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.     

Enzymatic properties of cloacin DF13 and kinetics of ribosome inactivation.

1. The cloacin DF13-induced inactivation of ribosomes in vitro can be described as an enzyme-catalyzed reaction according to the Michaelis-Menten equation. Most probably the cloacin acts as a unique endoribonuclease. 2. At pH 7.8 and 37 degrees C the Km value for the reaction of cloacin DF13 with ribosomes is 13.2 - 10(-6) M. If under these conditions the reaction mixture is supplemented with all components necessary for protein synthesis, the Km changes to 17.7 - 10(-6) M. 3. The in vitro activity of cloacin DF13 has a temperature optimum of 43 degrees C at pH 7.8 and a pH optimum of 8.4 at 37 degtees C. 4. Experiments with cloacin DF13-immunity protein as an inhibitor of the cloacin activity in vitro have indicated that the immunity protein might be considered as a non-competitive and virtually "irreversible" inhibitor.     

Purification and characterization of cloacin DF13 receptor from Enterobacter cloacae and its interaction with cloacin DF13 in vitro.

Extraction of the crude cell envelope fraction of cloacin DF13-susceptible Enterobacter cloacae strain 02 with Triton X-100 and ethylenediaminetetraacetate solubilized an outer membrane fraction which neutralized the lethal activity of cloacin DF13. A similar fraction could not be isolated from strains known to be lacking functional cloacin DF13 receptors. On this basis the isolated outer membrane fraction was assumed to contain the specific cloacin DF13 receptor. The receptor was purified to homogeneity by acetone precipitation and affinity chromatography, using cloacin DF13 as a ligand. The purified receptor was identified as a protein which consisted of a single polypeptide chain with an apparent molecular weight of 90,000 and a preponderance of acidic amino acids (pI = 5.0). The interaction of equimolar amounts of purified receptor and cloacin DF13 in vitro resulted in a complete, irreversible neutralization of the lethal activity of the bacteriocin. This interaction showed a temperature optimum at 43 degrees C but was only slightly affected by variation of the pH between 5.0 and 8.5 or by increasing the ionic strength of the incubation buffer. The receptor had no neutralizing activity towards other bacteriocins, such as colicin E1 or colicin E3.     

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.     

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.     

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.     

Sensitivity of Escherichia coli to cloacin DF13 involves the major outer membrane protein OmpF

Fourteen spontaneous cloacin DF13-insensitive mutants of an Escherichia coli strain expressing the aerobactin-cloacin DF13 receptor protein IutA were isolated. The mutants fell into three classes on the basis of outer membrane profiles analyzed by electrophoresis in denaturing polyacrylamide gels. The most frequent class lacked the IutA protein and was unable to bind cloacin DF13 or aerobactin. A second class of mutants had lost protein species corresponding in size to the porin proteins OmpF and OmpC. To determine which porin was required for the bactericidal activity of cloacin DF13, defined strains with mutations at the ompB (ompR envZ) locus were transformed with a recombinant plasmid carrying the iutA gene and screened for cloacin DF13 sensitivity. OmpF- strains, whether OmpC+ or OmpC-, were insensitive to cloacin DF13, indicating involvement of the OmpF protein in cloacin DF13 killing. An OmpC- OmpF+ strain, on the other hand, was more sensitive than the wild-type parent strain, probably because of compensatory overexpression of OmpF. The third class of cloacin DF13-insensitive mutant had lost an outer membrane protein of approximately 31 kDa. The nature and function of this protein are not yet known, but it is not the protease OmpT. Mutants of classes 2 and 3 bound cloacin DF13 and aerobactin as effectively as the cloacin DF13-sensitive parental strain, indicating that they remained IutA+. We propose that these mutants (more accurately described as cloacin DF13 tolerant) are defective in translocation of the active portion of cloacin DF13 across the bacterial membranes.     

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.     

Plasmid-determined cloacin DF13-susceptibility inEnterobacter cloacae andKlebsiella edwardsii; identification of the cloacin DF13/aerobactin outer membrane receptor proteins

BothEnterobacter cloacae H478 andKlebsiella edwardsii S15 were shown to harbour a relatively large conjugative plasmid that coded for cloacin DF13-susceptibility and the production and uptake of a hydroxamate iron chelator, most probably aerobactin. Protein-blotting experiments with antiserum raised against the purified cloacin DF13/aerobactin receptor protein fromEscherichia coli (Co1V-K30) revealed that the corresponding outer membrane receptor proteins ofEnt. cloacae H478 andK. edwardsii S15 had apparent mol wts of 85 000 and 76000, respectively.E. coli transconjugants harbouring either the plasmid fromEnt. cloacae H478 orK. edwardsii S15 expressed a cloacin DF13/aerobactin outer membrane receptor protein with a mol wt of 74000. The receptor protein encoded by theEnt. cloacae andK. edwardsii plasmids were immunologically more related to each other than to the pCo1V-K30-encoded receptor protein.     

Production and excretion of cloacin DF13 by Escherichia coli harboring plasmid CloDF13.

The production and the mechanism of excretion of cloacin DF13 were investigated in noninduced and mitomycin C-induced cell cultures. A mitomycin C concentration was selected which did not cause lysis of cloacinogenic cells, but at the same time induced a maximal production of cloacin DF13. Native cloacin DF13, possessing killing activity, was first released into the cytoplasm. Shortly thereafter, the bacteriocin was transported through the cytoplasmic membrane and accumulated in the periplasm. Finally, cloacin DF13 was excreted into the culture medium. A small amount of cloacin DF13 remained associated with the cell surface. Producing cells did not become permeable for the cytoplasmic enzyme beta-galactosidase. Apparently the cloacin DF13 leaves the producing cells by an excretion process which is not similar to the mechanism proposed for bacterial secretory proteins. The processes of excretion by producing cells and of uptake by susceptible cells were also not identical because mutant cloacin DF13, which was not transported through the outer membrane into susceptible cells, was excreted like the wild-type cloacin DF13. The composition of the culture medium greatly affected production of cloacin DF13. The presence of sugars known to cause catabolite repression not only inhibited the production but also strongly reduced the excretion of cloacin DF13 into the culture medium.     

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.     

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.     

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.     

Effects of cloacin DF13 on the functioning of the cytoplasmic membrane

Adsorption of cloacin DF13 onto sensitive cells induces a leakage of potassium ions after a lag of 15–20 min. The rate of the potassium efflux is proportional to the bacteriocin concentration used. The extent of the potassium loss depends on the concentrations of bacteriocin and of extracellular potassium. Leakage of potassium ions is accompanied by an uptake of sodium ions. Cloacin DF13 does not affect the transport of magnesium ions, phosphate ions or amino acids. About 10 min after addition of the bacteriocin the ATP content of the cells begins to decrease gradually to about 25% of the original level. Anaerobiosis, lack of energy source and inhibition of oxidative phosphorylation may protect the cells if applied within 10 min after addition of the bacteriocin. The results suggest that the action of cloacin DF13 proceeds through at least two distinct phases. Phase I is a period after bacteriocin adsorption which is reversible and in some way depends on oxidative phosphorylation. This phase does not cause any cellular damage. Phase II is irreversible and results in a disturbance of several cellular functions.     

Three immunity types of klebicins which use the cloacin DF13 receptor of Klebsiella pneumoniae

We have investigated a group of bacteriocinogenic strains used in the epidemiological investigation of Klebsiella infections. Transfer of plasmids from these strains to laboratory strains allowed the identification of three klebicins which use the cloacin DF13 receptor in Klebsiella, but are of three distinct immunity types. These klebicins use the ferric-aerobactin receptor determined by ColV-K30 in Escherichia coli, which is also used by cloacin DF13. We propose to call them group A klebicins, of immunity types A1, A2 and A3. On the basis of immunity, cloacin DF13 also belongs to the klebicin A1 group.     

Molecular structure and immunity specificity of colicin E6, an evolutionary intermediate between E-group colicins and cloacin DF13.

The primary structure of a 3.1-kilobase E6 or E3 segment carrying colicin and related genes was determined. Plasmid ColE6-CT14 showed striking homology to ColE3-CA38 throughout this segment, including homology to the secondary immunity gene, immE8, downstream of the E6 or E3 immunity gene. The ColE3-CA38 and ColE6-CT14 sequences, however, contained an exceptional hot spot region encoding both the colicin-active domain (RNase region) and the immunity protein, reflecting their different immunity specificities. On the other hand, some chimeric plasmids were constructed through homologous recombination between colicin E3 and cloacin DF13 operons. The resulting plasmids were deduced to produce chimeric colicins with a colicin E3-type N-terminal part, a cloacin DF13-type C-terminal-active domain, and the DF13 immunity protein. The killing spectra of the chimeric colicins and the immunities of the plasmids were identical to those of colicin E6 and ColE6-CT14, respectively, showing that the colicin E6 immunity specificity is completely equivalent to that of cloacin DF13. Nevertheless, colicin E6 has been found to show a sequence diversity from cloacin DF13 almost to the same extent as that from colicin E3 in their RNase and immunity regions, indicating that only a small number of amino acids defines the immunity specificity for discrimination between colicins E3 and E6 (or cloacin DF13).     

Siderophore production by Enterobacter cloacae and a common receptor protein for the uptake of aerobactin and cloacin DF13.

Iron-starved cultures of Enterobacter cloacae produced two siderophores, identified as enterochelin and aerobactin. The aerobactin was excreted in larger amounts than was enterochelin, and it was synthesized preferentially in the late logarithmic and stationary growth phases under iron-deficient conditions. Enterochelin was synthesized by cultures in the logarithmic phase of growth and preferentially in medium with 1 microM ferric chloride. Both siderophores appeared to be excreted immediately after their synthesis, since no intracellular aerobactin or enterochelin could be detected. The killing activity of the bacteriocin cloacin DF13 was inhibited by aerobactin. It was shown that aerobactin and cloacin DF13 bound to the same receptor sites located in the outer membrane. The synthesis of these receptor sites was induced by iron limitation. We conclude that the receptor for the uptake of aerobactin also functions as receptor for cloacin DF13.