Relaxation of stable RNA synthesis by a plasmid-borne locus

Summary The presence of Clo DF13 copy mutants in Escherichia coli (Flac) cells results, in contrast to the presence of Clo DF13 wt plasmids, in a decreased transfer of Flac and a decrease in the efficiency of plating (EOP) of male specific RNA phages.The degree of reduction of these processes is correlated to the number of Clo DF13 copies per cell as was found by the use of copy mutants and a thermosensitive copy control mutant of Clo DF13. For instance, the presence of the Clo DF13 cop3 plasmid results in a hundredfold decrease in EOP of RNA phages and a tenfold decrease in transfer of the F plasmid. No interaction with the efficiency of plating of male specific RNA phages was measured when the wild type Clo DF13, ColE1, ColE2, ColE3 or ColK plasmid is present in the cell. Studies with both, insertion and deletion mutants of CLo DF13 cop3 showed that these effects are not due to a high number of plasmid DNA molecules itself but due to a high amount of plasmid gene products in the cell. Furthermore these studies enabled us to locate the genes involved in these interactions on the Clo DF13 physical map. It turned out that two Clo DF13 genes are involved in the observed phenomena: one gene, coding for polypeptide B (molecular weight 61,000 daltons) which is also involved in the mobilisation of Clo DF13, and one gene coding for polypeptide D (molecular weight 21,000 daltons). The possible role of these Clo DF13 gene products, involved in the decrease in transfer of Flac as well as the decrease in efficiency of plating of male specific RNA phages, is discussed.     

Isolation and Characterization of a Copy Mutant of the Bacteriocinogenic Plasmid Clo DF13

After nitrosoguanidine mutagenesis, strain Escherichia coli P678-54, bacteriocinogenic for Clo DF13, yielded a mutant strain that showed an enhanced bacteriocin production. The results from conjugation experiments indicated that the mutation, responsible for the enhanced bacteriocin production, is located on the Clo DF13 plasmid. The following properties of strains harboring the mutant Clo DF13 plasmid could be observed. (i) The bacteriocin production in these strains can be further enhanced at least fourfold by mitomycin C. (ii) The fraction of spontaneously induced cells, as revealed by lacunae experiments, in cultures of these strains is about nine times higher than in cultures of wild-type Clo DF13-harboring strains. (iii) Chromosomeless minicells from strain P678-54 harboring the mutant Clo DF13 plasmid synthesize about six times more deoxyribonucleic acid, ribonucleic acid, and protein as compared to wild-type Clo DF13-harboring minicells. (iv) Analysis of this mutant Clo DF13-specific ribonucleic acid and protein on polyacrylamide gels revealed mainly the same ribonucleic acid and polypeptide species as synthesized by the wild-type Clo DF13 minicells, but in larger amounts (Kool et al., 1974). (v) Segregation experiments, using a strain with temperature-sensitive polymerase I, show that mutant Clo DF13-harboring cells contain an average of 70 Clo DF13 copies per cell, whereas wild-type Clo DF13-harboring cells contain only about 10 Clo DF13 copies per cell. The data presented in this paper indicate that the mutation on the Clo DF13 plasmid leads to an altered control of Clo DF13 replication and results in an enhanced number of Clo DF13 copies per cell. As a secondary effect, this enhanced number of Clo DF13 copies enhances the probability of "spontaneous" induction per cell. Since the mutation is plasmid specific and affects the number of plasmid copies produced, one can conclude that the Clo DF13 plasmid is not dependent solely on chromosomal information, but that at least plasmid base sequences are involved in Clo DF13 plasmid replication.     

In vitro construction of deletion mutants of the bacteriocinogenic plasmid Clo DF13.

The isolation and characterization of deletion mutants of the bacteriocinogenic plasmid Clo DF13 is described. To construct these deletion mutants, DNA of Clo DF13::Tn901 and Clo DF13-rep3::Tn901 plasmids was digested with restriction endonucleases, ligated with T4 ligase and introduced by transformation into Escherichia coli. The presence of the ampicilline transposon Tn901 facilitated the selection of plasmids. The resulting Clo DF13::Tn901 deletion mutants were analyzed by digestion with restriction endonucleases and electron microscopy. From the properties of the various deletion mutants it was concluded that a Clo DF13 DNA region, extending from 5 to 11.5% on the physical map, is essential for the replication of Clo DF13. This region, comprising about 600 base pairs, contains in addition to an origin of replication, DNA sequences which are involved in the regulation of Clo DF13 DNA replication. Furthermore it was observed that in case of the Clo DF13 copy mutant, Clo DF13-rep3, deletion of the 43% to 63% part of the plasmid genome, resulted in the generation of multimeric plasmid structures, accompanied with an impaired segregation of the plasmids to daughter cells.     

Isolation and characterization of a Clo DF13::Tn901 plasmid mutant with thermosensitive control of DNA replication.

After nitrosoguanidine mutagenesis, a mutant Escherichia coli strain harboring the Clo DF13::Tn901 plasmid pJN03 was isolated that is thermosensitive (Ts) for growth at 43 degrees C. The mutation responsible for this thermosensitive phenotype resides on the pJN03 plasmid genome. Cells harboring the pJN03 cop-1(Ts) plasmid mutant showed a large increase in plasmid copy number at 43 degrees C accompanied by an increase in the synthesis of plasmid-specified gene products like cloacin DF13 and beta-lactamase. The pJN03 cop-1(Ts) mutant showed uncontrolled plasmid DNA replication at the nonpermissive temperature. Analysis of plasmid deletions showed that the mutation is located in the Clo DF13 map interval from 0 to 12% or 29 to 45%. This implies that native cloacin DF13 and the Clo DF13-specified polypeptides B, C, D, E, and G are not involved in the pleiotropic phenotype of the plasmid mutant pJN03 cop-1(Ts).     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. I. Localisation and mutual interactions of four Clo DF13 incompatibility regions

Summary The incompatibility properties of the bacteriocinogenic plasmid Clo DF13 have been examined. By using Clo DF13, Clo DF13 deletion, and transposon insertion mutants as well as compatible R plasmids into which Clo DF13 fragments have been cloned, we could identify and localise four different incompatibility regions on the Clo DF13 genome. These regions, designated incA, incB, incC, and incD are located in the following positions: incA about incD between 1.8% and 9% of the Clo DF13 genome. We studied the contribution of each of the four inc regions, separately and/or in combination with each other, to the incompatibility between two plasmid replicons. Two types of incompatibility can be distinguished: Type I evoked by incD, that overlaps the replication control area of Clo DF13 and type II, caused by incA, B and C. From our observations we present a model for plasmid incompatibility based on a combination of the existing repressor dilution and membrane attachment models.     

Replication of the Bacteriocinogenic Plasmid Clo DF13 in Thermosensitive Escherichia coli Mutants Defective in Initiation or Elongation of Deoxyribonucleic Acid Replication

The replication of the bacteriocinogenic plasmid Clo DF13 has been studied in the seven temperature-sensitive Escherichia coli mutants defective in deoxyribonucleic acid (DNA) replication (dnaA-dnaG). Experiments with dna initiation mutants revealed that the replication of the Clo DF13 plasmid depends to a great extent on the host-determined dnaC (dnaD) gene product, but depends slightly on the dnaA gene product. The synthesis of Clo DF13 plasmid DNA also requires the dnaF and dnaG gene products, which are involved in the elongation of chromosomal DNA replication. In contrast, the Clo DF13 plasmid is able to replicate in the dnaB and dnaE elongation mutants at the restrictive temperature. When de novo protein synthesis is inhibited by chloramphenicol in wild-type cells, the Clo DF13 plasmid continues to replicate for at least 12 h, long after chromosomal DNA synthesis has ceased, resulting in an accumulation of Clo DF13 DNA molecules of about 500 copies per cell. After 3 h of chloramphenicol treatment, the Clo DF13 plasmid replicates at a rate approximately five times the rate in the absence of chloramphenicol. Inhibition of protein synthesis by chloramphenicol does not influence the level of Clo DF13 DNA synthesis at the restrictive temperature in the dna mutants, except for the dnaA mutant. Chloramphenicol abolishes the inhibition of Clo DF13 DNA synthesis in the dnaA mutant at the nonpermissive temperature. Under these conditions, Clo DF13 DNA synthesis was slightly stimulated in the first 30 min after the temperature shift, and continued for more than 3 h at an almost uninhibited level.     

Integration of a transposable DNA sequence which mediates ampicillin resistance into Clo DF13 plasmid DNA: determination of the site and orientation of TnA insertions.

The isolation and characterization of Clo DF13 plasmids containing a transposable DNA sequence (TnA) that specifies for ampicillin resistance is described. The particular transposon is derived from the R plasmid pRI30, and is designated Tn901. In order to determine the site and orientation of Tn901 insertions into the Clo DF13 genome, we made use of restriction endonucleases and heteroduplex mapping. For this purpose, Clo DF13 plasmid DNA and DNA of Clo DF13::Tn901 plasmids were digested with endonucleases HincII, PstI, BamH-I, SalI, and HpaI or with a combination of two of these enzymes. By analysis of the resulting fragmentation patterns, the physical maps of Clo DF13 DNA and Tn901 DNA could be derived. Furthermore, the site and orientation of Tn901 insertions into the Clo DF13 genome could be determined by this approach. The data obtained were verified by heteroduplex mapping. Analysis of 33 independently isolated Clo DF13 recombinant plasmids showed that insertion of Tn901 had occurred at 31 different sites. No preference with respect to the orientation of Tn901 was observed. Insertion of Tn901 into a segment of about 20% of the Clo DF13 genome resulted in the loss of cloacin production, indicating that the structural gene coding for cloacin is located in this area. The sites of Tn901 insertions within Clo DF13 were more or less scattered; however, no Tn901 insertion sites were found in two distinct areas comprising 11 and 17%, respectively, of the Clo DF13 genome. Transposition of Tn901 DNA to the copy mutant Clo DF13-rep3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.     

Influence of Protein and Ribonucleic Acid Synthesis on the Replication of the Bacteriocinogenic Factor Clo DF13 in Escherichia coli Cells and Minicells

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.     

Genetic analysis of the mobilization of the non-conjugative plasmid Clo DF13

Summary Insertion of the transposon Tn901 within a region of almost one third of the Clo DF13 genome is compared with the loss of its transfer (indicated as Mob^-) by a conjugative plasmid. By use of both insertion and deletion mutants of Clo DF13, this region was located on the Clo DF13 physical map. Studies with transfer mutants of the F plasmid showed that, in contrast with the traG gene product, the gene products of traI, traD and traM do not play an essential role in the transfer process of Clo DF13. Because Clo DF13 can be transferred under conditions in which the coningative plasmid is not transferred at all, it is obvious that normally Clo DF13 is not transferred to recipient cells as a cointegrate of the conjugative plasmid and Clo DF13. Characterization of the Mob^- Clo DF13:: Tn901 plasmids showed that the absence or alteration of the Clo DF13 specified polypeptide B (molecular weight 61,000 daltons) is correlated with the transfer deficiency of these plasmids. The existence of transfer deficient Clo DF13:: Tn901 plasmids, which direct the synthesis of polypeptide B, showed that other Clo DF13 genetic information is also involved in the transfer of this plasmid. On basis of the site of the mutation in the genome, the synthesis of polypeptide B in the minicell system and the behaviour of the Mob^- mutants in complementation studies, we preliminarily divide the Mob^- Clo DF13:: Tn901 plasmids into three different classes. The possible role of Clo DF13 genetic information involved in the transfer process of this plasmid is discussed.     

Origin and direction of replication of the bacteriocinogenic plasmid Clo DF13.

Cairn's type replicative intermediates of both the wildtype Clo DF13 plasmid and the copy mutant CLO DF13 cop3 were isolated by dye-buoyant density centrifugation. Replicative intermediates were linearized at the HpaI or Sa1I cleavage site, and examined with the electron-microscope. The data show that replication of both the Clo DF13 wild type plasmid and the Clo DF13 cop3 plasmid, initiates at about 2.8% on the physical map. Replication proceeds unindirectionally and counterclockwise on this map.     

Proteins synthesized by a non-induced bacteriocinogenic factor in minicells of Escherichia coli

Summary The cloacinogenic factor Clo DF13 from Enterobacter cloacae has been transferred to the minicell-producing strain P678-54 of Escherichia coli K12. The data presented show that this Clo DF13 factor segregates into minicells of P678-54 (Clo DF13) and that this factor is the only plasmid, present in these minicells. Proteins from purified P678-54 (Clo DF13) and P678-54 minicells, previously labelled with ¹⁴C-amino acids, were compared after electrophoresis on SDS-polyacrylamide gels. From this comparison it appeared that a noninduced Clo DF13 factor directs the synthesis of 4 proteins. The molecular weights of these proteins could be estimated to be about 72000, 32000, 18500 and 12000. In P678-54 (Clo DF13) minicells, one additional Clo DF13 protein was found to be unlabelled. Apparently this protein is not synthesized in P678-54 (Clo DF13) minicells, but is segregated into or is attached to the minicells after being synthesized in the P678-54 (Clo DF13) cells. The molecular weight of this protein is about 62000, which corresponds to the molecular weight of cloacin.     

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host

Summary We studied the expression of gene H, located between 9.3% and 11% on the Clo DF13 genome, as well as the functions of the gene product. We found that treatment of bacterial cells with mitomycin-C results in the induced synthesis of three Clo DF13 specified proteins namely cloacin DF13, immunity protein and protein H. Evidence was obtained that the genes encoding these proteins form one, mitomycin-C induceable, operon; the promoter at 32% in front of the cloacin gene is essential for the induced expression. Furthermore we could demonstrate that protein H is involved in the lethal effect of mitomycin-C treatment of bacteriocinogenic cells. The data in this paper show that a high concentration of protein H in cells, due either to an induced expression of gene H (mitomycin-C induction) or to a gene dosage effect (Clo DF13 cop1 Ts copy control mutant), results in the lysis of bacterial cells. The implication of these data are discussed.     

Genetic information of the bacteriocinogenic plasmid Clo DF13 involved in the inhibition of the multiplication of double stranded DNA phages

Summary The presence of plasmid Clo DF 13 in Escherichia coli cells alters the response of these cells to infection with the double stranded DNA phages P1 _vir , _vir or T1. The multiplication of these phages is reduced in Clo DF13 harbouring cells, resulting in an altered burstsize and plaque morphology. The degree of reduction is correlated to the amount of particular Clo DF13 gene product(s) in the cell. The genetic information of Clo DF13 involved in this plasmid-phage interaction could be located, using insertion and deletion mutants of Clo DF13 physical map. The genetic analysis of this region shows that at least two different genes, K and L, coding for polypeptides with a molecular weight of respectively 21 KD and 10.5 KD, are located in this region. The results presented, indicate that gene L and not gene K is involved in the interaction of Clo DF13 with the propagation of double stranded DNA phages.     

Identification of Messenger Ribonucleic Acids and Proteins Synthesized by the Bacteriocinogenic Factor Clo DF13 in Purified Minicells of Escherichia coli

It has previously been shown that the cloacinogenic factor Clo DF13 (Clo DF13) segregates into minicells of strain Escherichia coli P678-54 that harbors Clo DF13 and that this Clo DF13 factor is the only deoxyribonucleic acid (DNA) present in these otherwise chromosomeless minicells. The study reported here shows that minicells prepared from P678-54(Clo DF13) are able to incorporate radioactive precursors into ribonucleic acid (RNA) and protein. The RNA synthesized in these purified minicells is Clo DF13 specific, as shown by RNA-DNA hybridization experiments. The results indicate that all the de novo synthesized gene products in Clo DF13 minicells are Clo DF13 specific. Polyacrylamide gel electrophoretic patterns show that in these minicells at least three polypeptides (molecular weight about 70,000, 20,000, and 11,000) and one major species of messenger RNA (mRNA) (S value about 21.3) are synthesized. To investigate the factor in its induced state, we isolated a Clo DF13 mutant with an enhanced level of cloacin production. Minicells harboring this Clo DF13 mutant produce five additional polypeptides (molecular weight about 58,000, 44,000, 28,000, 16,000, and 14,000). Three additional mRNA species (S value about 19.5, 14, and 12) could be distinguished. The total molecular weight of the eight polypeptides corresponds to 85% of the total coding capacity of the mRNAs (303,000). The total molecular weight of the four mRNAs is 2.55 x 10(6), which covers 85% of the Clo DF13 DNA (molecular weight 6 x 10(6)).     

Escherichia coli host G668 stabilizes plasmids that are unstable in otherEscherichia coli strains

CloDF13 copy mutants that have their resolution site (crl) deleted accumulate as multimeric plasmid molecules in their host cells and are lost from severalEscherichia coli stains within 60 generations. Here we demonstrate that CloDF13cop3crl ^– mutants are stably maintained in theE.coli strain G668, although the plasmid copy number is not affected. Furthermore, we show that the stable maintenance of those plasmids is achieved even in the presence of multimeric molecules. Therefore, we conclude that a complete monomerization of multimeric molecules appears not to be a prerequisite for accurate partition of the plasmid molecules over daughter cells. The G668 strain may be applied as host for the stabilization of resolution-negative, unstable CloDF13 or related replicons.     

The use of mutants in the study of structure-function relationships in cloacin DF13

A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03· immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 · immunity protein complex and wild-type cloacin complex showed no significant differences.From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity.Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with “immunity” but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82.The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.     

The use of mutants in the study of structure-function relationships in cloacin DF13.

A bacteriocin from cells with a mutant Clo DF13 plasmid (cloacin clp03 . immunity protein complex) and a bacteriocin from cells containing the recombinant plasmic Clo DF13 :: Tn901 (cloacin pJN82) have been isolated. Both bacteriocins like wild-type cloacin DF13, are still able to inhibit in vitro protein synthesis, but their in vivo killing activity is absent. Comparison of some physicochemical characteristics of the cloacin clp03 . immunity protein complex and wild-type cloacin complex showed no significant differences. From a comparison of the binding capacity to specific receptors on sensitive cells, the translocation through the cell wall, and the interaction with cytoplasmic membranes, it could be concluded that the cloacin clp03 complex is hampered in its translocation from the outer membrane receptor site to the cytoplasmic membrane, resulting in the observed lack in killing activity. Cloacin pJN82 is shortened at the C-terminal of the molecule by approximately ten amino acid residues. Together with its loss of in vivo killing activity it has lost its capacity to bind immunity protein. Since the immunity protein probably not only provides cloacin-producing cells with "immunity" but is also involved in the translocation of the bacteriocin to the interior of sensitive cells, the absence of this protein is probably the reason for the lack of killing activity of cloacin pJN82. The implications of these findings for the topography of the cloacin molecule as suggested by de Graaf et al. (de Graaf, F.K., Stukart, M.J., Boogerd, F.C. and Metselaar, K. (1978) Biochemistry, in press) are discussed.     

Inhibition of cell division in Escherichia coli K-12 by the R-factor R1 and copy mutants of R1.

The effect of the copy number of plasmid R1drd-19 on cell division of Escherichia coli K-12 was studied in populations growing as steady-state cultures at different growth rates, the growth rate being varied by use of different carbon sources. The plasmid copy number was also varied by using copy mutants of the R-factor. The mean cell size was larger in populations carrying an R-factor than in R-factorless populations, an effect that was more pronounced at low growth rates and in populations carrying R-factor copy mutants. The increased cell size was due to formation of elongated cells in a fraction of the population and to an increase in the diameter of all cells. The majority of the cells divided at a normal cell length, but the presence of an R-factor caused some cells to elongate, probably by the uncoupling of chromosome replication and cell division. This can be explained as a competition between the chromosome and plasmid replicons for some replication factor(s), presumably acting on both initiation and elongation of replication. The formation of elongated cells was a reversible process, but occasionally some of the elongated cells reached lengths 20 times that of newborn cells. If cell division did not occur at the normal cell size, the septum was not formed until the cell size was four times that of a newborn cell. When an elongated cell divided, it usually formed a polar septum, thus producing a newborn cell of normal cell length. The ability of plasmid-containing cells to omit one cell division but to retain the capacity of dividing one mass doubling later is compatible with a mechanical model for septum formation and cell division.     

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance

Summary Clo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA^- and/or parB^- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.     

The role of DNA polymerase I, II and III in the replication of the bacteriocinogenic factor Clo DF13

Summary The replication of the bacteriocinogenic factor Clo DF13 was studied in Escherichia coli mutants which lack either DNA polymerase I (polA1 and resA1 mutants), DNA polymerase II (polB1 mutant) or DNA polymerase III (dnaE mutant). DNA polymerase I is required for Clo DF13 replication. The Clo DF13 factor, however, can be maintained in a strain carrying the polA107 mutation and thus lacking the 53 exonucleolytic activity of DNA polymerase I. DNA polymerase II is not required for transfer replication and maintenance of the Clo DF13 plasmid. In the temperature sensitive dnaE mutant, Clo DF13 can replicate at the nonpermissive temperature during the first two hours after the temperature shift from 30°C to 43°C. During this period DNA polymerase III seems not to be essential for Clo DF13 replication.