Role of the rom protein in copy number control of plasmid pBR322 at different growth rates in Escherichia coli K-12

The copy number per cell mass of plasmid pBR322 and a rom- derivative was measured as a function of generation time. In fast growing cells the copy number per cell mass was virtually identical for rom+ and rom- derivatives. However, the copy number of pBR322 only increased 3- to 4-fold from a 20- to 80-min generation time, whereas the copy number of the rom- derivative increased 7- to 10-fold. The copy number stayed constant for the rom+ and rom- plasmids at generation times longer than 80-100 min. Thus, the presence of the rom gene decreased the copy number of plasmid pBR322 in slowly growing cells at least 2-fold when compared with the rom- plasmid. To study the effect of the rom gene in trans we cloned the gene into the compatible P15A-derived rom- plasmid pACYC184. In cells carrying both pACYC184 rom+ and pBR322 rom- the presence of the rom gene in trans had little effect on the copy number of pBR322 rom- at fast growth, but it decreased its copy number at slow growth to the same level as found for pBR322, i.e., complemented the pBR322 rom- plasmid. The pACYC184 plasmid and its rom+ derivatives showed copy numbers similar to those of pBR322 rom- and pBR322 itself, respectively, at fast and slow growth. We conclude that the rom gene product-the Rom protein-is an important element in copy number control of ColE1-type plasmids especially in slowly growing cells.     

Increased stability of pBR322-related plasmids in Escherichia coli W3101 grown in carrageenan gel beads

Abstract The stability and the copy number of pBR322, pBR325 and pBR328 were studied during continous cultures of free and immobilized E. coli W3101 without selective pressure. In the free-cell system, it was found that pBR328 and pBR325-free E. coli cells appeared after a lag period. They rapidly overgrew the cultures and the plasmid copy number subsequently declined. On the other hand, an increase in the proportion of pBR322- carrying cells during a free continuous culture was observed. This increase correlated with that of plasmid copy number. By contrast, in the immobilized- cell system, plasmid free segregants were not detected in all the cases even after 250 generations. We have also shown that plasmid copy number remained constant and phenomena such as fluctuations or genetic modifications which occured after long term growth of bacteria in a free continuous culture could be avoided throughout cell immobilization.     

Amplification of bacterial plasmids without blocking protein biosynthesis

The effect of amino acids (presence or absence from the growth media) and metal ions on the replication of Escherichia coli plasmids in rel A+ strains was studied. It was found that: (i) The absence of one amino acid from the growth media had no effect on the plasmid copy number in prototrophic E. coli strains: (ii) The presence of only one amino acid in artificial media free of amino acids had a negligible effect on the plasmid copy number for the amino acids Ala, Arg, Glu, His, Leu, Phe, Thr, Trp, and Tyr: (iii) The combination of Met and Thr caused a rise in pBR322 plasmid copy number up to 90-100 plasmid copies per cell: (iv) The Fe3+ concentration had an amplification effect on E. coli plasmids. The pBR322 plasmid copy number for media free of amino acids and supplemented with 0.2-0.4 mM FeCl3 was 60-80 plasmid copies per cell: (v) The combination of Fe3+ with certain amino acids (Ala, Arg, Glu, Leu, Thr, and Trp) leads to a dramatic increase in the plasmid copy number reaching 180-270 plasmid copies per cell for the plasmid pBR322 and 20-24 for the plasmid pR100.     

Absolute and relative QPCR quantification of plasmid copy number in Escherichia coli

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5alpha harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid beta-lactamase gene (bla) and for the chromosomal d-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs. These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8-3.9% and 4.7-5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15-20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.     

Escherichia coli growth and plasmid copy numbers in continuous cultivations

Summary AnEscherichia coli K-12 strain harbouring either the plasmid pBR322, or the recombinant plasmid pKTH1220, a 14 kb derivative of pBR322, or no plasmid was grown in a chemostat. The cultivations were continued for 300–400 bacterial generations.E. coli hosts harbouring pBR322 or no plasmid grew in a similar way, but the growth of the host containing the big recombinant plasmid was slower. The plasmid copy numbers increased up to 2–3 fold as the dilution rate was increased from 0 to ca. 1 h^–1. After this point the increase in dilution rate seemed to induce a rapid decrease in the plasmid copy numbers. High copy numbers could be maintained using dilution rates resulting in good productivity of the cell mass.     

Adaptation of Escherichia coli growth rates to the presence of pBR322

Changes in the growth rate of Escherichia coli K12 J62-1 in response to the presence of plasmid pBR322 have been investigated. Plasmid-free and plasmid-containing strains were grown in batch culture and their maximum specific growth rate (μ_max) determined. The acquisition of pBR322 by the host resulted in a decreased μ_max. Following repeated subculturing of the plasmid-containing strain on selective medium, restoration in μ_max was observed. The copy number and structure of the plasmid were not significantly altered during the experiment Growth rate measurements for a series of strains constructed using a combination of host cells and plas-mids with and without culture histories, indicated that the site of the adaptive mutation was located on the host chromosome rather than on the plasmid.     

Thermodynamic analysis of the lactose repressor-operator DNA interaction

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.     

Simultaneous regulation of plasmid replication and heterologous gene expression in Escherichia coli.

An expression plasmid in which plasmid DNA replication and heterologous gene expression can be simultaneously regulated was constructed to avoid derepression prior to induction. This was achieved by placing a pBR322 origin of replication immediately downstream of an anthranilate synthase-human epidermal growth factor fusion gene (trpE-hEGF), both under the control of the promoter from the tryptophan biosynthetic operon. Regulation of plasmid copy number ensured tight repression of the trp promoter prior to induction. Upon induction, plasmid copy number increased up to six-fold and the fusion protein accumulated to approximately 12% of total cell protein. Induction experiments with a series of plasmid derivatives with sequentially lower copy numbers revealed that accumulation levels of the TrpE-hEGF fusion protein post-induction correlated well with plasmid copy number. Plasmid constructs where the native trp promoter had been replaced by derivatives deleted of the attenuator resulted in high levels of hEGF accumulation in the tryptophan-free medium prior to induction. Nevertheless, up to two-fold increase in TrpE-hEGF accumulation levels were obtained using the constructs lacking the attenuator compared to those bearing the native trp promoter.     

Homologies between the non-collagenous C-terminal (NC1) globular domains of the alpha 1 and alpha 2 subunits of type-IV collagen

Many promoter-fusion vectors contain an intact beta-lactamase (BLA) gene (bla) to allow measurement of BLA activity as an internal control for plasmid copy number. This approach rests on the assumption that bla is constitutively expressed. To use such vectors for comparison of promoter activity at different growth rates it was necessary to confirm that this is the case under all physiological conditions. The relationship between plasmid copy number and BLA activity at different steady-state growth rates in Escherichia coli HB101 transformed with a ColE1-type plasmid (pBR325) was examined. Both BLA activity and plasmid copy number decreased in a parallel fashion as growth rate increased. This finding was tested further by measuring the growth-rate-regulated expression of the chloramphenicol acetyltransferase (CAT) gene under the control of the rrnB P1 promoter in a plasmid pKK231-1 fusion. The results indicate that BLA activity is a reliable indicator of copy number at a wide range of growth rates and that CAT/BLA ratios can be employed as a valid measure of promoter-specific activity in such plasmid fusions under these different physiological conditions.     

Effects of genes exerting growth inhibition and plasmid stability on plasmid maintenance.

Plasmid stabilization mediated by the parA+ and parB+ genes of the R1 plasmid and the ccd+ and sop+ genes of the F plasmid was tested on a mini-R1 plasmid and a pBR322 plasmid derivative. The mini-R1 plasmid is thought to be unstably inherited owing to a low copy number and to random segregation of the plasmid at cell division, whereas cells harboring the pBR322 derivative used in this work are lost through competition with plasmid-free cells, mainly as a result of the shorter generation time of cells without plasmids. The pBR322 derivative carries a fusion between part of the atp operon of Escherichia coli and the bacteriophage lambda pR promoter, and the cI857 repressor gene. The insertion of sop+ from the F plasmid or parB+ from the R1 plasmid reduced the loss frequency by a factor of 10(3) for the pBR322 derivative and by at least a factor of 10(2) for the mini-R1 plasmid. Insertion of parA+ from the R1 plasmid decreased the loss frequency of the pBR322 derivative by a factor of 10 and that of the mini-R1 plasmid by a factor of 50. When ccd+ from the F plasmid was inserted, the loss frequency of the pBR322 derivative was decreased by a factor of 10, but it had only a marginal effect on the stability of the mini-R1 plasmid. In no case was any significant structural instability of the plasmids observed.     

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.     

Role of Nine Repeating Sequences of the Mini-F Genome for Expression of F-Specific Incompatibility Phenotype and Copy Number Control

An autonomously replicating 2,248-base-pair DNA segment of the mini-F plasmid carries nine 19-base-pair repeating sequences. Five of the repeats are arranged in one direction and form the right cluster, whereas the remaining four repeats are arranged in the opposite direction and form the left cluster (Murotsu et al., Gene 15:257-271, 1981). Each cluster, cloned separately into the multicopy plasmid vector pBR322, exhibited a strong F-specific incompatibility phenotype (FIP). These clusters were thought to be responsible for the expression of IncB and IncC phenotypes, causing a switchoff function on mini-F replication. Mini-F DNA fragments containing two, three, or more than four repeats were inserted into pBR322. Cells carrying these recombinant plasmids exhibited, respectively, no, intermediate, and strong FIP intensity. Cloning of five repeats into pSC101, whose copy number is about 6 in contrast to 20 for pBR322, resulted in an FIP of intermediate intensity. Thus, the intensity of FIP reflects the dosage of repeats in a cell. The five repeats in the right cluster were eliminated from the mini-F derivative without impairing its autonomous-replicating ability (Bergquist et al., J. Bacteriol. 147:888-889, 1981; Kline and Palchavdhuri, Plasmid 4:281-289). Such deletion, however, caused a sixfold elevation of the copy number. When the eliminated cluster of repeats was reinserted in the derivative, the copy number was lowered to the original value, viz., 1 to 2. The position and orientation of this insertion was not important in the copy number control. Thus, the repeats are also related to copy number control. A model to account for the role of the repeating sequences in the control of copy number and FIP is discussed.     

Construction of new vectors for cloning of promoters

Vectors for cloning promoter-DNA fragments were derived from plasmid pBR313 (Bolivar et al., 1977). These have several unique restriction sites and carry the trpA gene from Escherichia coli as a selective marker. The selection is based on an enhancement of the growth rate of those bacteria in which the expression of trpA is directed by the cloned promoter. The expression of trpA can be determined quantitatively, independently of the copy number of the vector, and should reflect the apparent strength of the promoter, since the DNA segment located before trpA contains translational stop signals in all three reading frames.     

Point mutations in a pBR322-based expression plasmid resulting in increased synthesis of bovine growth hormone in Escherichia coli

The bGH cDNA coding for bovine growth hormone (bGH) is expressed poorly in Escherichia coli using a pBR322-based expression plasmid. Random mutagenesis of the plasmid gave rise to two types of plasmid mutants which increased the expression of bGH. One class had single base changes in the first four codons of the bGH sequence. The second class had single base changes in regions of the plasmid involved in controlling plasmid replication but had little effect on plasmid copy number.     

A family of arabinose-inducible Escherichia coli expression vectors having pBR322 copy control

The pBAD series of expression vectors have been widely used in Escherichia coli, Salmonella enterica, and related bacteria. However, a complication with pBAD24, the most popular of these plasmids, is that it does not contain the complete functional replication origin of pBR322 as was depicted in the original paper. Instead, pBAD24 has a pBR322-derived origin that lacks the rop gene that negatively regulates copy number and thus pBAD24 has an appreciably higher copy number than that of pBR322, particularly at elevated growth temperatures. A rop-containing derivative of pBAD24 (called pBAD322) having the copy number of pBR322 is reported together with derivatives of pBAD322 that encode resistance to chloramphenicol, kanamycin, tetracycline, spectinomycin/streptomycin, gentamycin, or trimethoprim in place of ampicillin.