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.     

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.     

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.     

Purification and characterization of a complex between cloacin and its immunity protein isolated from Enterobacter cloacae (Clo DF13). Dissociation and reconstitution of the complex.

Cell of Enterobacter cloacae (Clo DF13) produce a bacteriocin which is characterized by its very effective killing activity against sensitive bacteria. Purification and characterization of the excreted bacteriocin has revealed that this bacteriocin consists of an equimolar complex of two plasmid-specific gene products: the cloacin and its inhibitor the immunity protein. Dissociation of the complex by treatment with sodium dodecylsulfate induces the endonucleolytic activity of the cloacin but strongly reduces the killing activity. The purified complex possesses no activity in vitro. Both cloacin and immunity protein isolated from the complex were functionally identical to cloacin and immunity protein purified from the bacteriocinogenic cells by other methods. Reconstitution of the complex results in a partial restoration of killing activity.     

The stable bacteriocin release protein signal peptide, expressed as a separate entity, functions in the release of cloacin DF13

Abstract The pCloDF1S encoded bacteriocin release protein (BRP) plays a role in the release of the bacteriocin cloacin DF13. The BRP signal peptide is stable after cleavage, and accumulates in the cytoplasmic membrane. A BRP which is correctly targeted by the unstable murein lipoprotein signal peptide (Lpp-BRP) is not capable of inducing the release of cloacin DF13. To investigate the role of the stable BRP signal peptide in the release of cloacin DF13, the stable BRP signal peptide and the Lpp-BRP were expressed in trans in cells also producing cloacin DF13. Expression and release experiments indicate that the stable signal peptide can complement the Lpp-BRP in the release of cloacin DF13.     

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.     

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.     

Functioning of the stable signal peptide of the pCloDF13-encoded bacteriocin release protein

The pCloDF13-encoded bacteriocin release protein (BRP) is a lipoprotein which is synthesized as a precursor with an amino-terminal signal peptide that appears to be stable after cleavage. The role of the stable signal peptide in the functioning of the BRP was studied with respect to the release of cloacin DF13, 'lysis' and leakage of periplasmic proteins. The BRP gene fragment encoding the stable signal peptide was replaced by a fragment encoding the unstable peptide of the murein lipoprotein (Lpp). The resulting hybrid protein was normally acylated and processed by signal peptidase II, leaving no stable signal peptide in the cells. Expression of the hybrid protein did not result in the specific release of cloacin DF13, whereas 'lysis' and the release of periplasmic enzymes were unaffected. These results indicated a role for the stable BRP signal peptide in the translocation of cloacin DF13 across the cytoplasmic membrane.     

Effect of a mutation preventing lipid modification on localization of the pCloDF13-encoded bacteriocin release protein and on release of cloacin DF13.

The pCloDF13-encoded bacteriocin release protein (BRP; Mr 2,871) is essential for the translocation of cloacin DF13 across the cell envelope of producing Escherichia coli cells. Overproduction of this BRP provokes lysis (quasilysis) of cells. Construction and analysis of a hybrid BRP-beta-lactamase protein (BRP-Bla) demonstrated that the BRP contains a lipid modified cysteine residue at its amino terminus and is mainly located in the outer membrane. The significance of lipid modification for the localization and functioning of the BRP was investigated. Site-directed mutagenesis was used to substitute the cysteine residue for a glycine residue in the lipobox of the BRP and the BRP-Bla protein. The mutated BRP was unable to bring about the release of cloacin DF13 and could not provide the lysis (quasilysis) of host cells. However, the mutated BRP strongly inhibited the colony-forming ability of the cells, indicating that induction of the mutated protein still affected cell viability. In contrast to the wild-type BRP-Bla protein, the mutated BRP-Bla protein was mainly located in the cytoplasmic membrane, indicating that the mutation prevented the proper localization of the protein. The results indicated that lipid modification of the BRP is required for its localization and release of cloacin DF13, but not for its lethality to host 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).     

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.     

In vitro binding of cloacin DF13 to its purified outer membrane receptor protein and effect of peptidoglycan on bacteriocin-receptor interaction.

The in vitro neutralization of the killing activity of cloacin DF13 by incubation with its purified receptor protein was shown to be the result of the formation of a direct and specific equimolar complex of both proteins. The binding of cloacin DF13 to its receptor protein did not result in a fragmentation of the cloacin molecules nor in the expulsion of immunity protein from the bacteriocin. The rate of the cloacin DF13-receptor interaction in vitro was found to be enhanced significantly in the presence of peptidoglycan, but lysozyme-treated peptidoglycan did not affect this interaction. Incubation of the cloacin DF13 as well as its receptor protein with peptidoglycan showed that the receptor protein but not the cloacin DF13 was able to bind to the peptidoglycan.     

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13 has been determined. The plasmid consists of 9957 base pairs (molecular weight 6.64 X 10(6] with a GC content of 54.4%. At this moment 16 identified biological functions can be assigned to the primary structure of the CloDF13 DNA. The functions include those of eight protein encoding genes, two untranslated RNA species, and six DNA sites. We discuss these functions in relation to the structure of CloDF13 DNA. For convenience we have divided the CloDF13 genome into five defined regions: region I (origin of vegetative replication, priming and control of replication, type I incompatibility), region II (cloacin DF13, cloacin immunity, cloacin release, cloacin operon control), region III (double-stranded DNA-phage interaction, type II incompatibility, multimer resolution), region IV (inhibition of male specific RNA phages and transfer of Flac), and region V (mobility proteins, basis of mobility).     

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.     

tolQ is required for cloacin DF13 susceptibility in Escherichia coli expressing the aerobactin/cloacin DF13 receptor IutA

We investigated the role of the tolQ gene in the import of cloacin DF13 across the outer membrane of Escherichia coli strains expressing the IutA receptor. The IutA outer-membrane protein is the receptor for the siderophore ferric aerobactin and also binds cloacin DF13, a bacteriocin produced by strains of Enterobacter aerogenes. In this report we present evidence that tolQ is required for the internalization of cloacin DF13 upon binding to IutA but it is not involved in the transport of ferric aerobactin.     

Effects of divalent cations and of phospholipase A activity on excretion of cloacin DF13 and lysis of host cells

Induction of cloacin DF13 synthesis in Escherichia coli harbouring plasmid CloDF13 results in the release of cloacin DF13, inhibition of growth and ultimately in lysis of the host cells. Expression of the pCloDF13-encoded protein H is essential for both the release of cloacin DF13 and the lysis of the cells. The divalent cations Mg2+ and Ca2+ interfered with the mitomycin C-induced protein H-dependent lysis, but hardly affected the release of cloacin DF13. Essentially all of the bacteriocin was released from the cells before a detectable degradation of the peptidoglycan occurred, independent of the presence of mitomycin C. Experiments with phospholipase A mutants revealed that activation of detergent-resistant phospholipase A was essential for the export of cloacin DF13 across the outer membrane and the lysis of induced cells. Transport of cloacin DF13 across the cytoplasmic membrane was mainly dependent on protein H. A revised model for the excretion of cloacin DF13 is presented.     

Role of tol genes in cloacin DF13 susceptibility of Escherichia coli K-12 strains expressing the cloacin DF13-aerobactin receptor IutA.

IutA is the outer membrane protein receptor for ferric aerobactin and the bacteriocin cloacin DF13. Although the same receptor is shared, ferric aerobactin transport across the outer membrane in Escherichia coli is TonB dependent, whereas cloacin DF13 transport is not. We have recently observed that tolQ is required for cloacin DF13 susceptibility (J.A. Thomas and M.A. Valvano, FEMS Microbiol. Lett. 91:107-112, 1992). In this study, we demonstrate that the genes tolQ, tolR, and tolA, but not tolB, tolC, and ompF, are required for the internalization of cloacin DF13 and they are not involved in the transport of ferric aerobactin.     

The stable BRP signal peptide causes lethality but is unable to provoke the translocation of cloacin DF13 across the cytoplasmic membrane of Escherichia coli

The bacteriocin release protein (BRP) mediates the secretion of cloacin DF13. The BRP precursor is slowly processed to yield the mature BRP and its stable signal peptide which is also involved in cloacin DF13 secretion. The function of the stable BRP signal peptide was analysed by constructing two plasmids. First, the stable BRP signal peptide was fused to the murein lipoprotein and, second, a stop codon was introduced after the BRP signal sequence. Exchange of the unstable murein lipoprotein signal peptide for the stable BRP signal peptide resulted in an accumulation of precursors of the hybrid murein lipoprotein. This indicated that the BRP signal peptide, as part of this hybrid precursor, is responsible for the slow processing. The stable BRP signal peptide itself was not able to direct the transfer of cloacin DF13 into the periplasmic space or into the culture medium. Over-expression of the BRP signal peptide was lethal and caused 'lysis'. Subcellular fractionation experiments revealed that the BRP signal peptide is located exclusively in the cytoplasmic membrane whereas the mature BRP, targeted by either the stable BRP signal peptide or the unstable Lpp signal peptide, is located in both the cytoplasmic and outer membrane. These results are in agreement with the hypothesis that the stable signal peptide and the mature BRP together are required for the passage of cloacin DF13 across the cell envelope.