the interactions between the three molecules Rop, RNAI and RNAII that are involved in the regulatory mechanism controlling the replication of ColE1 plasmids. We show that it is possible to purify the two RNA molecules by passing an RNA mixture through an affinity column containing Rop immobilized to a solid support. The dissociation constants of the Rop-RNAI and Rop-RNAII complexes are of the order of 10(-4) M, several orders of magnitude higher than dissociation constants of stable protein-nucleic acid complexes (10(-10) M in the lambda repressor system). Although complete RNAI molecules have higher affinity, stem-and-loop I alone can also bind Rop, suggesting that this structure plays an important role in the interaction. Rop protects the stems of RNAI and RNAII from digestion by RNases while the sensitivity of the loops to digestion by RNase T1 is not affected by high concentrations of Rop. We propose a model for Rop-RNAI/RNAII interaction in which the dimeric protein acts as an adaptor between stem structures to position the two RNAs in the correct position for loop interaction.     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.     

Structural and functional organization of ColE2 and ColE3 replicons

Summary The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined. They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation. An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication. The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%). The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33 bp for ColE3. They are the smallest of all the prokaryotic replication origins so far reported. They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins. Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3. These results indicate that these plasmids share a common IncA determinant. A possibility that a small antisense RNA is involved in copy number control and incompatibility (IncA function) was suggested.     

Lateral transfer of rfb genes: a mobilizable ColE1-type plasmid carries the rfbO:54 (O:54 antigen biosynthesis) gene cluster from Salmonella enterica serovar Borreze.

Plasmid pWQ799 is a 6.9-kb plasmid isolated from Salmonella enterica serovar Borreze. Our previous studies have shown that the plasmid contains a functional biosynthetic gene cluster for the expression of the O:54 lipopolysaccharide O-antigen of this serovar. The minimal replicon functions of pWQ799 have been defined, and a comparison with nucleotide and protein databases revealed this replicon to be virtually identical to ColE1. This is the first report of involvement of ColE1-related plasmids in O-antigen expression. The replicon of pWQ799 is predicted to encode two RNA molecules, typical of other ColE1-type plasmids. RNAII, the putative replication primer from pWQ799, shares regions of homology with RNAII from ColE1. RNA1 is an antisense regulator of DNA replication in ColE1-related plasmids. The coding region for RNAI from pWQ799 shares no homology with any other known RNAI sequence but is predicted to adopt a secondary structure characteristic of RNAI molecules. pWQ799 may therefore represent a new incompatibility group within this family. pWQ799 also possesses cer, rom, and mob determinants, and these differ minimally from those of ColE1. The plasmid is mobilizable in the presence of either the broad-host-range helper plasmid pRK2013 or the IncI1 plasmid R64drd86. Mobilization and transfer of pWQ799 to other organisms provides the first defined mechanism for lateral transfer of O-antigen biosynthesis genes in S. enterica and explains both the distribution of related plasmids and coexpression of the O:54 factor with other O-factors in different Salmonella serovars. The base composition of the pWQ799 replicon sequences gives an average percent G+C value typical of Salmonella spp. In contrast, the percent G+C value is dramatically lower with rfb0:54, consistent with the possibility that the cluster was acquired from an organism with much lower G+C composition.     

Comparative analysis of the replicon regions of eleven ColE2-related plasmids.

The incA gene product of ColE2-P9 and ColE3-CA38 plasmids is an antisense RNA that regulates the production of the plasmid-coded Rep protein essential for replication. The Rep protein specifically binds to the origin and synthesizes a unique primer RNA at the origin. The IncB incompatibility is due to competition for the Rep protein among the origins of the same binding specificity. We localized the regions sufficient for autonomous replication of 15 ColE plasmids related to ColE2-P9 and ColE3-CA38 (ColE2-related plasmids), analyzed their incompatibility properties, and determined the nucleotide sequences of the replicon regions of 9 representative plasmids. The results suggest that all of these plasmids share common mechanisms for initiation of DNA replication and its control. Five IncA specificity types, 4 IncB specificity types, and 9 of the 20 possible combinations of the IncA and IncB types were found. The specificity of interaction of the Rep proteins and the origins might be determined by insertion or deletion of single nucleotides and substitution of several nucleotides at specific sites in the origins and by apparently corresponding insertion or deletion and substitution of amino acid sequences at specific regions in the C-terminal portions of the Rep proteins. For plasmids of four IncA specificity types, the nine-nucleotide sequences at the loop regions of the stem-loop structures of antisense RNAs are identical, suggesting an evolutionary significance of the sequence. The mosaic structures of the replicon regions with homologous and nonhomologous segments suggest that some of them were generated by exchanging functional parts through homologous recombination.     

Replication of plasmids in gram-negative bacteria.

Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical data about the initial priming reaction of DNA synthesis exist. Detailed models have been developed for the initiation and regulation of ColE1 replication. For other plasmids, such as pSC101, some hypotheses for priming mechanisms and replication initiation are presented. These hypotheses are based on experimental evidence and speculative comparisons with other systems, e.g., the chromosomal origin of E. coli. In most cases, knowledge concerning plasmid replication is limited to regulation mechanisms. These mechanisms coordinate plasmid replication to the host cell cycle, and they also seem to determine the host range of a plasmid. Most plasmids studied exhibit a narrow host range, limited to E. coli and related bacteria. In contrast, some others, such as the IncP plasmid RK2 and the IncQ plasmid RSF1010, are able to replicate in nearly all gram-negative bacteria. This broad host range may depend on the correct expression of the essential rep genes, which may be mediated by a complex regulatory mechanism (RK2) or by the use of different promoters (RSF1010). Alternatively or additionally, owing to the structure of their origin and/or to different forms of their replication initiation proteins, broad-host-range plasmids may adapt better to the host enzymes that participate in initiation. Furthermore, a broad host range can result when replication initiation is independent of host proteins, as is found in the priming reaction of RSF1010.     

ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.

Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.