Homology between Escherichia coli plasmids ColE1 and p15A.

The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented.     

Incompatibility properties of Col E1 and pMB1 derivative plasmids: Random replication of multicopy replicons

The incompatibility properties of Col E1-like plasmids have been examined in Rec⁺ and RecA^? bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.     

Phasmids: hybrids between ColE1 plasmids and E. coli bacteriophage lambda

Plasmids carrying cloned lambda att sites may be integrated into the bacteriophage genome by the site-specific recombination mechanism of lambda. The cross, referred to as "lifting" the plasmid, requires mixed infection of an Escherichia coli strain carrying the plasmid with two appropriately constructed "lifting" lambda phages. One phage donates a short left arm and the other donates a short right arm. These two short arms are of insufficient length to produce a viable phage genome and yield no recombinants when crossed on standard bacteria. However, viable recombinants are obtained when the genome length is extended by integration of one or more plasmids. We call these recombinants phasmids. They contain multiple att sites introduced at the ends of the integrated plasmids, and in the presence of integrase, recombination between these att sites can be exploited to effect release of the plasmid components. These novel genetic elements can be used in a variety of ways as vectors in genetic manipulation experiments. Sequences cloned in phasmids may be studied as a component of either a plasmid and or of a phage, and easily interconverted between the two states.     

Incompatibility and transforming efficiency of ColE1 and related plasmids

Summary Replicons derived from the ColE1 plasmid are incompatible with one another, but are compatible with their naturally occurring relatives ColK and CloDF13. The incompatibility results in loss, by segregation, of one or the other ColE1 plasmid. In most cases, the smaller derivatives tend to displace the larger ones, and the rate of displacement depends on the difference in size. One mini-plasmid retains only 19% of the sequences of ColE1, yet it exrrts strong incompatibility: other ColE1 plasmids are rapidly lost when it is introduced into the host. The region essential for ColE1 incompatibility is deduced to lie within 700 base pairs of the origin of replication.The transforming efficiency of any ColE1 plasmid is markedly lowered when another incompatible replicon is resident in the competent cells, even when the transforming plasmid is much smaller than the resident.A model of incompatibility is proposed to account for these effects.     

Molecular homology and incompatibility relationships between F and IncH1 plasmids

IncH1 plasmids and the F plasmid of Escherichia coli display one-way compatibility. An entering IncH1 plasmid is incompatible with a resident F plasmid, but is compatible when it is the resident plasmid. There is little molecular homology between IncH1 plasmids and the F plasmid. A single 5 MDal EcoRI restriction enzyme fragment from digests of several IncH1 plasmids hybridizes with probes constructed from the primary replication region of F. Homology can be demonstrated only with the gene for the essential replication protein of F (gene E), but the expression of incompatibility behaviour appears to be associated with the presence of the secondary replicon of the F plasmid. Thus R27 and F are compatible under growth conditions allowing replication and maintenance of F by the secondary replicon. However, a mutant F plasmid which lacks the secondary replicon of F is incompatible with R27 in both directions, irrespective of the growth conditions used.     

Replication of ColE2 and ColE3 plasmids: Two ColE2 incompatibility functions

Summary We have identified and localized two incompatibility determinants (IncA and IncB) within a 1.3 kb segment of ColE2 sufficient for autonomous replication. The IncA determinant is localized in a region shorter than 250 bp and expresses incompatibility against both ColE2 and ColE3. The region which determines sensitivity to the IncA determinant seems to overlap with the region specifying the IncA determinant. The expression of the trans-acting factor(s) specifically required for replication of ColE2 interferes with expression of the IncA determinant against ColE2 but not against ColE3. The IncA determinant might be at least partly responsible for the copy number control of the plasmid. The IncB determinant is localized in a 50 bp region (origin) which is sufficient for initiation of replication in the presence of the trans-acting factor(s). The IncB determinant is specific for ColE2 and seems to be due to titration of the trans-acting essential replication factor(s) by binding.     

Isolation and characterization of replication-deficient mutants of ColE1 plasmids.

Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.     

An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.     

Maintenance of some ColE1-type plasmids in chemostat culture

Summary When cells carrying the plasmids RP1, pDS4101 (a ColK derivative) or pDS1109 (a ColE1 derivative) were maintained in chemostat culture in the absence of antibiotic selection, plasmid-free segregants were not detected after 120 generations of nutrient-limited growth. By contrast, plasmid-free segregants of pMB9- and pBR322-containing cells arose after approximately 30 generations, irrespective of the host genetic background. However, even though pDS1109 was maintained its copy-number fell five-fold during 80 generations of limited growth. It is suggested that loss of pBR322 occurs following a similar copy-number decrease which results in defective segregation of the plasmid to daughter host cells. This defective segregation was not complemented in trans by either RP1 or pDS4101.     

Genetic homology between independently isolated chlorobenzoate-degradative plasmids

Two chlorobenzoate-degradative plasmids were studied by the hybridization of the restriction endonuclease-generated fragments of one plasmid after transfer to a nitrocellulose filter with nick-translated radioactive DNA of the other plasmid as a probe. Two strains harboring the 3-chlorobenzoic acid-degradative plasmids were isolated in two different parts of the world at two different times. The plasmids are now found to be closely related to each other by hybridization studies. The chlorobenzoate-degradative plasmid from Pseudomonas sp. strain B13 (termed pB13) has a 6-kilobase deletion but otherwise is homologous with previously described plasmid pAC25.     

Sequence homology between Inc N group plasmids

DNA-DNA hybridization combined with "Southern blotting" was used to analyse the genetic organization and the nucleotide sequence homology between different regions of a previously characterized Inc N group plasmid pCUI and nine other Inc N group plasmids. The following conclusions could be reached: (1) N plasmids isolated from different parts of the world share substantial DNA sequence homology and also some similarity of overall genetic organization, (2) the majority of the N plasmids used in this study showed conservation of distribution of BglII and KpnI cleavage sites. Often, restriction endonuclease fragments of similar electrophoretic mobility encoded the same genetic function, (3) in one case, the N-specific properties appear to be integrated into the bacterial chromosome. (4) the plasmid DNA in strains carrying two Inc N plasmids, R199 and R113 were each composed of two molecular species only one of which constituted an N group plasmid.     

Functional relationship between parts of the replication region of plasmid ColE1.

The inhibition of plasmid ColE1 replication caused by a deletion of the ColE1 plasmid replication origin has been previously reported (T. Hashimoto-Gotoh and J. Inselburg, J. Bacteriol. 139:597-619). Evidence is presented showing that restoration of the deleted nucleotide sequence in the precise relationship it normally has to the rest of the replication region is essential for restoration of ColE1 replication capability to the deletion mutant.     

Analysis of the physiological control of replication of ColE1-type plasmids

The physiology of ColE1-type plasmid replication in a growing host has been examined both theoretically, using computer simulation, and experimentally, by observing replication of the plasmid pBR322 after a nutritional shift-up from glycerol minimal medium (doubling time 71 min) to LB medium (doubling time 24 min). The theory was based on a negative control model and uses three rate equations: for the accumulation of cell mass, for the accumulation of the replication inhibitor, and for the rate of plasmid synthesis. The implications of the theory were explored by simulating the effects of changes in the expression of replication control genes. The nutritional shift-up experiment showed that plasmid replication was blocked immediately after the shift for about half a mass doubling time; after that time, replication rapidly increased until plasmid numbers per unit volume of culture parallelled the increase in culture mass. After the establishment of steady-state growth in the post-shift medium, the plasmid concentration (plasmids per cell mass) was reduced in comparison to pre-shift growth in the same proportion as the culture doubling time. The results showed that plasmid replication factors are under metabolic control and that the changes in the control of these factors compensate one another during steady-state growth, but not immediately after the medium shift.     

pMB9 plasmids bearing the Salmonella typhimurium his operon and gnd gene

A plasmid containing the entire Salmonella typhimurium his operon was constructed from plasmid pM89 and an EcoRI fragment of phi 80 his imm lambda DNA. The recombinant pST41 also includes the glucose 6-phosphate dehydrogenase (gnd) gene and has one EcoRI endonuclease cleavage site in the integrated fragment. This plasmid served as a source for the construction of two additional plasmids, one carrying the OGDC-region of the his operon and the other a CBHAFIE segment of the his gene along with the gnd gene. The presence of the his operon in the constructed plasmids was confirmed by hybridization to S. typhimurium his RNA. The location of the gnd gene in the CBHAFIE fragment of the his gene was confirmed genetically: after transfection with the plasmid bearing the gnd gene, a gnd recipient gained the capacity to utilize gluconate as a sole carbon source. The DNAs of the three hybrid plasmids were analyzed by gel electrophoresis. By comparing the EcoRI endonuclease cleavage pattern of these three hybrid plasmids with the DNA cleavage pattern of phi 80 his imm lambda, phi 80 imm lambda and lambda phages, the EcoRI cleavage map of phi 80 his imm lambda was obtained.     

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other transposon insertions near polA. Reciprocal transduction to Met(+) Pol(+) or to Fad(+) Pol(+) ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.     

Salmonella enterica subsp. enterica serovar Enteritidis harbours ColE1, ColE2, and rolling-circle-like replicating plasmids

Using DNA hybridization, at least three distinct groups of low molecular mass plasmids were identified in Salmonella enterica subsp. enterica serovar Enteritidis. After sequencing representative plasmids from each group, we concluded that they belonged to ColE1, ColE2, and rolling-circle-like replicating plasmids. Plasmid pK (4245 bp) is a representative of widely distributed ColE1 plasmids. Plasmid pP (4301 bp) is homologous to ColE2 plasmids and was present predominantly in single-stranded DNA form. The smallest plasmids pJ (2096 bp) and pB (1983 bp) were classified as rolling-circle-like replicating plasmids. Both encoded only a single protein essential for their own replication, and they must have existed in an unusual molecular structure, as (i) they were capable of hybridization without denaturation, (ii) their DNA could be linearized with S1 nuclease, and (iii) even after such treatment, the ability to hybridize without denaturation did not disappear.     

Stability of ColE1-like and pBR322-like plasmids in Escherichia coli

The average copy number, the level of ampicillin resistance conferred by one plasmid, and the degree of plasmid multimerization were determined for several ColE1-like and pBR322-like plasmids. From the results obtained, the variance of the units of partition corresponding to each plasmid studied was calculated. Experimentally determined plasmid stability was compared with that calculated using the variance of the units of partition and the ratio between the generation times of plasmid-free and of plasmid-carrying cells, assuming that the units of partition are distributed randomly between daughter cells. Stability of the pBR322-like plasmids present mainly as monomers in the bacterial host was consistent with random partitioning, whereas pBR322-like plasmids, present mainly as dimers, and the ColE1-like plasmid showed greater stability than that predicted with random partitioning at cell division.     

Characterization of the ColE1 mobilization region and its protein products

Summary A third of the 6.6 kb genome of ColE1 is devoted to mobilization (mob) genes necessary to promote its specific transfer in the presence of conjugative plasmids. Themob region is genetically complex: twomob genes are entirely overlapped by a third. Oligonucleotide-directed mutagenesis was used to insert an amber codon into one of the overlapped genes and make possible a full complementation analysis ofmob. Fourmob genes essential for mobilization by R64drd11 were thus identified. Fragments ofmob were subcloned under control of the P_tac promoter in a suitable vector, overexpressed in minicells and the mobilization proteins visualized. A comprehensive alignment of themob region of ColE1 with those of its close relatives ColK and ColA demonstrating that the four essentialmob genes are conserved is also presented.     

Replication of ColE2 and ColE3 plasmids: The regions sufficient for autonomous replication

Summary We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions. A 1.3 kb segment of each plasmid is sufficient for autonomous replication. Plasmids carrying this segment retain the replication properties of the original plasmid. The 1.3 kb segment consists of three functional portions. Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid. Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter. The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid. The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s). The action of the trans-acting factor(s) on the origin is plasmid specific. The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.     

Replication of ColE2 and ColE3 plasmids In vitro replication dependent on plasmid-coded proteins

Summary We developed an in vitro replication system for ColE2 and ColE3 plasmids using cell extracts prepared from bacteria with or without these plasmids. DNA synthesis depended on host DNA polymerase I and was sensitive to rifampicin and chloramphenicol. Preincubation of the extracts with plasmid DNA, however, allowed replication of template DNA added subsequently in a plasmid-specific manner in the presence of rifampicin and chloramphenicol. The plasmid-specified trans-acting factor(s) was detected in cell extracts from bacteria carrying a recombinant plasmid with the region of ColE2 or ColE3 encoding the Rep protein. The plasmid-specified factor(s) consisted at least in part of protein, probably the Rep protein. In vitro replication started within a region of ColE2 or ColE3 containing the smallest cis-acting segment essential for in vivo replication and proceeded in a fixed direction.