A 76-residue polypeptide of colicin E9 confers receptor specificity and inhibits the growth of vitamin B12-dependent Escherichia coli 113/3 cells.

The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.

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

The pCloDF13 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 murecin 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 DF 13.

Effect of precultivation conditions on colicin susceptibility in Escherichia coli.

The effect of 20 colicins and cloacin was studied after various precultivations. Nutrient agar supplemented with subinhibitory concentration of EDTA used for precultivation or elevating the growth-temperature of the inoculum from 37 degrees C to 42 degrees C increased the susceptibility of wild-type (smooth) Escherichia coli strains to the inhibitory action of some colicins. There were great differences among the colicins in respect to these effects. In case of rough mutants, their sensitivities did not change or eventually decrease after EDTA or heat pretreatment. The LPS pattern in SDS-PAGE of smooth cells grown in EDTA-containing nutrient medium changed in some degree towards the rough character. In case of precultivation at 42 degrees C this change was less considerable. It is supposed that both factors applied during precultivation have influence on colicin sensitivity by means of the change of receptor activity caused by LPS modification.

Diphenylamine increases cloacin DF13 sensitivity in avian septicemic strains of Escherichia coli.

Thirteen avian septicemic isolates of Escherichia coli were examined for the presence of the aerobactin iron transport system. All of the strains possessed a functional aerobactin system and hybridization experiments showed that the aerobactin genes were located on ColV-type plasmids in all cases. The expression of the aerobactin receptor IutA was also studied by determining the bacterial susceptibility to the bacteriocin cloacin DF13. Twelve of the 13 isolates were cloacin-resistant but became sensitive to this bacteriocin upon treatment with diphenylamine which caused a reduction in the amount of O-side chain lipopolysaccharide.

Application and assessment of cloacin typing of Enterobacter cloacae.

Three methods, O-serotyping, phage typing and susceptibility to bacteriocins, were used to type 357 clinical isolates of Enterobacter cloacae cultured from 219 patients. One hundred and sixty isolates were typed by serology and phage typing. When these two methods were used, primary classification of isolates was based on serology (65.7% typable) and phage typing for further subdivision (94.1% typable). When all the isolates were typed by cloacin susceptibility, 81.5% of them were typable. Maximum discrimination between cultures was achieved when the three methods were used together; no single method was sufficiently discriminatory. There was a close parallel between serotyping and bacteriocin lysis pattern. The latter was easy to perform and the results were achieved within 48 h. By applying this typing system two episodes of cross-infection were identified in a haematology/oncology unit and intensive care unit.

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.

Siderophore production by Salmonella species isolated from different sources.

A total of 230 Salmonella strains were screened for enterobactin and aerobactin production, sensitivity to bacteriocins and resistance to antibiotics. All the isolates produced the phenolate siderophore enterobactin. Amongst these, 74 strains, most belonging to S. enteritidis, were sensitive to colicin B. Only 26 isolates, all belonging to S. wien, produced an additional iron chelator, i.e. the siderophore aerobactin, and 22 out of these were sensitive to cloacin DF13. Analysis of iron repressible outer membrane proteins and plasmid profiles in S. wien strains showed that the expression of a 74-kDa iron-repressible outer membrane protein and the presence of large plasmids were associated with multiple antibiotic resistance, aerobactin production and sensitivity to cloacin DF13. The incidence of aerobactin-producing strains among S. wien isolates was higher during years 1974-1985; the epidemiological implications of these results are discussed.

Cloacin tolerance and aspecific colicin insensitivity of human Escherichia coli strains.

Of 182 wild-type human, aerobactin producer Escherichia coli strains 86.3% were insensitive to cloacin. All randomly chosen 51 strains were relatively cloacin tolerant. Cloacin tolerant strains were not considerably more sensitive to hydrophobic drugs than the cloacin sensitive descendant strains. Pathogenicity of the cloacin sensitive strains was significantly lower (p less than 0.05) in intraperitoneal mice infection than that of the cloacin tolerant ones. Suggesting a new aspect of the uptake mechanism of colicins, cloacin tolerance was very frequently associated with an aspecific insensitivity to a broad spectrum of colicins.

Polymorphism in the aerobactin-cloacin DF13 receptor genes from an enteroinvasive strain of Escherichia coli and pColV-K30 is associated only with a decrease in cloacin susceptibility.

We have cloned chromosomal genes mediating the aerobactin iron transport system from the enteroinvasive strain Escherichia coli 978-77. The physical map of the region spanning the siderophore biosynthesis genes and the upstream portion of the receptor gene in strain 978-77-derived clones was identical to the corresponding regions in pColV-K30, while the downstream portion was different. Recombinant plasmids derived from strain 978-77 encoded a 76-kDa outer membrane protein, in contrast to the 74-kDa polypeptide encoded by similar clones derived from pColV-K30. No differences were found in the uptake of ferric aerobactin mediated by either the 76-kDa- or the 74-kDa-encoding plasmids. In contrast, cells containing the 76-kDa-encoding plasmids showed a 16-fold decrease in susceptibility to cloacin compared with cells harboring the 74-kDa-encoding plasmids. Two classes of chimeric aerobactin receptor genes were constructed by exchanging sequences corresponding to the downstream portion from the aerobactin receptor gene of both systems. The pColV-K30-978-77 chimeric gene encoded a 76-kDa outer membrane protein which mediated a low level of cloacin susceptibility, whereas the 978-77-pColV-K30 type encoded a protein of 74 kDa determining a level of cloacin susceptibility identical to that mediated by pColV-K30.

Novel aerobactin receptor in Klebsiella pneumoniae.

Several Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype K1:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not K1 antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the K1:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.

Expression in Escherichia coli K-12 of the 76,000-dalton iron-regulated outer membrane protein of Shigella flexneri confers sensitivity to cloacin DF13 in the absence of Shigella O antigen.

One of the chromosomal segments associated with virulence in Shigella flexneri encodes the production of aerobactin and the synthesis of an iron-regulated 76-kilodalton outer membrane protein believed to be the ferric-aerobactin receptor. However, S. flexneri expressing this putative aerobactin receptor, which is slightly larger than that encoded by pColV, is insensitive to the killing action of cloacin DF13, a bacteriocin which binds to other aerobactin receptor proteins and kills the cells. In this paper we show that the conjugal transfer of DNA encoding the iron-regulated 76-kilodalton protein from S. flexneri to Escherichia coli K-12 conferred cloacin DF13 sensitivity on the recipients. However, E. coli K-12 which had also inherited genes specifying Shigella O-antigen biosynthesis remained cloacin insensitive. The data suggest that it is unwise to use cloacin DF13 sensitivity alone to screen transconjugants or clinical isolates for the expression of aerobactin receptor proteins.

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.

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 amino-terminal 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 carboxyl-terminal 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.

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.

Alterations in the carboxy-terminal half of cloacin destabilize the protein and prevent its export by Escherichia coli.

Several overlapping carboxy-terminal and internal deletions were constructed in the cloacin structural gene. The expression, the binding of the cloacin DF13 immunity protein and the release into the culture medium of the mutant cloacin polypeptides were studied by immunoblotting and ELISAs. Minor alterations at the carboxy-terminal end of the cloacin did not affect protein expression, stability or release to a large extent, but larger carboxy-terminal deletions strongly destabilized the protein and no release was observed. The removal of a particular region within the carboxy-terminal portion of cloacin strongly destabilized the polypeptide and made it a target for proteolytic degradation. Binding of immunity protein did not affect stability and release of the mutant polypeptides. By using immunoelectron microscopy, the polypeptides that were not exported were located in the cytoplasm of producing cells. Large aggregates of these mutant polypeptides were not observed in the cytoplasm: the polypeptides were present in a soluble form.

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.

Uncoupling of synthesis and release of cloacin DF13 and its immunity protein by Escherichia coli.

The synthesis of the bacteriocin cloacin DF13 and its release into the culture medium were genetically uncoupled by subcloning the gene encoding the bacteriocin release protein (BRP) from pCloDF13. The gene was cloned under the control of the IPTG-inducible lpp-lac promoter-operator system on the expression vector pINIIIA1, giving pJL1. A 4 kb DNA fragment of pJL1, containing the tandem lpp-lac promoter, the BRP gene and lacI (BRP cassette), was cloned into the pCloDF13 derivative plasmid pJN67, which encodes cloacin DF13 but not the release protein. Furthermore, the pCloDF13 immunity protein gene was subcloned downstream of the temperature-inducible PL promoter of the expression vector pPLc236, together with the BRP cassette. Growth, induction and excretion experiments with Escherichia coli cells harbouring the constructed plasmids revealed that: the BRP is the only pCloDF13-derived gene product responsible for the observed growth inhibition and apparent lysis of strongly induced cells. This growth inhibition and lysis can be prevented by Mg2+ ions added to the culture medium, and involves induction of phospholipase A activity. The expression of the BRP gene can be regulated by varying the IPTG concentration. A separately controlled and moderate induced BRP synthesis can be used to bring about the release of large amounts of cloacin DF13 under conditions that allow a strong induction of the bacteriocin and which do not result in lysis of cells. Preliminary results indicated that the BRP can stimulate the release of immunity protein in the absence of cloacin or cloacin fragments.

Presence and expression of aerobactin genes in virulent avian strains of Escherichia coli.

Virulent and nonvirulent isolates of avian Escherichia coli were tested for the presence of aerobactin genes by colony hybridization with a specific gene probe constructed from plasmid pABN1 (A. Bindereif and J. B. Neilands, J. Bacteriol. 153:1111-1113, 1983). Positive hybridization with the gene probe was highly correlated with virulence, as measured by the 50% lethal dose of the strains for chicks. Evidence for the expression of aerobactin genes in the virulent strains was obtained by demonstrating their susceptibility to cloacin DF13, which binds to the same receptor that binds aerobactin, and their ability to produce aerobactin, as revealed by cross-feeding the E. coli mutant WO987 (aroB fepA iuc iut+), which is unable to synthesize but capable of taking up aerobactin. We suggest that the production of aerobactin is involved in the virulence of avian septicemic E. coli.

Escherichia coli ribosomal protein S1 does not protect the 49-nucleotide 3' terminal cloacin fragment of 16 S rRNA from nuclease S1.

Footprinting of ribosomal protein S1 on the 49-nucleotide 3' terminal cloacin DF13 fragment of 16 S rRNA at physiological ionic strength, pH and temperature yielded no detectable protection of any nucleotides from subsequent attack by the single strand specific nuclease S1, even at large excesses of ribosomal protein S1.

Methylation-dependent transcription controls plasmid replication of the CloDF13 cop-1(Ts) mutant.

The CloDF13 cop-1(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42 degrees C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-1(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42 degrees C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA II synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.