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.     

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.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.     

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).     

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.     

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.     

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.     

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.     

Mutants of the Clo DF13 plasmid inEscherichia coli with a decreased bacteriocinogenic activity

Summary Three Clo DF13 mutant plasmids (designated asclp03, clp05 andclp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by theclp03 and theclp21 mutant plasmids have a strongly decreased killing activityin vivo in comparison with the wild type cloacin and the cloacin of theclp05 mutant plasmid. Furthermore no lacunae could be observed fromclp03 orclp21 harbouring strains, while strains harbouring theclp05 plasmid showed a 50–100 times decreased frequency of lacunae. In addition theclp05 mutant showed a decreased rate of RNA synthesis inclp05 harbouringEscherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that theclp03 andclp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by theclp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

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.     

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.     

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.     

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.     

Transposable plasmid deoxyribonucleic acid sequence in Pseudomonas aeruginosa which mediates resistance to gentamicin and four other antimicrobial agents.

A 9.1 x 10(6)-dalton transposable deoxyribonucleic acid sequence resides within Pseudomonas aeruginosa plasmid R1033 and mediates resistance to gentamicin, streptomycin, sulfamethoxazole, chloramphenicol, and mercuric chloride. Transposability was demonstrated in Escherichia coli when this sequence, designated Tn1696, excised from R1033 and integrated into plasmid pMB8. Excision and insertion of Tn1696 occurred independently of the host Rec phenotype and may involve the 140-base pair, inverted deoxyribonucleic acid repeated region that flanks this sequence. Occurrence of a multiresistance transposon on a transferrable plasmid that has a broad host range may have serious epidemiological and therapeutic consequences.     

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.