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.     

Characterization of plasmids from Haemophilus parainfluenzae and Haemophilus haemolyticus.

The laboratory strains of Haemophilus parainfluenzae and Haemophilus haemolyticus that are commonly used as restriction enzyme sources carry several small multicopy plasmids. H. parainfluenzae carries plasmids pKC1, pKC2, and pKC3 of sizes 1.50, 2.86, and 3.84 kb, respectively, as determined by gel electrophoresis and electron microscopy. H. haemolyticus carries plasmids pKC4 and pKC5 of sizes 1.3 and 1.7 kb as determined by gel electrophoresis. At least two of the plasmids pKC1 and pKC4 were successfully transferred into E. coli by cotransfection with plasmid pBR322. They are compatible with pBR322 and have a comparable copy number.     

Copy number control and incompatibility of plasmid R1: Identification of a protein that seems to be involved in both processes

Summary Investigations into the genetic determinants for incompatibility of miniplasmids and hybrid replicons constructed from wild type and mutant R1 revealed the presence of an incompatibility function at the junction of two small PstI fragments. These two fragments were not distinguished in earlier experiments since they have the same mobility on agarose gels. This incompatibility function is distinct from other inc-determinants of R1 (Kollek and Goebel 1979; Molin and Nordström, 1980) and independent of R1-type replication. By means of specific deletions and subcloning of DNA fragments, the location of this new inc-determinant could be determined further. After deletion of this inc-determinant from miniplasmids, a 5-fold increase in copy number was observed which could then be reduced to a copy number of about 1 plasmid per cell by complementation with hybrid plasmids having this function. Incompatibility of miniplasmids deleted in this determinant is not reduced, whereas analogous deletions introduced into recombinant plasmids nearly abolish their incompatibility. This determinant seems to exert strong incompatibility only when cloned on pBR322. Therefore, its main function in plasmid R1 is probably restricted to copy control. The appearance of low copy numbers of miniplasmids carrying this determinant and of trans-acting copy control and strong incompatibility exerted by hybrid plasmids is consistently correlated with the presence of a protein of 11,000 molecular weight, synthesized in relatively large amounts in Escherichia coli minicells.     

Cloning of a Bacillus subtilis restriction fragment complementing auxotrophic mutants of eight Escherichia coli genes of arginine biosynthesis

Summary Following shotgun cloning of EcoRI fragments of Bacillus subtilis 168 chromosomal DNA in pBR322 a hybrid plasmid, pUL720, was isolated which complements Escherichia coli K12 mutants defective for argA, B, C, D, E, F/I, carA and carB. Restriction analysis revealed that the insert of pUL720 comprises four EcoRI fragments, of sizes 12.0, 6.0, 5.0 and 0.8 kbp. Evidence was obtained from subcloning, Southern blot hybridisation, enzyme stability studies and transformation of B. subtilis arginine auxotrophs that the 12 kbp EcoRI fragment carries all the arg genes. It proved impossible to subclone the intact fragment in isolation in the multicopy vectors pBR322, pBR325 or pACYC184, and although it could be subcloned in the low copy vector pGV1106, propagation of the hybrid rapidly resulted in the selection of stable derivatives carrying, near one end, an insertion of 1 kbp of DNA originating from the E. coli chromosome. These and other stable derivatives resulting from subcloning the 12 kbp EcoRI fragment have lost only the ability to complement for E. coli argC, and it is suggested that sequences located close to the equivalent of argC are involved in destabilising plasmids bearing the 12 kbp fragment in E. coli in a copy number dependent manner.Abbreviations kop kilobase pairs - OcTase ornithine carbamoyl transferase - CPSase carbamoyl phosphate synthetase     

Boundaries of the Origin of Replication: Creation of a pET-28a-Derived Vector with p15A Copy Control Allowing Compatible Coexistence with pET Vectors

During our studies involving protein-DNA interactions, we constructed plasmid pSAM to fulfill two requirements: 1) to facilitate transfer of cloned sequences from widely used expression vector pET-28a(+), and 2) to provide a vector compatible with pBR322-derived plasmids for use in cells harboring two different plasmids. Vector pSAM is a pET-28a(+)-derived plasmid with the p15A origin of replication (ori); pET-28a(+) contains the pBR322 replicon that is incompatible with other pBR322-derived plasmids. By replacing the original pET-28a(+) replicon–comprising the ori, RNAI, RNAII, and Rom–with the p15A replicon, we generated pSAM, which contains the pET-28a(+) multiple cloning site and is now compatible with pBR322-derived vectors. Plasmid copy number was assessed using quantitative PCR: pSAM copy number was maintained at 18±4 copies per cell, consistent with that of other p15A-type vectors. Compatibility with pBR322-derived vectors was tested with pGEX-6p-1 and pSAM, which maintained their copy numbers of 49±10 and 14±4, respectively, when both were present within the same cell. Swapping of the ori is a common practice; however, it is vital that all regions of the original replicon be removed. Additional vector pSAMRNAI illustrated that incompatibility remains when portions of the replicon, such as RNAI and/or Rom, are retained; pSAMRNAI, which contains the intact RNAI but not ROM, lowered the copy number of pGEX-6p-1 to 18±2 in doubly transformed cells due to retention of the pET-28a(+)-derived RNAI. Thus, pSAMRNAI is incompatible with vectors controlled by the pBR322 replicon and further demonstrates the need to remove all portions of the original replicon and to quantitatively assess copy number, both individually and in combination, to ensure vector compatibility. To our knowledge, this is the first instance where the nascent vector has been quantitatively assessed for both plasmid copy number and compatibility. New vector pSAM provides ease of transferring sequences from commonly used pET-28a(+) into a vector compatible with the pBR322 family of plasmids. This essential need is currently not filled.     

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.     

The Agrobacterium rhizogenes pRi TL-DNA segment as a gene vector system for transformation of plants

Summary A plant gene transfer system was developed from the Agrobacterium rhizogenes pRi15834 TL-DNA region. Intermediate integration vectors constructed from ColE1-derived plasmids served as cloning vectors in Escherichia coli and formed cointegrates into the TL-DNA after transfer to A. rhizogenes. An A. rhizogenes strain with pBR322 plasmid sequences replacing part of the TL-DNA was also constructed. Plasmids unable to replicate in Agrobacterium can integrate into this TL-DNA by homologous recombination through pBR322 sequences. No loss of pathogenicity was observed with the strains formed after integration of intermediate vectors or strains carrying pBR322 in the TL-DNA segment. Up to 15 kb of DNA have been transferred to plant cells with these systems. The T-DNA from a binary vector was cotransformed into hairy roots which developed after transfer of the wild-type pRi T-DNA. Tested on Lotus corniculatus the TL-derived vector system transformed 90% of the developed roots and the T-DNA from the binary vector was cotransformed into 60% of the roots. Minimum copy numbers of one to five were found. Both constitutive and organ-specific plant genes were faithfully expressed after transfer to the legume L. corniculatus.     

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.     

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives

Summary We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.     

An F-derived conjugative cosmid: Analysis of tra polypeptides in cosmid-infected cells

The genes involved in the conjugational transfer of F plasmid DNA are organized into three closely linked operons spanning an overall length of approximately 33 kilobase pairs of F. The entire transfer (tra) region comprising all three operons has been cloned into the cosmid vector pHC79 by in vitro recombination and packaging techniques. The transfer-proficient chimeric cosmid pRS2405 was packaged into λ capsids, and uv-irradiated E. coli cells were infected with these DNA-filled particles. A number of polypeptides programmed by the infecting DNA were identified as tra-specified products; a traJ90 mutation on pRS2405 resulted in the significant reduction of synthesis of all detectable pRS2405-specified tra polypeptides, with the exception of TraTp.     

Evaluation of several enrichment procedures for the isolation of recombinant plasmid DNA

A number of methods for the selective enrichment of recombinant plasmids were examined; these include alkaline phosphatase treatment of the restricted pBR322 vector, as well as a combination of this and S1 nuclease treatment of the ligated mixture of pBR322 and pCR1 plasmids orS. griseus DNA followed by D-cycloserine treatment to enrich for cells carrying recombinant molecules. The relative efficiencies of these methods were compared.     

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Elimination of ColE1 (pBR322 and pBR329) plasmids in Escherichia coli by α-santonin

alpha-Santonin, a compound extracted from the flower heads of Arthemisia maritima plant, was effective in elimination of small, multicopy, relaxed plasmids (pBR322 and pBR329 with CoLE1 origin of replication in Escherichia coli, whereas plasmids of IncF1, H1 and X group were totally refractory under similar conditions, suggesting that this agent was specific in curing the CoLE1 group of plasmids.     

Recombination between short direct repeats in a RecA host

Summary Spontaneous deletion derivitives of plasmid pHV14, a recombinant of pBR322, have been isolated in the recA strain HB101. About 2.5 kilobases (Kb) of DNA has been lost in each of the four plasmids described including, in two cases the region involved in control of copy number and the initiation of replication.Sequence analysis of each deletion junction has revealed that in 3 out of 4 cases the deletion event occured by recombination between short direct repeats of as little as 7 base pairs (bp).     

Cloning, restriction endonuclease mapping and post-transcriptional regulation of rpsA, the structural gene for ribosomal protein S1

Transducing lambda phages have been isolated that carry segments of the Escherichia coli chromosome in the aspC region, 20.5 min on the E. coli map. One of these phages, lambda aspC2, carries rpsA, the structural gene for the ribosomal protein S1. A three kilobase fragment from this phage, cloned into either the plasmid pACYC184 or the plasmid pBR322, was found to express S1. In cells carrying the rpsA gene on the high copy number plasmid pBR322 the rate of rpsA mRNA synthesis was increased 40-fold, whereas the rate of protein S1 synthesis was doubled, in comparison with these rates in an rpsA haploid.     

Observations on the formation of deletions on monomeric and dimeric plasmids in Escherichia coli

We have studied the formation of spontaneous mutations on plasmids present In the monomeric and dimeric states in a recF strain of Escherichia coli. Two test systems were employed: (i) the precise excision of Tn5 from the tetA gene of the plasmid pBR322 and (ii) operator constitutive (O^c) mutations on the pBR322-derived plasmid pPY97. The rate of O^c mutations was increased by a factor of three when this plasmid was present in the dimeric state compared to the monomeric state and the O^c phenotype was caused by small deletions in the operator sequence. No apparent mutational hot-spot was found. The rate of Tn5 excision was increased on dimeric compared to monomeric plasmids. Excision from a dimeric plasmid usually resulted in two types of mutant plasmids; a dimeric plasmid, where the Tn5 had excised from one of the plasmid units, and a monomeric parental pBR322. A mechanism to account for this is suggested. Complementation tests revealed that the increased mutation rate on dimeric plasmids is the result of dimers being mutaphilic per se, rather than the result of a general, trans-acting increase in mutation rates of the host, induced by the presence of the dimeric plasmid. Furthermore, it was found that the rate of Tn5 excision from plasmids in the monomeric state was increased when the region carrying the inserted Tn5 was duplicated.     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

Role of DnaA protein during replication of plasmid pBR322 in Escherichia coli

Summary The in vivo role of the Escherichia coli protein DnaA in the replication of plasmid pBR322 was investigated, using a plasmid derivative carrying an inducible dnaA ⁺ gene. In LB medium without inducer, the replication of this plasmid, like that of pBR322, was inhibited by heat inactivation of chromosomal DnaA46 protein so that plasmid accumulation ceased 1 to 2 h after the temperature shift. This inhibition did not occur when the plasmid dnaA ⁺ gene was expressed in the presence of the inducer isopropyl-1-thin--d-galactopyranoside (IPTG). Inhibition was also not observed in glycerol minimal medium or in the presence of low concentrations of rifampicin or chloramphenicol. Deletion of the DnaA binding site and the primosome assembly sites (pas, rri) downstream of the replication origin did not affect the plasmid copy number during exponential growth at 30° C, or after inactivation of DnaA by a shift to 42° C in a dnaA46 host, or after oversupply of DnaA, indicating that these sites are not involved in a rate-limiting step for replication in vivo. The accumulation of the replication inhibitor, RNAI, was independent of DnaA activity, ruling out the possibility that DnaA acts as a repressor of RNAI synthesis, as has been suggested in the literature. Changes in the rate of plasmid replication in response to changes in DnaA activity (in LB medium) could be resolved into an early, rom-dependent, and a late, rom-independent component. Rom ^– plasmids show only the late effect. After heat inactivation of DnaC, plasmid replication ceased immediately. These results, together with previously published reports, suggest that DnaA plays no specific role during in vivo replication of ColE1 plasmids and that the gradual cessation of plasmid replication after heat inactivation of DnaA in LB medium results from indirect effects of the inhibition of chromosome replication and the ensuing saturation of promoters with RNA polymerase under nonpermissive growth conditions.