Characterization of copy number mutants of plasmid pSC101

We have characterized three copy number mutants of the plasmid pSC101. These mutations caused single amino acid substitutions at the 46th, 83rd and 115th codons in the rep gene and an increase in the copy number by 4- to 8-fold. Although the in vivo and in vitro repressor activities of these mutated Rep proteins were quite different from each other, the intracellular concentrations of the proteins were maintained at higher levels than the wild-type protein. It has been reported that excess amounts of Rep inhibit pSC101 replication (Ingmer and Cohen, 1993). This inhibitory activity of Rep was markedly decreased in all three mutants. When both the wild-type and one of the mutated rep genes were retained in the same plasmids, the copy number of these plasmids was decreased compared with plasmids retaining a single mutated rep gene. These results support the theory that the inhibitory activity of Rep for its own replication plays an important role in copy number regulation.     

Impact of targeted vector design on Co/E1 plasmid replication

The demands for recombinant proteins, in addition to plasmid DNA, for therapeutic use are steadily increasing. Bacterial fermentation processes have long been and still are the major tool for production of these molecules. The key objective of process optimization is to attain a high yield of the required quality, which is determined, to a large extent, by plasmid replication rates, metabolic capacity and the properties of the specific gene construct. When high copy number plasmids are used, the metabolic capacity of the host cell is often overstrained and efficient protein production is impaired. The plasmid copy number is the key parameter in the exploitation of the host cell, and can be maximized by optimal control of the flux ratios between biosynthesis of host cell proteins and recombinant proteins.     

Isolation of chromosomal mutations that affect carotenoid production in Escherichia coli: mutations alter copy number of ColE1-type plasmids

Chromosomal mutants were isolated in Escherichia coli that altered carotenoid production from transformed carotenoid biosynthesis genes on a pACYC-derived plasmid (pPCB15). The mutations were mapped by sequencing. One group of mutations appeared to affect the cell metabolism without changing the copy number of the carotenoid synthesis plasmid. The other group of mutations either increased or decreased the copy number of the pPCB15 plasmid as determined by real-time PCR. The copy number change in most mutants was likely specific for ColE1-type plasmids for which copy number is controlled by a small antisense RNA. This collection of host strains would be useful for fine tuning expression of proteins and adjusting production of desired molecules without recloning to different vectors.     

Identification and molecular genetic analysis of replication functions of the bacteriocinogenic plasmid pIP404 from Clostridium perfringens

The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis. This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences. The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication. By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences. A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region. In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C. perfringens or B. subtilis.     

Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli

Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two-dimensional gel electrophoresis patterns. The plasmids investigated had very similar sequences except for small domains encoding the represser of plasmid replication. At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased. At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed. Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation. Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses. These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.     

Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy

The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC lo the PT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.     

Nucleotide sequence analysis of cryptic plasmid pAM5 from Acidiphilium multivorum

Plasmid pAM5 of Acidiphilium multivorum JCM-8867 has been completely sequenced by initial cloning of HindIII-PstI fragments followed by primer walking. It has a size of 5161bp and single site for several restriction enzymes as revealed by DNA sequencing. Sequence analysis predicts five putative open reading frames. ORF1 and ORF3 show significant identity with various plasmid encoded mobilization (Mob) and replication initiation (Rep) proteins, respectively. The putative Mob protein has several characteristics of the MOB(Q) family having the motifs with conserved amino acid residues. Upstream of the Mob ORF, there exists a 34bp oriT region having a nic consensus sequence. The constructed plasmid pSK1 bearing pAM5 mob region can be mobilized to Escherichia coli in presence of conjugative plasmid pRK2013. The replication module comprises of several DnaA like boxes, several perfect direct and inverted repeats, a potential prokaryotic promoter and putative rep gene. The rep module is very similar to several theta replicating iteron family plasmids, suggesting pAM5 replication to follow the same course. Any phenotypic character determinant (e.g., metal resistance, antibiotic resistance etc.) gene is absent in pAM5, suggesting this plasmid to be cryptic in nature. However, a pAM5 derivative plasmid named pSK2, containing the putative pAM5 rep region, can replicate and be stably maintained in Acidiphilium, Acidocella, and E. coli strains; it can also carry foreign DNA fragments. Thus, pSK2 could serve as a cloning shuttle vector between these bacteria. It was observed that pAM5 Rep is essential for pSK2 to replicate in acidophiles. In its natural host, A. multivorum JCM-8867, pAM5 maintains a copy number of 50-60, and its derivative pSK2 maintains a comparatively, higher copy number in E. coli than in acidophiles.     

A 37 X 10(3) molecular weight plasmid-encoded protein is required for replication and copy number control in the plasmid pSC101 and its temperature-sensitive derivative pHS1

Nucleotide sequences were determined for a region essential for autonomous replication and partitioning of pSC101, a plasmid whose replication is dependent on the Escherichia coli dnaA gene product. The essential replication region contains one long coding sequence, rep101 , for a protein composed of 316 amino acids, and a polypeptide approximately 37 X 10(3) Mr in size was identified as the rep101 gene product. rep101 is preceded by two inverted repeat sequences, three directly repeated sequences and a region of high A + T content containing a sequence similar to the E. coli oriC consensus sequence. Because the lesions in seven replication-deficient insertion mutants, four mutants with increased copy number and one temperature-sensitive replication mutant occur within rep101 , the rep101 gene product must control pSC101 replication and copy number. par, a region adjacent to the replication region, which functions in stable plasmid inheritance, contains several inverted repeat sequences.     

Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA.

The trfA gene, encoding the essential replication initiation protein of the broad-host-range plasmid RK2, possesses an in-frame overlapping arrangement. This results in the production of TrfA proteins of 33 and 44 kDa, respectively. Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone. TrfA-44/98L is identical to wild-type TrfA-44 with the exception of a single conservative amino acid alteration from methionine to leucine at codon 98; this alteration removes the translational start codon for the TrfA-33 protein. Copy number and stability were virtually identical for plasmids containing both TrfA-44 and TrfA-33 proteins or TrfA-44/98L alone in Pseudomonas aeruginosa and Agrobacterium tumefaciens, two unrelated bacteria in which TrfA-33 is poorly functional. This, along with recent in vitro studies comparing TrfA-44, TrfA-33, and TrfA-44/98L, suggests that the functional activity of TrfA-44 is not significantly affected by the 98L mutation. Analysis of minimal RK2 derivatives in certain gram-negative bacterial hosts suggests a role of the overlapping arrangement of trfA in facilitating the broad host range of RK2. RK2 derivatives encoding TrfA-44/98L alone demonstrated decreased copy number and stability in Escherichia coli and Azotobacter vinelandii when compared with derivatives specifying both TrfA-44 and TrfA-33. A strategy employing the trfA-44/98L mutant gene and in vivo homologous recombination was used to eliminate the internal translational start codon of trfA in the intact RK2 plasmid. The mutant intact RK2 plasmid produced only TrfA-44/98L. A small reduction in copy number and beta-lactamase expression resulted in E. coli, suggesting that overlapping trfA genes also enhance the efficiency of replication of the intact RK2 plasmid.     

Maintenance of pBR322-derived plasmids without functional RNAI

pBR322-derived plasmids that lack the bla gene and 40% of the gene for the replication inhibitor, RNAI, have been constructed. Since the RNAI gene totally overlaps with the gene for the replication primer, RNAII, this primer is similarly defective and also lacks its normal promoter. The primer is presumed to by synthesized either from the counter-tet promoter (plasmid pCL59) or from an inserted lacUV5 promoter (plasmid pCL59-65). Based mainly on the observation that the plasmid Rom protein, which normally assists in the RNAI/RNAII interaction, has no effect on the replication of the RNAI/RNAII-defective plasmids, we suggest that the defective RNAI is not functional while the defective RNAII primer, although less efficient, still allows plasmid replication. The defective plasmids are fully compatible with the intact parent plasmid, indicating that they do not share a common control of replication. In the absence of antibiotics, the bacteria lose the defective plasmid, beginning after 80 generations; under the same conditions, the parent plasmid is retained even after 140 generations. During exponential growth of their host, the number of defective plasmids in a culture increases exponentially with a doubling time either smaller or greater than that of the host cell growth, depending on the growth medium and, in the case of pCL59-65, on the presence or absence of lac inducer IPTG. As a result of these differences in host cell growth and plasmid replication, the plasmids are either gradually diluted out or their copy number continually increases. This shows that, without RNAI, plasmid replication is uncoupled from the host cell growth and not, as usual, adjusted to it. It also implies that the RNAI mechanism is the only means of replication control for ColE1-type plasmids that senses and adjusts the copy number; limiting host factors cannot provide a back-up control to stabilize copy numbers.     

Measurement of gene expression by translational coupling: effect of copy mutations on pT181 initiator synthesis.

We have prepared and analyzed two types of gene fusion between the replication initiator gene, repC, and the reporter gene, blaZ, in order to investigate the relationship between pT181 plasmid copy number and RepC initiator protein production. A series of pT181 copy mutant plasmids, with copy numbers ranging from 70 to 800 copies per cell, were analyzed. In one type of gene fusion used in this study, blaZ was translationally coupled to the C-terminal end of the repC coding sequence such that native forms of both proteins were produced. This gene fusion arrangement, which permitted monitoring of RepC production (as BlaZ activity) by plasmids using the protein for their own replication, demonstrated a linear relationship, with one exception, between RepC production and plasmid copy number over a 20-fold range. In the second type of fusion, blaZ was translationally fused to the C-terminal end of repC. As the translational fusion did not produce active RepC protein, the fusion-containing pT181 derivatives were maintained in a strain which provided RepC in trans, and were thus analyzed at constant copy number. In contrast to previous analyses of this type, our translational fusion constructs expressed repC at levels proportional to the copy numbers of the plasmids from which the fusions were prepared. Using these data, we have calculated a minimum figure for the number of RepC molecules synthesized per replication event.     

Nucleotide sequence analysis of small cryptic plasmid pGP2 from <Emphasis Type="Italic">Acetobacter estunensis</Emphasis>

Complete nucleotide sequence of plasmid pGP2 from Acetobacter estunensis GP2 was identified after initial cloning of EcoRI fragment followed by preparation of deletion derivatives. Its size was defined to 2,797 bp and several sites for several restriction enzymes were revealed by DNA sequencing. Sequence analysis predicts three putative open reading frames (ORFs). ORF1 shows significant identity with the bacterial excinuclease α-subunit, ORF2 is a putative replication protein with low similarity with other Acetobacter plasmid’s replication proteins, and ORF3 encodes a class B acid phosphatase/phosphotransferase. The replication module comprises a DnaA box like sequence, direct repeats, a potential prokaryotic promoter and a rep gene. The rep module is similar with several θ-replicating, iteron-containing modules from plasmids, suggesting pGP2 replication may follow the same course. Any phenotypic character determinant gene is absent in pGP2, suggesting this plasmid to be cryptic. However, a pGP2 derivative plasmid, containing the putative pGP2 rep region, can replicate and is stably maintained in Acetobacter and Escherichia coli strains; it can also carry foreign DNA fragments. Thus, pGP2-X could serve as a cloning shuttle vector between these bacteria. Prepared deletion derivatives of plasmid pGP2 suggested that Rep protein is essential for plasmid replication in host bacteria. In its natural host, A. estunensis GP2, pGP2 maintains a four-times lower copy number than in E. coli.     

A DNA segment conferring stable maintenance on R6K gamma-origin core replicons.

The plasmid R6K gamma origin consists of two adjacent modules, the enhancer and the core, and requires R6K initiator protein pi for replication. While the core alone can replicate at a low level of wild-type pi protein, we show here that host cells do not stably maintain core plasmids. The presence of the enhancer segment confers stable inheritance on core plasmids without a significant change in average plasmid copy number. Deletions and site-directed mutagenesis indicated that the stability of core plasmids is not mediated by binding sites or consensus sequences in the enhancer for DnaA, pi protein, gyrase, Fis, or Dcm methylase. Proper segregation of core plasmids requires only the R6K stb or stability-related region, which includes the 20-bp segment of the 100-bp enhancer adjacent to the core. The use of the pi 116 mutant protein, which increases plasmid copy number fourfold, does not stabilize core plasmids lacking the enhancer. We also show that at an elevated level of wild-type pi, the gamma-origin plasmid is unstable, even in the presence of the enhancer. We discuss the differences and similarities between the R6K stability system and those found in other plasmids.     

Sociobiological Control of Plasmid Copy Number in Bacteria

All genes critical for plasmid replication regulation are located on the plasmid rather than on the host chromosome. It is possible therefore that there can be copy-up “cheater” mutants. In spite of this possibility, low copy number plasmids appear to exist stably in host populations. We examined this paradox using a multilevel selection model. Simulations showed that, a slightly higher copy number mutant could out-compete the wild type. Consequently, another mutant with still higher copy number could invade the first invader. However, the realized benefit of increasing intra-host fitness was saturating whereas that of inter-host fitness was exponential. As a result, above a threshold, intra-host selection was overcompensated by inter-host selection and the low copy number wild type plasmid could back invade a very high copy number plasmid. This led to a rock-paper-scissor (RPS) like situation that allowed the coexistence of plasmids with varied copy numbers. Furthermore, another type of cheater that had lost the genes required for conjugation but could hitchhike on a conjugal plasmid, could further reduce the advantage of copy-up mutants. These sociobiological interactions may compliment molecular mechanisms of replication regulation in stabilizing the copy numbers.     

Replication mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B resistance plasmid pT48

Copy-number mutants of Staphylococcus aureus macrolide-lincosamide-streptogramin B (MLS) resistance plasmid pT48 were isolated by their resistance to the non-inducing macrolide, tylosin. One mutant plasmid, pcopD3, showed a three- to five-fold cis-dominant increase in copy number, and nucleotide sequence analysis revealed that the mutant had a single base change within the replication region. All other pT48 mutants examined had the unusual phenotype of increased plasmid multimerization and elevated copy number. These mutants were effective in trans and DNA sequencing showed that plasmids with this phenotype were deleted in one of two ways. The deletions caused similar alterations to the C-terminus of the wild-type pT48 Rep protein. The two types of mutant Rep proteins terminate with the same pentapeptide sequence: Ala-Asn-Glu-Ile-Asp. The multimerization phenotype of these mutants can be explained by defective termination of rolling-circle type replication.     

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.     

Negative control of plasmid pSC101 replication by increased concentrations of both initiator protein and iterons

Increased intracellular concentrations of the initiator protein Rep (or RepA) interfere with pSC101 DNA replication, and mutated Rep proteins that result in an increase in plasmid copy numbers do not inhibit the replication. A rep mutant (rep(inh)) defective in the inhibitory activity was isolated and found to be a new high copy number mutant. The inhibitory function of Rep was enhanced by the coexistence of directly repeated sequences (DR; iterons) in the replication origin region (ori), but not by the inverted repeat sequences (IR) in ori and the rep promoter. This synergistic effect of Rep and DR sequences for the replication inhibition was dependent on their intracellular concentrations. Considering that DR sequences are the specific binding sites of the Rep monomer form, the Rep monomer-DR complex might be responsible for the inhibition of the plasmid replication. Furthermore, the Rep monomer in the crude cell extracts facilitated dimerization of DR DNA fragments by DNA ligase. Neither synergistic inhibitory function with DR nor Rep mediated dimerization of DR DNA was observed in high copy number mutant Rep proteins. The role of the Rep-iteron complex in the copy number control of pSC101 is discussed.     

Translational control by antisense RNA in control of plasmid replication

Control of replication of plasmids involves two processes: measurement of the copy number of the plasmid and adjustment of the replication frequency accordingly. For both these processes IncFII plasmids use an antisense RNA (CopA RNA) that forms a duplex with the upstream region (CopT) of the mRNA of the rate-limiting RepA protein. The kinetics of duplex formation was measured in vitro for the wild type and for a cop mutant plasmid; the mutant showed a reduction in the second-order rate constant for the formation of the RNA duplex and a similar increase in copy number. Hence, the kinetics of duplex formation and the concentration of CopA RNA determines the copy number of the plasmid.