Isolation and characterization of ColE2 plasmid mutants unable to kill colicin-sensitive cells

Summary After transfer from a mutagenized host, twenty one ColE2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene for colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors.Studies with a nonsense mutant producing only a small colicin E2 fragment (ColE2-421) suggest that colicin E2 is not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33°, 37° and 43°. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI I. Isolation and Specificity of Host and Plasmid Mutations

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.     

Comparative Study of the Events Associated with Colicin Induction

Colicinogenic factors ColI and ColV, which have been shown to behave as sex factors, could not be induced with mitomycin C. In contrast, the ColE(1), ColE(2), and ColE(3) factors, which do not exhibit any fertility factor characteristics, are inducible by this agent. The induced production of colicins E(1), E(2), and E(3) was accompanied by a loss in viability at a concentration of mitomycin C which was bacteriostatic to noncolicinogenic cells or to cells carrying the ColV or ColI factors. The loss in viability accompanying the mitomycin C induction of the ColE(1), ColE(2), or ColE(3) factors also occurred when colicin synthesis was blocked by chloramphenicol or amino acid starvation. However, chloramphenicol was able to block the loss of viability of a recipient cell after mitomycin C induction of a newly acquired Col factor if the antibiotic was present throughout the mating period. No detectable internal colicin or colicin precursor could be demonstrated during the lag period prior to the appearance of colicin outside the cell 20 to 30 min after the addition of mitomycin C. If chloramphenicol was present during the lag period following the addition of mitomycin C, colicin synthesis began immediately after the removal of these antibiotics. The synthesis of tryptophan synthetase and induced beta-galactosidase proceeded normally throughout the lag period and well into the period of colicin production. Regulation of beta-galactosidase synthesis did not seem to be profoundly affected during the lag period subsequent to mitomycin C addition. Induced colicin synthesis, like bacterial or induced prophage protein synthesis, was subject to inhibition by virulent phage infection.     

Temperature-Sensitive Mutants for the Replication of Plasmids in Escherichia coli: Requirement for Deoxyribonucleic Acid Polymerase I in the Replication of the Plasmid ColE1

An Escherichia coli mutant (polA1), defective in deoxyribonucleic acid (DNA) polymerase I, (EC 2.7.7.7) is unable to maintain colicinogenic factor E1 (ColE1), whereas several sex factor plasmids are maintained normally in this strain. polA1 mutant strains containing these sex factor plasmids do not exhibit a readily detectable plasmid-induced polymerase activity. A series of E. coli mutants that are temperature sensitive for ColE1 maintenance, but able to maintain other plasmids, were isolated and shown to fall into two phenotypic groups. Mutants in one group are defective specifically in ColE1 maintenance at 43 C, but exhibit normal DNA polymerase I activity. Mutations in the second group map in the polA gene of E. coli, and bacteria carrying these mutations are sensitive to methylmethanesulfonate (MMS). Revertants that were selected either for MMS resistance or the ability to maintain ColE1 were normal for both properties. The DNA polymerase I enzyme of two of these mutants shows a pronounced temperature sensitivity when compared to the wild-type enzyme. An examination of the role of DNA polymerase I in ColE1 maintenance indicates that it is essential for normal replication of the plasmid. In addition, the presence of a functional DNA polymerase I in both the donor and recipient cell is required for the ColV-promoted conjugal transfer of ColE1 and establishment of the plasmid in the recipient cell.     

cea-kil operon of the ColE1 plasmid.

We isolated a series of Tn5 transposon insertion mutants and chemically induced mutants with mutations in the region of the ColE1 plasmid that includes the cea (colicin) and imm (immunity) genes. Bacterial cells harboring each of the mutant plasmids were tested for their response to the colicin-inducing agent mitomycin C. All insertion mutations within the cea gene failed to bring about cell killing after mitomycin C treatment. A cea- amber mutation exerted a polar effect on killing by mitomycin C. Two insertions beyond the cea gene but within or near the imm gene also prevented the lethal response to mitomycin C. These findings suggest the presence in the ColE1 plasmid of an operon containing the cea and kil genes whose product is needed for mitomycin C-induced lethality. Bacteria carrying ColE1 plasmids with Tn5 inserted within the cea gene produced serologically cross-reacting fragments of the colicin E1 molecule, the lengths of which were proportional to the distance between the insertion and the promoter end of the cea gene.     

Effect of Rec− Mutations on the Activity of Colicinogenic Factors

The effect of two Rec(-) mutations (AB2463 and JC1553) on the ability of a cell to accept, maintain, and express the colicinogenic factors ColE(1), ColE(2), and ColV was examined in Escherichia coli. These mutations had no observable effect on the colicinogenic properties of the ColV factor, but prevented the spontaneous and induced production of colicins E(1) and E(2) which are determined by the ColE(1) and ColE(2) factors, respectively. The two Rec(-) mutations had no apparent effect on the ability of the cells to acquire, maintain, or transfer the ColE(1) and ColE(2) factors. These mutations did not affect the expression of immunity by any of the three Col factors. ColE(1) and ColE(2) were also shown to be indirectly induced by mating F(-) cells carrying these Col factors with ultraviolet-irradiated, non-colicinogenic, Hfr and F(+) cells. Indirect induction of colicin production occurred when either an irradiated F(+) Rec(+) or F(+) Rec(-) strain was employed as the donor strain.     

RNase H and replication of ColE1 DNA in Escherichia coli

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.     

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     

Plasmid ColE1 DNA replication in Escherichia coli strains temperature-sensitive for DNA replication

Mutants of the dnaA, dnaC, dnaD, polC, dnaF and dnaG gene loci were tested for their capacity for colicinogenic plasmid E1 (ColE1) replication at a non-permissive temperature. It was found that ColE1 replication was independent of the dnaA gene function and dependent on dnaC, D, F and G. ColE1 replication in the polC mutant E486 continued for several hours but at a greatly reduced rate. No effect was found of the dnaG mutation on thymine-deprivation-induced "priming" of ColE1 replication at the non-permissive temperature. The mutants also were tested for aberrant replication intermediates of plasmid DNA as well as a temperature sensitive supercoiled DNA-protein relaxation complex. RNA-containing supercoils were found to accumulate in a poIC mutant also blocked for protein synthesis.     

Colicin synthesis and cell death.

Colicin E1 is a small plasmid, containing the cea gene for colicin, the most prominent product of the plasmid. Colicin is a 56-kilodalton bacteriocin which is especially toxic to Escherichia coli cells that do not contain the plasmid. Under normal growth conditions very low levels of the plasmid are produced as a result of cea gene repression by the host LexA protein. Conditions that lower the concentration of LexA protein result in elevated levels of colicin synthesis. The LexA protein concentration can be lowered by exposing the cells to DNA-damaging reagents such as UV light or mitomycin C. This is because DNA damage signals the host SOS response; the response leads to activation of the RecA protease which degrades the LexA protein. DNA-damaging reagents result in very high levels of colicin synthesis and subsequent death of plasmid-bearing cells. Elevated levels of colicin are also produced in mutants of E. coli that are deficient in LexA protein. We found that comparably high levels of colicin can be produced in such mutants in the absence of cell death. In lexA strains carrying a defective LexA repressor, colicin synthesis shows a strong temperature dependence. Ten to twenty times more colicin is synthesized at 42 degrees C. This sharp dependence of synthesis on temperature suggests that there are factors other than the LexA protein which regulate colicin synthesis.     

Structuro-functional organization of segments of Co1A plasmid DNA, determining its stable inheritance

Two par regions were localized within the structure of a small colicinogenic plasmid ColA. One of them functions at the expense of plasmid multimere resolution. Analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid ColE1. As compared to E. coli C600, the function of multimere forms' resolution of plasmid DNA in E. coli C is reduced or absent due to par regions of the ColE1 type. Par regions of various degrees of homology with the par locus of ColE1 were localized by Southern hybridization within the structure of colicinogenic plasmids ColN and ColD. The stabilization of the colicinogenic plasmids is believed to be also determined by the functioning of genes connected with the synthesis and action of colicin.     

Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

Characteristics of replication of small colicinogenic plasmids

Specificity of small multicopy colicinogenic plasmids ColA, ColD, ColE2 and ColK replication has been compared with the one of ColE1 plasmid. Copy number for these plasmids per host cell has been estimated under the normal conditions of cellular growth and under the conditions of chloramphenicol-inhibited growth. DNA polymerase I and dnaB protein, an obligatory component for elongation step in replication, have been shown to be necessary for the plasmids replication. Initiation of plasmids replication has been demonstrated to be independent of dnaA and dnaC proteins. Replication of plasmid ColE2, being similar in its main features to replication of other plasmids from this group, has an important distinction. It requires de novo protein synthesis implying that ColE2 replicon may be different from ColA, ColD, ColK, ColE1 replicons. Thus study of the inducible A, D, K, El colicin synthesis coded by the corresponding plasmids has revealed the similarity regulation of genes, determining the synthesis of each of the mentioned colicins.     

Inhibition of colicin synthesis during integration of transposons into the ColE1 plasmid in Escherichia coli

Insertions of transposons into ColE1 plasmid have been shown to influence the plasmid-specified colicin synthesis. The quantity of colicin produced by a single bacterium being unchanged, a portion of colicin-producing cells in the population of those containing insertion mutants was 10(1)-10(4)-fold lower than in the case of ColE1. The effect of transposon was only observed in cis. Insertions were located in different sites of plasmid in both orientations. No secondary DNA rearrangements were formed in regions adjacent to the points of insertions. On the basis of data obtained. It is concluded that the phenomenon can be connected with neither of known types of mutations induced by transposons. A possibility of existence of a new type of such mutations is discussed.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Rifampicin-induced replication of the plasmid pBR322 in Escherichia coli strains carrying dnaA mutations

The replication pattern of the plasmid pBR322 was examined in the dnaA mutants of Escherichia coli. The rate of pBR322 DNA synthesis is markedly decreased after dnaA cells are shifted to the restrictive temperature of 42 degrees C. However, addition of rifampicin (RIF) to cultures of dnaA strains incubated at 42 degrees C after a lag of 90 min results in a burst of pBR322 synthesis. This RIF-induced pBR322 replication remains dependent on DNA polymerase I activity. Efficient plasmid pBR322 replication is observed at 42 degrees C in the double mutant dnaA46cos bearing an intragenic suppressor of dnaA46. Though replication of pBR322 in dnaA46cos growing at 42 degrees C is initially sensitive to RIF plasmid synthesis is restored after 90 min incubation in the presence of the drug. RIF-induced replication of the plasmid pBR327, lacking the rriB site implicated in RIF-resistant synthesis of the L strand of ColE1-like plasmids (Nomura and Ray 1981; Zipursky and Marians 1981), was observed also in dnaA46 at 42 degrees C.     

Effect of dna mutations on the replication of plasmid pSC101 in Escherichia coli K-12.

Escherichia coli strains with mutations in genes dnaB, dnaC, and dnaG were tested for their capacity to replicate pSC101 deoxyribonucleic acid (DNA) at a nonpermissive temperature. Only a small amount of radioactive thymine was incorporated into pSC101 DNA in the dna mutants at 42 degrees C, whereas active incorporation into plasmid DNA took place in wild-type strains under the same conditions. The effects of the dnaB and dnaC mutations were greater on plasmid DNA synthesis than on host chromosomal DNA synthesis, suggesting that these gene products are directly involved in the process of pSC101 DNA replication. In dnaG mutants, both plasmid and chromosomal DNA synthesis were blocked soon after the shift to high temperature; although the extent of inhibition of the plasmid DNA synthesis was greater during the early period of temperature shift to 42 degrees C as compared with that of the host DNA synthesis, during the later period it was less. It was found that the number of copies of pSC101 per chromosome in dnaA and dnaC strains, grown at 30 degrees C, was considerably lower than that in wildtype strains, suggesting that the replication of pSC101 in these mutant strains was partially suppressed even under the permissive conditions. No correlation was found between the number of plasmid copies and the tetracycline resistance level of the host bacterium.     

Isolation and characterization of dnaJ null mutants of Escherichia coli.

Bacteriophage lambda requires the lambda O and P proteins for its DNA replication. The rest of the replication proteins are provided by the Escherichia coli host. Some of these host proteins, such as DnaK, DnaJ, and GrpE, are heat shock proteins. Certain mutations in the dnaK, dnaJ, or grpE gene block lambda growth at all temperatures and E. coli growth above 43 degrees C. We have isolated bacterial mutants that were shown by Southern analysis to contain a defective, mini-Tn10 transposon inserted into either of two locations and in both orientations within the dnaJ gene. We have shown that these dnaJ-insertion mutants did not grow as well as the wild type at temperatures above 30 degrees C, although they blocked lambda DNA replication at all temperatures. The dnaJ-insertion mutants formed progressively smaller colonies at higher temperatures, up to 42 degrees C, and did not form colonies at 43 degrees C. The accumulation of frequent, uncharacterized suppressor mutations allowed these insertion mutants to grow better at all temperatures and to form colonies at 43 degrees C. None of these suppressor mutations restored the ability of the host to propagate phage lambda. Radioactive labeling of proteins synthesized in vivo followed by immunoprecipitation or immunoblotting with anti-DnaJ antibodies demonstrated that no DnaJ protein could be detected in these mutants. Labeling studies at different temperatures demonstrated that these dnaJ-insertion mutations resulted in altered kinetics of heat shock protein synthesis. An additional eight dnaJ mutant isolates, selected spontaneously on the basis of blocking phage lambda growth at 42 degrees C, were shown not to synthesize DnaJ protein as well. Three of these eight spontaneous mutants had gross DNA alterations in the dnaJ gene. Our data provide evidence that the DnaJ protein is not absolutely essential for E. coli growth at temperatures up to 42 degrees C under standard laboratory conditions but is essential for growth at 43 degrees C. However, the accumulation of extragenic suppressors is necessary for rapid bacterial growth at higher temperatures.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.