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.     

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.     

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.     

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.     

Effects of temperature on the activity of cloacin DF13 and cloacin DF13-immunity protein.

During the interaction of cloacin DF13 and sensitive cells the cloacin molecules display different functions which can be distinguished on the basis of their heat-sensitivity. Binding to cell envelope receptors, binding of immunity protein and in vitro inactivation of ribosomes are heat-stable functions in contrast with the entire killing action in vivo. Cloacin DF13-immunity protein appears to be a heat-stable inhibitor of the fibosome inactivation caused by 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.     

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.     

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.     

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.     

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.     

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.     

Characterization of the pColV-K30 encoded cloacin DF13/aerobactin outer membrane receptor protein of Escherichia coli; isolation and purification of the protein and analysis of its nucleotide sequence and primary structure

Abstract The cloacin DF13/aerobactin receptor protein from Escherichia coli (pFS8) and from Klebsiella edwardsii were isolated by repeated Triton X-100 extractions and purified by affinity chromatography. Both receptor proteins ran as a single protein band on SDS-PAGE. Their apparent M_r values were 74 000 and 76 000, respectively. The binding constants of the purified receptor proteins from E. coli (pFS8) and K. edwardsii and cloacin DF13 were determined. Values of 2.0 × 10⁸ M⁻¹ and 1.0 × 10⁹ M⁻¹, respectively, were found.
The nucleotide sequence of the pColV-K30 gene, contained on pFS8 and encoding the cloacin DF13/aerobactin receptor protein, was determined and the primary structure of the protein as well as its secondary structure were deduced. The results revealed that the pColV-K30-specified receptor protein might be synthesized as a precursor, with a signal sequence of 25 amino acid residues. The mature protein has an M_r of 77 345.     

Subcloning of the cloacin DF13/aerobactin receptor protein and identification of a pColV-K30-determined polypeptide involved in ferric-aerobactin uptake.

A plasmid containing a pColV-K30 fragment that encoded only for the cloacin DF13/aerobactin receptor protein was constructed. Escherichia coli cells harboring this plasmid were sensitive to cloacin DF13 but were unable to take up ferric-aerobactin. Another pColV-K30-determined polypeptide (molecular weight, 50,000), localized in the membrane fraction, was essential for the uptake of ferric-aerobactin.     

Cloning and expression of the cloacin DF13/aerobactin receptor of Escherichia coli (ColV-K30)

A DNA fragment derived from the ColV-K30 plasmid and coding for both sensitivity to cloacin DF13 and Fe3+-aerobactin uptake was cloned into pBR322. The cloned fragment coded for two polypeptides with molecular masses of 74,000 (the cloacin DF13/aerobactin receptor protein) and 50,000 daltons, respectively. When grown with sufficient iron, cells harboring pFS8 (with this fragment) possessed about 10 times as many receptor protein molecules as compared with cells of Escherichia coli (ColV-K30). The synthesis of the receptor protein specified by pFS8, however, was independent of the availability of iron, in contrast to strains harboring the intact ColV-K30 plasmid. Aerobactin was taken up but not synthesized by cells harboring pFS8. No growth occurred when iron-starved cultures of these cells were incubated with Fe3+-aerobactin, suggesting that expression of other ColV-K30-encoded genes is necessary to remove the iron from the Fe3+-aerobactin complex.     

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.