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.     

pCloDF13-encoded bacteriocin release proteins with shortened carboxyl-terminal segments are lipid modified and processed and function in release of cloacin DF13 and apparent host cell lysis.

By oligonucleotide-directed mutagenesis, stop codon mutations were introduced at various sites in the pCloDF13-derived bacteriocin release protein (BRP) structural gene. The expression, lipid modification (incorporation of [3H]palmitate), and processing (in the presence and absence of globomycin) of the various carboxyl-terminal shortened BRPs were analyzed by a special electrophoresis system and immunoblotting with an antiserum raised against a synthetic BRP peptide, and their functioning with respect to release of cloacin DF13, lethality, and apparent host cell lysis were studied in Sup-, supF, and supP strains of Escherichia coli. All mutant BRPs were stably expressed, lipid modified, and processed by signal peptidase II, albeit with different efficiencies. The BRP signal peptide appeared to be extremely stable and accumulated in induced cells. Full induction of the mutant BRPs, including the shortest containing only 4 amino acid residues of the mature polypeptide, resulted in phospholipase A-dependent and Mg2+-suppressible apparent cell lysis. The extent of this lysis varied with the mutant BRP used. Induction of all mutant BRPs also prevented colony formation, which appeared to be phospholipase A independent. One shortened BRP, containing 20 amino acid residues of the mature polypeptide, was still able to bring about the release of cloacin DF13. The results indicated that the 8-amino-acid carboxyl-terminal segment of the BRP contains a strong antigenic determinant and that a small segment between amino acid residues 17 and 21, located in the carboxyl-terminal half of the BRP, is important for release of cloacin DF13. Either the stable signal peptide or the acylated amino-terminal BRP fragments (or both) are involved in host cell lysis and lethality.     

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.     

Functioning of a hybrid BRP-β-lactamase protein in the release of cloacin DF13 and lysis of Escherichia coli cells

The gene encoding a hybrid BRP-Bla protein consisting of the pCloDF13 encoded BRP signal sequence, 25 of the 28 amino acid residues of the mature bacteriocin release protein (BRP) and the mature portion of beta-lactamase (Bla) was subcloned in the expression vector pEB112. A similar construct was made using a mutant gene encoding a BRP-Bla protein in which the cysteine residue at the +1 position was changed into a glycine residue. The expression, processing, functioning and subcellular localization of the 'wild-type' and mutant hybrid protein at high-level expression conditions were studied. The 'wild-type' BRP-Bla protein was mainly found in the outer membranes and possessed all the activities of the BRP itself; the protein was able to bring about the release of cloacin DF13 and caused apparent cell-lysis after high-level synthesis. The mutant hybrid protein was predominantly located in the inner membranes, was inactive in the release of cloacin DF13, but caused apparent cell-lysis only after strong induction.     

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.     

Expression of the pCloDF13 encoded bacteriocin release protein or its stable signal peptide causes early effects on protein biosynthesis and Mg2+ transport

The effect of the pCloDF13 encoded bacteriocin release protein (BRP) onEscherichia coli cell lethality was studied. Induction of the BRP resulted in a strong inhibition of the incorporation of radioactive labeled amino acids and affected the transport of Mg²⁺ ions. Similar effects were obtained when the BRP stable signal peptide was expressed as a separate entity. Kinetic studies revealed that these effects occurred prior to quasi-lysis and release of cloacin DF13. The results indicated that the BRP induced cell lethality is caused by early effects on protein synthesis and Mg²⁺ transport, due to the accumulation of stable BRP signal peptides in the cytoplasmic membrane.     

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.     

Optimization of Bacteriocin Release Protein (BRP)-Mediated Protein Release by Escherichia coli: Random Mutagenesis of the pCloDF13-Derived BRP Gene To Uncouple Lethality and Quasi-Lysis from Protein Release

Bacteriocin release proteins (BRPs) can be used for the release of heterologous proteins from the Escherichia coli periplasm into the culture medium. However, high-level expression of BRP causes apparent lysis of the host cells in liquid cultures (quasi-lysis) and inhibition of growth on broth agar plates (lethality). To optimize BRP-mediated protein release, the pCloDF13 BRP gene was subjected to random mutagenesis by using PCR techniques. Mutated BRPs with a strongly reduced capacity to cause growth inhibition on broth agar plates were selected, analyzed by nucleotide sequencing, and further characterized by performing growth and release experiments in liquid cultures. A subset of these BRP derivatives did not cause quasi-lysis and had only a small effect on growth but still functioned in the release of the periplasmic protein β-lactamase and the periplasmic K88 molecular chaperone FaeE and in the release of the bacteriocin cloacin DF13 into the culture medium. These BRP derivatives can be more efficiently used for extracellular production of proteins by E. coli than can the original BRP.     

Cloning,expression and release of native and mutant cloacin DF13 immunity protein

The pCloDF13 encoded immunity protein gene was subcloned in the expression vector pINIIIA1 and several deletion, insertion and point mutations were constructed in the aminoterminal and carboxyl-terminal regions of the protein. The expression, stability, BRP-dependent export and protective capacity of the native and mutant immunity proteins were studied by SDS-PAGE, immunoblotting and an in vivo activity assay. In the absence of cloacin the unbound, native immunity protein was stable produced by E. coli cells and released after BRP induction. The expression of most of the mutant immunity proteins was strongly reduced and non of the proteins were found to be released. All mutations in the carboxyl-terminal region strongly affected expression of the proteins, probably by causing protein instability and proteolytic degradation. One of these mutant immunity proteins, with an insertion mutation in its carboxylterminal region, still caused an intermediate immunity of susceptible cells against extracellularly added cloacin DF13. Mutations in the amino-terminal region of the immunity protein had less effect on its expression and did not affect the protective capacity of the protein.     

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 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.     

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.     

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.     

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 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.     

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.     

Uptake of cloacin DF13 by susceptible cells: removal of immunity protein and fragmentation of cloacin molecules.

Monoclonal antibodies (MAb) directed against different epitopes on the equimolar complex of cloacin and immunity protein (cloacin DF13) were isolated, characterized, and used to study the uptake of cloacin DF13 by susceptible cells. Four MAbs recognized the amino-terminal part, one MAb recognized the central part, and three MAbs recognized the carboxyl-terminal part of the cloacin molecule. Three MAbs reacted with the immunity protein. Five MAbs inhibited the lethal action of cloacin DF13, but none of the MAbs inhibited the binding of cloacin DF13 to its purified outer membrane receptor protein or the in vitro inactivation of ribosomes. Binding of cloacin DF13 to susceptible cells cultured in broth resulted in a specific, time-dependent dissociation of the complex and a fragmentation of the cloacin molecules. Increasing amounts of immunity protein were detected in the culture medium from about 20 min after the addition of cloacin DF13. Cloacin was fragmented into two carboxyl-terminal fragments with relative molecular masses of 50,000 and 10,000. The larger fragment was detected 5 min after the binding of the bacteriocin complex to the cells. The smaller fragment was detected after 10 min. Both fragments were associated with the cells and could not be detected in the culture supernatant fraction. Cells grown in brain heart infusion were much less susceptible to cloacin DF13 than cells grown in broth, although they possessed a similar number of outer membrane receptor molecules. This decreased susceptibility correlated with a decreased translocation, dissociation, and fragmentation of cloacin DF13.     

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.     

Molecular characterisation of the colicin E2 operon and identification of its products

Summary The DNA sequence of the entire colicin E2 operon was determined. The operon comprises the colicin activity gene, ceaB, the colicin immunity gene, ceiB, and the lysis gene, celB, which is essential for colicin release from producing cells. A potential LexA binding site is located immediately upstream from ceaB, and a rho-independent terminator structure is located immediately downstream from celB. A comparison of the predicted amino acid sequences of colicin E2 and cloacin DF13 revealed extensive stretches of homology. These colicins have different modes of action and recognise different cell surface receptors; the two major regions of heterology at the carboxy terminus, and in the carboxy-terminal end of the central region probably correspond to the catalytic and receptor-recognition domains, respectively. Sequence homologies between colicins E2, A and E1 were less striking, and the colicin E2 immunity protein was not found to share extensive homology with the colicin E3 or cloacin DF13 immunity proteins. The lysis proteins of the ColE2, ColE1 and CloDF13 plasmids are almost identical except in the aminoterminal regions, which themselves have overall similarity with lipoprotein signal peptides. Processing of the ColE2 prolysis protein to the mature form was prevented by globomycin, a specific inhibitor of the lipoprotein signal peptidase. The mature ColE2 lysis protein was located in the cell envelope. The results are discussed in terms of the functional organisation of the colicin operons and the colicin proteins, and the way in which colicins are released from producing cells.