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.     

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.     

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.     

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 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).     

Early development of bacteriophages SP01 and SP82G in minicells of Bacillus subtilis.

SP01- and SP82G-infected Bacillus subtilis CU403 divIVBI minicells synthesize 13 easily detectable early RNA species with molecular weights ranging from 60 × 10³ to 430 × 10³. Comparison of in vivo and in vitro translation of early messenger RNA indicates that five early mRNAs of SP01 are synthesized but not translated unless protein synthesis has been permitted in the infected minicell, providing evidence for a translation control mechanism. A sequential appearance of 48 polypeptides has been determined in SP01-infected minicells. The polypeptides have been grouped into two classes of early polypeptides, i.e. those encoded by early mRNA and three subsequent classes as demonstrated by the analysis of polypeptides synthesized in minicells infected with the SP01 mutants, susF21, susF4 and susF14. Phage capsid proteins are not synthesized in minicells. RNA synthesized in infected minicells is subject to turnover. The individual mRNA species have differing functional stabilities ranging from a loss of only 50% functional activity, in 20 minutes at 37 °C, to loss of over 99% activity.Infection of anucleate minicells has been shown to be a very simple method for comparison of closely related phages (slight differences are detected between SP01- and SP82G-encoded mRNA and polypeptides), detection of polypeptides affected by amber mutations and the analysis of early events in phage development in the absence of host syntheses.     

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.     

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 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.     

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.     

R Factor Deoxyribonucleic Acid in Chromosomeless Progeny of Escherichia coli

The deoxyribonucleic acid (DNA) of resistance (R) factor 222 carried by Escherichia coli strain P678-54 was found in the normally chromosomeless progeny (minicells) of that strain. The entry of the R222 DNA into minicells appears to be via segregation at the time of their formation from normal cells. The R222 DNA can replicate in minicells although the extent of its replication appears to be limited. An analysis of the R222 DNA structure indicates that it exists in minicells as double-stranded linear, open circular, and twisted circular monomers (molecular weight, about 6.2 x 10(7) daltons). The monomers visualized by electron microscopy are 31.0 +/- 0.5 mum in length. An examination of the effect of acridine orange on the replication of R222 and colicin E1 DNA indicates the dye intereferes with plasmid DNA replication.     

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.     

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.     

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.     

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.     

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.     

Expression of bacteriophage M13 dna in vivo. I. Synthesis of phage-specific RNA and protein in minicells.

It is demonstrated that after infection of the appropriate minicell-producing strain of Escherichia coli with the filamentous bacteriophage M13, its replicative form DNA is segregated into minicells. Consequently these minicells have acquired the capability to direct the synthesis of phage-specific RNA and protein. Comparision of the electrophoretic mobilities of phage-specific RNA species made in vitro with those made in M13 replicative form DNA harbouring minicells, have indicated that almost all in vitro synthesized G-start RNAs have an equivalent among the in vivo synthesized RNA products. Furthermore it could be demonstrated that in M13 replicative form DNA harbouring minicells the phage-specific proteins encoded by genes III, IV, V and VIII are made. In addition the synthesis of a phage-specific polypeptide (molecular weight approx. 3000) co-migrating with the recently discovered capsid protein (designated C-protein) could be demonstrated. The meaning of these results for the resolution of the regulatory mechanisms operative during the life cycle of this phage will be discussed.     

Synthesis of high molecular weight polypeptides in Escherichia coli minicells directed by cloned Saccharomyces cerevisiae 2-μm DNA

The minicell-producing Escherichia coli strain P 678-54 was transformed with a series of defined PTY chimeric plasmids consisting of yeast 2-μm DNA and E. coli plasmid pCR1. In minicells the integrated 2-μm DNA from yeast directed specifically the synthesis of six polypeptides with apparent molecular weights of 15 000, 17 500, 20 000, 22 000, 37 000, and 48 000. The specificity of five other polypeptides, which cover a molecular weight range of 19 000 to 28 000, has not yet been established with certainty. Neither the orientation of the integrated DNA, nor the inversion which distinguishes the two structural forms of 2-μm DNA affected the polypeptides synthesized. However, integration at a given EcoRI site appeared to be correlated with the absence of one particular polypeptide band; this suggests that at least one of these sites is located in an expressed region of the DNA.     

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.     

Trehalose Metabolism in Germinating Spores of Streptomyces hygroscopicus

Catenated deoxyribonucleic acid molecules of colicin factor E1 (Col E1) were found with nearly equal frequency in minicells derived from Escherichia coli strain P678-54 recA(+) (Col E1) and P678-54 recA(-) (Col E1). The result suggests that the recombination function controlled by the recA gene does not influence the formation of catenated Col E1.