Eduard Veltkamp

Genetic map of the bacteriocinogenic plasmid CLO DF13 derived by insertion of the transposon Tn901

SummaryAn ampicillin transposon Tn901 was used as a “mutagen” to isolate insertion mutants of the bacteriocinogenic plasmid Clo DF13. By combining the obtained heteroduplex and restriction maps of the Clo DF13::Tn901 plasmids (van Embden et al., 1977b) with their polypeptide pattern in minicells, we were able to map five genes on the Clo DF13 genome. These five genes designated A (cloacin gene), B, C, D and G cover 55% of the coding capacity of Clo DF13 DNA. Since integration of Tn901 within these five genes did not result in a loss of the Clo DF13::Tn901 plasmids involved, it is suggested that these genes do not play an essential role in the maintenance of these plasmid insertion mutants. In addition, the described methods allowed us to indicate the initiation site of cloacin synthesis and to propose the counter-clockwise direction of transcription of the cloacin gene. The Tn901 DNA directed the synthesis of at least three polypeptides one of which is shown to be a TEM-1 beta-lactamase.

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

SummaryInsertion 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.

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

Abstract 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 Tn 901 . In order to determine the site and orientation of Tn 901 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::Tn 901 plasmids were digested with endonucleases Hin cII, Pst I, Bam H-I, Sal I, and Hpa I or with a combination of two of these enzymes. By analysis of the resulting fragmentation patterns, the physical maps of Clo DF13 DNA and Tn 901 DNA could be derived. Furthermore, the site and orientation of Tn 901 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 Tn 901 had occurred at 31 different sites. No preference with respect to the orientation of Tn 901 was observed. Insertion of Tn 901 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 Tn 901 insertions within Clo DF13 were more or less scattered; however, no Tn 901 insertion sites were found in two distinct areas comprising 11 and 17%, respectively, of the Clo DF13 genome. Transposition of Tn 901 DNA to the copy mutant Clo DF13- rep 3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.

Characterization of a replication mutant of the bacteriocinogenic plasmid Clo DF13.

In a previous paper (Kool, A.J. and Nijkamp, H.J.J. (1974) J. Bacteriol. 120, 569--578) the isolation of a mutant of the bacteriocinogenic plasmid Clo DF13-Rep3, has been described. It was observed that cells harbouring the wild type plasmid synthesize more plasmid DNA cells harbouring the wild type plasmid. This paper deals with the characterization of the nature of this plasmid-specific mutation. The following properties of the Clo DF13-Rep3 mutant plasmid could be observed: 1. The plasmid-specific mutation did not lead to a significant change in the sedimentation value of Clo DF13 DNA. 2. The specific rate of Clo DF13-Rep3 DNA synthesis (expressed as the number of plasmid DNA molecules synthesized per min) is on average seven times the specific rate of wild type Clo DF13 DNA synthesis. 3. Also chromosomeless minicells, harbouring the Clo DF 13-Rep 3 plasmid, contain about seven times more plasmid DNA as wild type Clo DF13 harbouring minicells. 4. The replication time of the Clo DF13-Rep3 plasmid is approx. 90 s at 30 degrees C and does not differ significantly from the replication time of the wild type plasmid. 5. The Rep3 mutation did not alter the dependence of Clo DF13 plasmid replication on the dnaA and dnaC gene products. 6. The plasmid-specific mutation is cis-dominant over wild type. The data presented in this paper indicate that this mutant plasmid is not affected in the elongation but in the initiation of plasmid DNA replication.

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

SummaryThe 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 5′→3′ 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.

Clo DF13 plasmid genes affecting Flac transfer and propagation of male specific RNA phages

SummaryThe 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.

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

SummaryThe presence of plasmid Clo DF 13 in Escherichia coli cells alters the response of these cells to infection with the double stranded DNA phages P1vir, λvir 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.

Replication of the bacteriocinogenic plasmid Clo DF13: Action of the plasmid protein cloacin DF13 on Clo DF13 DNA

Abstract The bacteriocinogenic plasmid Clo DF13 can be isolated from Escherichia coli cells and minicells as a complex of Clo DF13 DNA and one plasmid-specific protein, cloacin DF13 (Veltkamp et al. , 1975). The construction of a physical map of Clo DF13 DNA on the basis of specific cleavage of Clo DF13 DNA by Hin dII restriction endonuclease (from Hemophilus influenzae ), allowed us to determine the Clo DF13 DNA region(s) involved in the binding of cloacin DF13. It turned out that cloacin DF13 does not bind at random but to a specific Clo DF13 DNA region located on one of the Hin dII-generated Clo DF13 fragments. Circular Clo DF13 DNA can be cleaved under suitable conditions by in vivo as well as in vitro synthesized cloacin DF13 into linear DNA molecules of full Clo DF13 genome length. Cloacin DF13, which binds to supercoiled and open circular Clo DF13 DNA, remains bound to the DNA after cloacin-induced conversion of the circular form to the linear form. By using Bam H-I restriction enzyme (from Bacillus amyloliquefaciens ) it was demonstrated that cloacin DF13 acts on a very specific Clo DF13 DNA region, probably at one specific site, which is located on the Bam H-I-generated Clo DF13 DNA fragment B. The optimal conditions for the endonuclease activity of cloacin DF13 are described. About 60% of the cloacin-generated linear Clo DF13 DNA molecules can be recircularized in vitro , indicating that cloacin DF13 makes two staggered, single-stranded endonucleolytic cuts in duplex Clo DF13 DNA. The binding as well as the endonucleolytic action of cloacin DF13 on Clo DF13 can be abolished by another Clo DF13 plasmid-specific protein, namely the immunity protein. Clo DF13 immunity protein itself has no affinity for Clo DF13 DNA but neutralizes the action of cloacin DF13 by forming a complex with this protein. The possible role of cloacin DF13 and Clo DF13 immunity protein in the replication of Clo DF13 plasmid DNA is discussed.