Plasmid Clo DF13 Research Articles

Localization and nucleotide sequence of the bom region of Clo DF13.

Mobilization of the non-conjugative plasmid Clo DF13 requires both gene products of a conjugative plasmid and Clo DF13 encoded proteins as well as a cis-acting Clo DF13 DNA region, termed bom (basis of mobility). The bom region was located within a 264 bp fragment around the unique HpaI site. Comparison with the corresponding ColE1 and pBR322 sequences showed similarities with respect to the secondary structure. With respect to a possible relationship between the origin of vegetative replication (oriV) and the origin of transfer (oriT), we found that neither distance nor orientation of the Clo DF13 bom region with respect to oriV had any significant influence on the mobilization frequency. Surprisingly, after cloning of the 264 bp HpaII fragment in a bom- vector, restoration of the bom+ phenotype was only observed in one orientation. From these observations and from the sequence analysis of the bom region we suggest that transcription into this cloned bom fragment is essential for effective mobilization of the plasmid.

Structure and regulation of gene expression of a Clo DF13 plasmid DNA region involved in plasmid segregation and incompatibility.

The bacteriocinogenic plasmid Clo DF13 contains genetic information involved in the accurate partitioning of the plasmid (parA and parB) as well as in incompatibility phenomena (incA, B, C and D). In this paper we report on the primary structure and regulation of gene expression of the 29% - 50% part of Clo DF13, containing the DNA regions incA, incB and parB as well as genes K and L. According to the results of our DNA sequence analysis, mapping of transposon insertions, RNA blotting and S1 mapping experiments, we conclude that: a) genes K and L are transcribed as one operon; transcription of this operon is initiated at a promoter (P2) located at 32.5% and proceeds in a clockwise direction. b) treatment of cells with mitomycin-C, significantly enhances transcription from P2, although this promoter is probably not directly repressed by lexA protein. c) Termination of transcription of this operon occurs between genes K and L, as well as distal to gene L. The possible role of gene products and/or sites, located within the 29-50% DNA region, in plasmid incompatibility and segregation is discussed.

Molecular structure and function of the bacteriocin gene and bacteriocin protein of plasmid Clo DF13.

In this paper we present the complete nucleotide sequence of the bacteriocin gene of plasmid Clo DF13. According to the predicted aminoacid sequence the bacteriocin, cloacin DF13, consists of 561 aminoacids and has a molecular weight of 59,293 D. To obtain insight into the structure and function of specific parts of the cloacin molecule, we constructed a hydration profile and we predicted the secondary structure of the protein. According to our predictions, the N-terminus of cloacin DF13 (corresponding to the first 150-180 aminoacids) is relatively hydrophobic and is rich in glycine residues. The data obtained support previous findings that the N-terminal part of cloacin DF13 is involved in translocation of this protein across the cell membrane. The C-terminal part of the cloacin protein is rich in positively charged aminoacids; this might reflect the RNase activity located within this domain. A comparison of the bacteriocin genes and corresponding proteins of Clo DF13 and Col E1 did not reveal any homology at the level of either the nucleotide or the aminoacid sequence. The codon usage of both genes, however, exhibits striking similarities. The sequence data obtained during this study enabled us to present the nucleotide sequence of the entire cloacin operon. The structure of this operon and the regulation of expression of the genes, located within this operon, is discussed.

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

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

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

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 32%, incB between 45% and 50%; incC about 97% and 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.

Protein H encoded by plasmid CloDF13 is involved in excretion of cloacin DF13.

Excretion of cloacin DF13 was studied in Escherichia coli cells harboring different CloDF13 insertion and deletion mutant plasmids. Insertions of a transposon at position 9.8 or 11.5% of the CloDF13 plasmid blocked the expression of gene H and strongly reduced the specific excretion of cloacin DF13 into the culture medium, but had no effect on the production of cloacin DF13. Insertions in or deletions of regions of the CloDF13 DNA upstream the cloacin operon did not affect the excretion or production of the bacteriocin. Introduction of a CloDF13 plasmid that encodes for the gene H product in cells harboring a CloDF13 plasmid with an insertion in gene H stimulated the excretion of cloacin DF13 significantly in mitomycin C-induced and in noninduced cultures. Cloacin DF13 in cloacinogenic cells that did not produce the gene H protein was found to be about 90% located in the cytoplasm. In cells that did produce the gene H product, about 30% of the cloacin DF13 molecules were found in the cytoplasm, about 18% were found in the periplasm, about 2% were in the membranes, and about 50% were located in the culture supernatant. Cyclic AMP stimulated the production but not the excretion of cloacin DF13 in cells cultivated in the presence of glucose.

The nucleotide sequence of the bacteriocin promoters of plasmids Clo DF13 and Co1 E1: role of lexA repressor and cAMP in the regulation of promoter activity.

Treatment of cells, harbouring the bacteriocinogenic plasmic Clo DF13 with mitomycin-C, which induces the cellular SOS response, results in a significantly increased transcription of the operon encoding the bacteriocin cloacin DF13, the immunity protein and the lysis protein H. The nucleotide sequences of the promoter regions and N-terminal parts of the bacteriocin genes of Clo DF13, Col E1 and the pMB1 derivative pBR324 have been determined. A comparison of these sequences with those of corresponding regions of the lexA, recA and uvrB genes revealed that the promoter regions of the bacteriocin genes studied contain binding sites for the lexA protein, which is the repressor of the E. coli DNA-repair system. Using both, a thermosensitive lexA host strain and a host with pACYC184 into which the lexA gene had been cloned, we were able to demonstrate, that in vivo the lexA protein is involved in the regulation of bacteriocin synthesis. From the data presented, we conclude that bacteriocin synthesis is controlled at least by the lexA repressor. It has been reported that also catabolite repression might play an essential role in the control of bacteriocin synthesis. Computer analysis of the DNA sequence data indicated that the promoter regions of both, the cloacin DF13 and colicin E1 genes contain potential binding sites for the cyclic AMP-cyclic AMP Receptor Protein complex.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. I. Localisation of the gene and identification and subcellular localisation of the gene H product.

The gene expression of the Clo DF13 "replication region", located between 1.8% and 12% on the plasmid genome, was studied using newly constructed Clo DF13 insertion and deletion mutants. We were able to detect a Clo DF13 specified protein of 6 kilodaltons (kd) by electrophoretic analysis of plasmid proteins, synthesized in Escherichia coli minicells, on 14-25% gradient polyacrylamide gels. The gene encoding this protein was mapped between 1.8% and 12% on the Clo DF13 genome. The nucleotide sequence of this region, as determined by Stuitje et al. (1980), revealed three open reading frames each potentially coding for a protein of 6 kd. Since these three proteins differ in amino acid composition we could distinguish which of these proteins was actually synthesized, by labeling Clo DF13 proteins with specific 14C-labeled amino acids. We found that gene H, located between 9.3% (bp 744) and 11% (bp 893), encodes the observed protein of 6 kd (denominated protein H). With respect to the subcellular localization we observed that protein H, which contains a large hydrophobic region at its C-terminal part, is predominantly present in the bacterial membrane. Although gene H is located close to the region known to be involved in Clo DF13 replication, its gene product, protein H, is not essential for the plasmid DNA replication process. The possibility of the existence of a comparable protein encoded by the related plasmid Col E1 will be discussed.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host. II. Functions and regulation of synthesis of the gene H product.

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 demontrate 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 copl Ts copy control mutant), results in the lysis of bacterial cells. The implication of these data are discussed.

Identification of mutations affecting replication control of plasmid Clo DF13.

The bacteriocinogenic plasmid Clo DF13, originally isolated from Escherichia cloacae, is stably maintained in Escherichia coli to the extent of about 10 copies per cell. Its replication resembles that of many other small, multicopy plasmids; plasmid-encoded protein is not required but plasmid-specific genetic information is involved in regulation of replication as both conditional and nonconditional copy-number mutants of Clo DF13, and transcomplementable copy-number mutants of plasmid Col E1 have been described. The sequences essential for replication of Col E1 (refs 16, 17) and Clo DF13 (refs 18, 19) have been identified within a region surrounding the replication origin. Initiation of Col E1 replication is preceded by transcription of the origin region, providing the RNA primer at the origin. However, transcription in the opposite direction results in a small transcript of approximately 100 nucleotides (RNA-100) for both Col E1 (refs 21, 22) and Clo DF13 (ref. 23). Data suggest that Col E1 RNA-100 acts as a negative control element for the initiation of replication. We show here that single base transitions in the RNA-100 cistron of Clo DF13 can result in a nonconditional increase in plasmid copy-number. Also, sequence analysis has revealed that a specific base transition in a DNA region, apparently involved in both termination and initiation of transcription towards the replication origin, results in a thermosensitive plasmid copy-number.

Transcription of bacteriocinogenic plasmid CloDF13 in vivo and in vitro: structure of the cloacin immunity operon.

Escherichia coli minicells harboring plasmid CloDF13 synthesized at least 25 messenger ribonucleic acid (RNA) species; three of these RNAs, a 2,400-, a 2,200-, and a 100-nucleotide RNA, were synthesized in relatively large amounts. Using insertion and deletion mutants of CloDF13 as well as an RNA blotting technique, we could demonstrate that these three RNAs are transcripts from the CloDF13 DNA region from 0 to 40%. This region contains the cloacin and immunity genes and the genetic information involved in plasmid DNA replication. A transcription map of this region is presented and discussed. The data indicate that the cloacin and immunity genes were coordinately transcribed into messenger RNAs of about 2,400 and 2,200 nucleotides, which differ in length at their 3' terminus. RNA polymerase binding studies and in vitro transcription assays indicated that transcription of these genes initiates at a promoter located around 32% on the CloDF13 map. Furthermore, it is shown that a 100-nucleotide RNA is encoded by the CloDF13 DNA region between 7.7 and 8.8% on the plasmid genome; the synthesis of this RNA proceeds in a direction opposite to the transcription of the cloacin and immunity genes.

Interactions between the F conjugal transfer system and CloDF13::Tna plasmids.

It was confirmed that all the F transfer genes required for the formation of stable mating pairs, including traN and traG, are essential for transfer of the two small multicopy plasmids ColE1 and cloDF13, whereas the traM, traI and traZ genes that are required for F conjugal DNA metabolism, are not. Differences between ColE1 and CloDF13 were that the F traD gene was needed for transfer of ColE1 but not of CloDF13, and that R100-1 efficiently transferred CloDF13 but not ColE1. A copy number mutant of CloDF13 inhibited F transfer and reduced plaque formation by the F-specific RNA phage f2, but not by Q beta or by the single-strand DNA phage f1. This phenotype suggests that the inhibition system (FinC) acts on traD, mutations in which give a similar phenotype. Hybridisation experiments with lambda tra phage DNA showed that transcription of traD was not reduced, and FinC probably inhibits function of the traD product rather than its synthesis. FinC-insensitive Flac mutants were isolated and characterised. One was shown to have an uppromoter mutation resulting in increased transcription of traJ and hence of the traY leads to Z operon including traD: the raised level of the traD product presumably then counteracted FinC inhibition.

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

The presence of plasmid Clo DF13 in Escherichia coli cells alters the response of these cells to infection with the double stranded DNA phages P1vir, lambda 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, between 29 an 62% on the 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.

Molecular structure of the immunity gene and immunity protein of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence of the Clo DF13 DNA region comprising the immunity gene has been determined. We also elucidated the aminoacid sequence of the 40 N-terminal and 7 C-terminal aminoacids of the purified immunity protein. From analysis of the data obtained we were able to locate the immunity gene between 11.7 and 14.5% on the Clo DF13 map, and to determine the complete aminoacid sequence of the immunity protein. It was observed that the Clo DF13 immunity gene encodes an 85 aminoacid protein and is transcribed in the same direction as the cloacin gene. These experimental data support our model, presented elsewhere, which implicates that the cloacin and immunity genes of Clo DF13 are coordinately transcribed from the cloacin promoter. We also present DNA sequence data indicating that an extra ribosome binding site precedes the immunity gene on the polycistronic mRNA. This ribosome binding site might explain the fact that in cloacinogenic cells more immunity protein than cloacin is synthesized. The comparison of the complete aminoacid sequence of the Clo DF13 immunity protein, with the aminoacid sequence data of the purified, comparable Col E3 immunity protein revealed that both proteins have extensive homologies in primary and secondary structure, although they are exchangeable only to a low extent in vivo and in vitro. It was also observed that a lysine residue was modified in immunity protein isolated from excreted bacteriocin complexes.

The nucleotide sequence surrounding the replication origin of the cop3 mutant of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence from about 100 base-pairs downstream to about 600 base pairs upstream the CloDF13 replication origin has been determined. A comparison of this sequence with the corresponding ColE1 origin sequence reveals that: The sequence at the origin of replication is conserved. There are large differences in the nucleotide sequence downstream the replication origin, whereas there is a large homology in the region of about 410 base-pairs upstream the replication origin. This conserved region might code for a largely homologous basic, arginine rich polypeptide of about 45 amino-acids, for both ColE1 and CloDF13. Although there are large differences in the primary structure of the region coding for the 100 nucleotide RNA, the secondary structure of this region seems to be conserved.

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

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.

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.

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.

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 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 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 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- rep3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.

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

An 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 Emboden 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.