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.     

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.     

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.     

A DNA segment within the colicin E1 structural gene on ColE1 affecting immunity to colicin

Summary Insertion of DNA at the EcoRI site of ColE1 results in increase of immunity to colicin killing in E. coli harboring such recombinant ColE1 plasmid as compared to E. coli (ColE1). This effect is neither due to cis or trans interactions originating from the inserted foreign DNA fragment, nor to changes in plasmid copy number. This defect in the immunity mechanism is not trans complemented for by wild type ColE1. Increase in immunity can also be obtained by deleting a DNA segment from the ColE1 genome. This segment is 120 bp left to the EcoRI site within the colicin structural gene. It is concluded that the structure of DNA per se, around the EcoRI site, within colicin structural gene, is the structure which affects immunity expression.     

Nucleotide sequences from the colicin E5, E6 and E9 operons: Presence of a degenerate transposon-like structure in the ColE9-J plasmid

Summary The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, theircis- ortrans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys^*) present in the ColE9-J plasmid. The ColE6 gene organisation, in the ordercol-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%–94% homologous. In ColE9-J, the genes are organised ascol-imm-lys ^*-E5imm-lys. The E9col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which istrans-acting. Neither the predicted structures of E5Imm[E9] nor thecis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conservedbtuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene,lys ^*. The presence of a transposon-like element in the ColE9 plasmid exemplifies a new phenomenon relevant to the evolution of colicin E plasmids. Issued as NRCC publication no. 30065     

Sequence heterogeneity and differential expression of the α-Amy2 gene family in wheat

Summary Within plasmid pUB110 we have identified a 1.2 kb segment necessary and sufficient for driving autonomous replication in Rec⁺ cells at a wild-type copy number. This region can be divided into three functionally discrete segments: a 24 base pair (bp) region that acts as an origin, a 949 bp determinant of an essential replication protein, repU, and a 358 bp incompatibility region, incA, overlapping with the repU gene. The synthesis of the IncA determinant/s proceeds in the direction opposite to that of RepU. The positively (RepU) and negatively (IncA) trans-acting products seem to be involved in the control of plasmid replication. The RepU product has an M_r of 39 kDa, could be overproduced in Escherichia coli, and binds to the pUB110 origin region. Outside the minimal replicon a cis-acting, orientation dependent, 516 bp determinant is required (i) to compete with a coexisting incompatible plasmid and (ii) for segregational stability.     

Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment

Summary An analysis was carried out on the replication functions within a 2.3 kilobase (kb) segment of the F plasmid which contains an origin (ori S) of replication and is capable of autonomous replication inEscherichia coli. Two separable regions were delineated for this segment: an origin region of approximately 1,140 bp in length and a segment of approximately 1,400 bp that functionsin trans to support replication of the origin region. The trans-acting segment is functional as part of an F replicon or when inserted into theE. coli chromosome. A prominent feature of the trans-acting segment is a coding sequence for a 29 K protein (Murotsu et al. 1981).     

Excision of a DNA sequence determining kanamycin resistance from a ColE1-Km recombinant plasmid

Summary A 4.8×10⁶ dalton ECoRI-generated fragment of the R-factor R6-5 carrying the gene for kanamycin resistance (Km) was joined in vitro to ECoRI-treated ColE1 plasmid DNA. Transformation ofE. coli with the ColE1-Km recombinant plasmid yielded clones, which were immune to colicin E1, resistant to kanamycin and failed to produce colicin E1. During multiplication of this recombinant plasmid in the presence of chloramphenicol, cells expressed an increased resistance to kanamycin. Transformation studies with the recombinant DNA molecule showed very frequent loss of Km resistance in those cells harbouring a preexisting F'gal plasmid. Since colicin immunity is not affected and the col^- phenotype is still present, one has to test for a remaining DNA sequence further existing in ColE1 DNA by cleaving the plasmid DNA with the ECoRI restriction endonuclease. The full length of ColE1 DNA (6.2 kb) was restored, which confirmed that no deletion of ColE1 DNA sequences had occured. The remaining DNA sequence was identified as a 2.0 or 2.2 kb segment. On the basis of the length of the excised fragment it is proposed that the insertion sequence IS1 and a part of the inverted repeat sequence with coordinates 21.0 to 22.0 of the R6-5 DNA are recognised by a nucleolytic function.     

ColE1 plasmid mobility: Essential and conditional functions

Summary Sequences essential for the conjugal transfer of ColE1 can be divided into a cis-acting site and a region encoding trans-acting products. Each of these was successively cloned into a non-transmissible plasmid vector. The resulting chimera was transmissible by the conjugative plasmids F'lac,pro (incFI) and R64drd11 (incI). The sequences encoding colicin E1, immunity, and incompatibility were absent from this chimera: therefore they are not essential for the conjugal transmission of the ColE1 plasmid.In contrast to ColE1, however, the same chimera was deficient in conjugal transfer initiated by R751 (incP) and R388 (incW). This suggests that ColEl sequences other than those cloned in the chimeric plasmid are necessary for its mobilization by R751 and R388. Three such regions were revealed by screening a series of ColE1 insertion mutants for transfer by R751 and R388. Two of these regions encode no other known function while the third is encoded by a region which overlaps the gene for colicin E1 itself.     

Cloning of the ColE3-CA38 colicin and immunity genes and identification of a plasmid region which enhances colicin production

The colicin and immunity genes of plasmid ColE3-CA38 have been localized by characterization of bacteria carrying its cloned restriction fragments. They are within a 3.14-kb EcoRI segment, such that the immunity gene contains the KpnI site, and the colicin gene is adjacent to it within a 2.1-kb KpnI-HincII segment. The immunity gene and one end of the colicin gene are in the region of ColE3-CA38 which is not homologous to the closely related plasmid ColE2-P9. A 0.64-kb PvuI-EcoRI segment of the plasmid adjacent to that containing the colicin and immunity genes was found to augment colicin production on solid media, and also affected the morphology of clearing zones produced by the cells when used as indicators in overlays of stabs of colicin E2 or E7 producers. The 0.64-kb segment was required in its native orientation relative to the 3.14-kb EcoRI segment to cause its effects.     

Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin

Summary Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5 end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.     

Replication of the Streptomyces plasmid pSN22 through single-stranded intermediates

The replication of the 11 kb conjugative multicopy Streptomyces plasmid pSN22 was analyzed. Mutation and complementation analyses indicated that the minimal region essential for plasmid replication was located on a 1.9 kb fragment of pSN22, containing a trans-acting element encoding a replication protein and a cis-acting sequence acting as a replication origin. Southern hybridization showed that minimal replicon plasmids accumulated much more single-stranded plasmid molecules than did wild-type pSN22. Only one strand was accumulated. A 500 by fragment from the pSN22 transfer region was identified which reduced the relative amount of single-stranded DNA, when added in the native orientation to minimal replicon plasmids. This 500 by DNA sequence may be an origin for second-strand synthesis. It had no effect on the efficiency of co-transformation, plasmid incompatibility, or stability. The results indicate that pSN22 replicates via single-stranded intermediates by a rolling circle mechanism.     

Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1

It has previously been concluded that regions tentatively designated trfA and trfB, located at 16–18.7 and 54–56 kb, respectively, on the genome of broad host range plasmid RK2 provide trans-acting functions involved in plasmid replication and maintenance in Escherichia coli (Thomas et al., 1980). A third region, the replication origin, ori_RK2, located at 12 kb on the genome, is also required. A segment of DNA containing ori_RK2 can be linked to a nonreplicating selective marker and can replicate as an autonomous plasmid so long as DNA of RK2 carrying the gene for one or more trans-acting replication functions is present in the same cell on an independent plasmid or integrated into the chromosome. It is demonstrated here that the trfA region alone can provide the trans-acting functions necessary for replication from ori_RK2. Deletion of the trfB region in trans to an ori_RK2 plasmid does not correlate with alteration in copy number or stability of the ori_RK2 plasmid. Temperature-sensitive mutants defective in plasmid maintenance can apparently arise from mutations in both the trfA and trfB regions as indicated by complementation analysis of three different mutants. The trfA and trfB regions from two mutant plasmids have been cloned and used to allow a physically separate but functionally dependent ori_RK2 plasmid to replicate at 30 °C. When the source of trfA and trfB is a trfB mutant the ori_RK2 plasmid is temperature stable but is temperature sensitive when the source is a trfA mutant. This confirms that only trfA is essential for initiation at and elongation from ori_RK2 which is probably the primary event in RK2 replication and suggests that the trfB region plays some other role in plasmid maintenance in plasmids carrying all three regions, ori_RK2, trfA, and trfB.     

Identification of functional regions of the colicinogenic plasmid ColA

Summary ColA is a colicinogenic plasmid of 6.72 kb. It is compatible with ColE1 but not with ColK. Transposon insertion mutagenesis as well as complementation studies have been carried out to investigate the location of the various functional regions of this plasmid. Four independent ColA::Tn1 and one ColA::Tn3 plasmids were isolated and the locations of insertions were determined. From these plasmids, six different deletion mutants were constructed. In addition, various restriction fragments of ColA have been cloned into pUC8 to carry out complementation studies. We have thus confirmed the location of the DNA regions involved in colicin production, colicin release and immunity function. The DNA region involved in conjugal mobility promoted by R64 drd11 has been identified and we have demonstrated that the ColE1 mobility proteins can act in trans on the bom (basis of mobility) site of ColA. The location of this site, as well as the region involved in stable maintenance of ColA, have also been determined. These results are discussed with regard to the homology in nucleotide sequence between ColA and ColE1.     

Nucleotide sequence of small ColE1 derivatives: Structure of the regions essential for autonomous replication and colicin E1 immunity

Summary A small ColE1 derivative, pAO2, which replicates like the original ColE1 and confers immunity to colicin E1 on its host cell has been constructed from a quarter region of ColE1 DNA (Oka, 1978). The entire nucleotide sequence of pAO2 (1,613 base pairs) was determined based on its fine cleavage map. The sequence of a similar plasmid, pAO3, carrying additional 70 base pairs was also deduced.The sequence in the region covering the replication initiation site on these plasmids was consistent with those reported for ColE1 by Tomizawa et al. (1977) and by Bastia (1977). DNA sequences indispensable for autonomous replication were examined by constructing plasmids from various restriction fragments of pAO2 DNA. As a result, a region of 436 base pairs was found to contain sufficient information to permit replication. The occurrence of initiation and termination codons and of the ribosome-binding sequence on pAO2 DNA suggests that a polypeptide chain consisting of 113 amino acid residues may be encoded by the region in which the colicin E1 immunity gene has been mapped.Abbreviations ColE1 colicin E1 plasmid - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediaminetetraacetate - dNTP deoxyribonucleoside triphosphates - ATP adenosine 5-triphosphate     

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.     

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene

Summary A 37.5 kb region encompassing a set of the naphthalene degrading genes on the Pseudomonas plasmid NAH7 was found to be transposable only in the presence of the transposase encoded by the Tn1721 subgroup of the class II transposons. This newly identified mobile element, designated Tn4655, contained short (38 bp) terminal inverted repeats which shared extensive sequence homology with those of members of the Tn1721 subgroup. Tn4655 transposed by a two-step process involving formation of the cointegrate followed by its subsequent resolution. In contrast to the defect in the trans-acting factor for the first step, a functional system for the latter step was encoded within a 2.4 kb region in Tn4655. Analysis of deletion and insertion mutants demonstrated that the 2.4 kb region contained the cis-acting (res) site and the gene for a trans-acting factor (resolvase); complementation analysis indicated that Tn4655 resolvase function was not interchangeable with those of other well-studied class 11 transposons, including the Tn1721 subgroup. Tn4655 had no DNA sequences that were hybridizable with the transposase or resolvase genes of the Tn1721 subgroup.Abbreviations Ap ampicillin - Cb carbenicillin - Cm chloramphenicol - Km kanamycin - Sm streptomycin - Tc tetracycline - Tp trimethoprim     

Re-examination of the ColE1 DNA regions essential for autonomous replication and their functions

Starting from pAO3, a plasmid consisting of a quarter of colicinogenic factor E1 (ColE1) DNA, various small ColE1 derivatives were constructed by in vitro recombination and their ability to achieve autonomous replication was examined. The 436 base pair HaeIII-C fragment of pAO3 contained information for replication when it was recombined with the non-replicating Amp fragment. However, when it was connected to other DNA fragments, the resulting hybrid molecules were not isolated as plasmids. The present results indicate that the additional region of about 240 base pairs next to the HaeIII-C fragment of ColE1 is also essential for the maintenance of a plasmid state. Moreover, using various small ColE1 derivatives, the DNA region responsible for the interference and incompatibility functions of ColE1 DNAs was located. The results indicate that the interference and incompatibility functions are coded by the same ColE1 DNA segment and are not essential for the maintenance of a plasmid state.     

The transposon Tn1 as a probe for studying ColE1 structure and function.

Summary Insertion of the transposable genetic element Tn1 into different sites of plasmid ColE1 results in a number of mutant phenotypes. Whereas all plasmids examined were present in normal amount, all showed reduced immunity to killing by colicin E1. Of six insertions isolated after conjugation, five fail to produce colicin, are conjugally proficient (transmissible), and map within a 500 nucleotide region of the genome. The other is conjugally deficient, produces colicin normally and maps close to two others with a similar phenotype isolated after transformation. Of four others isolated after transformation, two have similar properties to the original five transmissible plasmids. The other two are nontransmissible and produce colicin. Non-transmissibility is correlated with reduced relaxation complex. Patterns of protein synthesis in minicels by ColE1 and ColE1:: Tn1 plasmids have been examined: all ColE1 plasmids containing Tn1 show an altered pattern of ColE1 protein synthesis in addition to three presumptive Tn1-specified proteins, one of which is shown to be -lactamase. ColE1:: Tn1 plasmids can be inserted into the conjugative plasmid R64drd11 to form a cointegrate in which ColE1 and Tn1 function can be expressed.