Processing of DNase domain during translocation of colicin E7 across the membrane of Escherichia coli

Translocation of colicin across the membrane of sensitive cells has been studied extensively. However, processing of the toxicity domain of colicin during translocation has been the subject of much controversy. To investigate the final translocation product of colicin across the membrane of Escherichia coli, an endogenously expressed His-tagged Im7 protein was constructed to detect any translocation product containing the DNase domain traversed the inner membrane into cytoplasm of the E. coli cells. As a result, a final processed DNase domain of ColE7 was identified in the intracellular space of the cells treated with Col-Im complex. In the presence of periplasmic extracts, in vitro processing of DNase domain of ColE7 was also observed. These results suggest that the processing of ColE7 has occurred for translocation of the DNase-type colicin across the membrane and the process is probably taking place in the periplasmic space of the membrane.     

Detection of induced synthesis of colicin E9 using ColE9p::gfpmut2 based reporter system

The majority of colicin operons are regulated by an SOS response inducible promoter (SOS promoter), located at upstream of the colicin operons. Therefore, colicin synthesis is induced by DNA damaging agents like mitomycin C (MMC) because the resulting DNA damage switches on the SOS response in bacteria. In this study, we have described the strategy for fusion of the SOS promoter of the colicin E9 operon (ColE9p) with a promoterless green fluorescent reporter gene (gfpmut2). We observed that the ColE9p–gfpmut2 is inducible by MMC which confirmed that the ColE9p–gfpmut2 is sensitive to SOS response inducing agents. The data implies that the ColE9p–gfpmut2 based reporter system is suitable for monitoring the ColE9 synthesis induced by SOS response inducing agents including antibiotics. Using green fluorescent protein expression from the ColE9p–gfpmut2 as an indicator of ColE9 synthesis; we have investigated, first time, the inducing effects of cephalexin antibiotic on ColE9 synthesis. Our data demonstrated that the cephalexin has potential to induce ColE9 synthesis from E. coli JM83 host cells albeit the level of this induction is very low hence its detection required a highly sensitive method.     

Investigating early events in receptor binding and translocation of colicin E9 using synchronized cell killing and proteolytic cleavage

Enzymatic colicins such as colicin E9 (ColE9) bind to BtuB on the cell surface of Escherichia coli and rapidly recruit a second coreceptor, either OmpF or OmpC, through which the N-terminal natively disordered region (NDR) of their translocation domain gains entry into the cell periplasm and interacts with TolB. Previously, we constructed an inactive disulfide-locked mutant ColE9 (ColE9(s-s)) that binds to BtuB and can be reduced with dithiothreitol (DTT) to synchronize cell killing. By introducing unique enterokinase (EK) cleavage sites in ColE9(s-s), we showed that the first 61 residues of the NDR were inaccessible to cleavage when bound to BtuB, whereas an EK cleavage site inserted at residue 82 of the NDR remained accessible. This suggests that most of the NDR is occluded by OmpF shortly after binding to BtuB, whereas the extreme distal region of the NDR is surface exposed before unfolding of the receptor-binding domain occurs. EK cleavage of unique cleavage sites located in the ordered region of the translocation domain or in the distal region of the receptor-binding domain confirmed that these regions of ColE9 remained accessible at the E. coli cell surface. Lack of EK cleavage of the DNase domain of the cell-bound, oxidized ColE9/Im9 complex, and the rapid detection of Alexa Fluor 594-labeled Im9 (Im9(AF)) in the cell supernatant following treatment of cells with DTT, suggested that immunity release occurred immediately after unfolding of the colicin and was not driven by binding to BtuB.     

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.     

Immunity protein protects colicin E2 from OmpT protease

The endonuclease colicin E2 (ColE2), a bacteriocidal protein, and the associated cognate immunity protein (Im2) are released from producing Escherichia coli cells. ColE2 interaction with the target cell outer membrane BtuB protein and Tol import machinery allows the dissociation of Im2 from its colicin at the outer membrane surface. Here, we use in vivo approaches to show that a small amount of ColE2-Im2 protein complex bound to sensitive cells is susceptible to proteolytic cleavage by the outer membrane protease, OmpT. The presence of BtuB is required for ColE-Im2 cleavage by OmpT. The amount of colicin cleaved by OmpT is greatly enhanced when ColE2 is dissociated from Im2. We further demonstrate that OmpT cleaves the C-terminal DNase domain of the toxin. As expected, strains that over-produce OmpT are less susceptible to infection by ColE2 than by ColE2-Im2. Our findings reveal an additional function for the immunity protein beside protection of producing cells against their own colicin in the cytoplasm. Im2 protects ColE2 against OmpT-mediated proteolytic attack.     

Flexibility in the receptor-binding domain of the enzymatic colicin E9 is required for toxicity against Escherichia coli cells

The events that occur after the binding of the enzymatic E colicins to Escherichia coli BtuB receptors that lead to translocation of the cytotoxic domain into the periplasmic space and, ultimately, cell killing are poorly understood. It has been suggested that unfolding of the coiled-coil BtuB receptor binding domain of the E colicins may be an essential step that leads to the loss of immunity protein from the colicin and immunity protein complex and then triggers the events of translocation. We introduced pairs of cysteine mutations into the receptor binding domain of colicin E9 (ColE9) that resulted in the formation of a disulfide bond located near the middle or the top of the R domain. After dithiothreitol reduction, the ColE9 protein with the mutations L359C and F412C (ColE9 L359C-F412C) and the ColE9 protein with the mutations Y324C and L447C (ColE9 Y324C-L447C) were slightly less active than equivalent concentrations of ColE9. On oxidation with diamide, no significant biological activity was seen with the ColE9 L359C-F412C and the ColE9 Y324C-L447C mutant proteins; however diamide had no effect on the activity of ColE9. The presence of a disulfide bond was confirmed in both of the oxidized, mutant proteins by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The loss of biological activity of the disulfide-containing mutant proteins was not due to an indirect effect on the properties of the translocation or DNase domains of the mutant colicins. The data are consistent with a requirement for the flexibility of the coiled-coil R domain after binding to BtuB.     

The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein

Background: Colicin E7 (ColE7) is one of the bacterial toxins classified as a DNase-type E-group colicin. The cytotoxic activity of a colicin in a colicin-producing cell can be counteracted by binding of the colicin to a highly specific immunity protein. This biological event is a good model system for the investigation of protein recognition.Results: The crystal structure of a one-to-one complex between the DNase domain of colicin E7 and its cognate immunity protein Im7 has been determined at 2.3 Å resolution. Im7 in the complex is a varied four-helix bundle that is identical to the structure previously determined for uncomplexed Im7. The structure of the DNase domain of ColE7 displays a novel α/β fold and contains a Zn²⁺ ion bound to three histidine residues and one water molecule in a distorted tetrahedron geometry. Im7 has a V-shaped structure, extending two arms to clamp the DNase domain of ColE7. One arm (α1¹–loop12–α2¹; where ¹ represents helices in Im7) is located in the region that displays the greatest sequence variation among members of the immunity proteins in the same subfamily. This arm mainly uses acidic sidechains to interact with the basic sidechains in the DNase domain of ColE7. The other arm (loop 23–α3¹–loop 34) is more conserved and it interacts not only with the sidechain but also with the mainchain atoms of the DNase domain of ColE7.Conclusions: The protein interfaces between the DNase domain of ColE7 and Im7 are charge-complementary and charge interactions contribute significantly to the tight and specific binding between the two proteins. The more variable arm in Im7 dominates the binding specificity of the immunity protein to its cognate colicin. Biological and structural data suggest that the DNase active site for ColE7 is probably near the metal-binding site.     

Nucleotide sequences from the colicin E8 operon: homology with plasmid ColE2-P9

The primary structures of the immunity (Imm) and lysis (Lys) proteins, and the C-terminal 205 amino acid residues of colicin E8 were deduced from nucleotide sequencing of the 1,265 bp ClaI-PvuI DNA fragment of plasmid ColE8-J. The gene order is col-imm-lys confirming previous genetic data. A comparison of the colicin E8 peptide sequence with the available colicin E2-P9 sequence shows an identical receptor-binding domain but 20 amino acid replacements and a clustering of synonymous codon usage in the nuclease-active region. Sequence homology of the two colicins indicates that they are descended from a common ancestral gene and that colicin E8, like colicin E2, may also function as a DNA endonuclease. The native ColE8 imm (resident copy) is 258 bp long and is predicted to encode an acidic protein of 9,604 mol. wt. The six amino acid replacements between the resident imm and the previously reported non-resident copy of the ColE8 imm ([E8 imm]) found in the ribonuclease-producing ColE3-CA38 plasmid offer an explanation for the incomplete protection conferred by [E8 Imm] to exogenously added colicin E8. Except for one nucleotide and amino acid change in the putative signal peptide sequence, the ColE8 lys structure is identical to that present in ColE2-P9 and ColE3-CA38.     

Colicinogenic mutant of ColE1 plasmid that fails to confer immunity to colicin E1.

Insertion of the ampicillin transposon (Tn3) into ColE1 DNAs causes various mutations in the plasmids. Escherichia coli K-12 cells carrying one of these mutants showed novel properties; they were sensitive to colicin E1 and were able to produce active colicin E1. The site and the orientation of Tn3 insertion in this mutant ColE1 DNA were determined by heteroduplex analysis and by enzymatic digestion with restriction endonucleases. The potential usefulness of this mutant ColE1 DNA as a cloning vehicle is discussed.     

Colicin E8, a DNase which indicates an evolutionary relationship between colicins E2 and E3.

Colicin E8-J and its immunity protein were characterized with regard to their activities and gene structures. Colicin E8 is a complex of proteins A and B; protein A (the naked E8) exhibits an apparently nonspecific DNase activity that is inhibited by protein B (the immunity protein), as in the case of colicin E2. The nucleotide sequence of the downstream half of the colicin operon of ColE8-J was determined to be highly homologous to that of ColE2-P9, with the exception of the hot spot region of the 3'-terminal segment of the colicin gene and the adjacent immunity gene. The immE2-like gene of ColE3-CA38 was, as assumed previously, extensively homologous to the immE8 gene of ColE8-J, and thus, ColE8-J was shown to be situated between ColE2-P9 and ColE3-CA38 in the evolution of the E-group Col plasmids.     

Involvement of colicin in the limited protection of the colicin producing cells against bacteriophage

The restriction/modification system is considered to be the most common machinery of microorganisms for protection against bacteriophage infection. However, we found that mitomycin C induced Escherichia coli containing ColE7-K317 can confer limited protection against bacteriophage M13K07 and lambda infection. Our study showed that degree of protection is correlated with the expression level of the ColE7 operon, indicating that colicin E7 alone or the colicin E7-immunity protein complex is directly involved in this protection mechanism. It was also noted that the degree of protection is greater against the single-strand DNA bacteriophage M13K07 than the double-strand bacteriophage(lambda). Coincidently, the K(A) value of ColE7-Im either interacting with single-strand DNA (2.94x10(5)M(-1)) or double-strand DNA (1.75x10(5)M(-1)) reveals that the binding affinity of ColE7-Im with ssDNA is 1.68-fold stronger than that of the protein complex interacting with dsDNA. Interaction between colicin and the DNA may play a central role in this limited protection of the colicin-producing cell against bacteriophages. Based on these observations, we suggest that the colicin exporting pathway may interact to some extent with the bacteriophage infection pathway leading to a limited selective advantage for and limited protection of colicin-producing cells against different bacteriophages.     

Nucleotide polymorphism in colicin E2 gene clusters: evidence for nonneutral evolution

To explore the molecular mechanisms behind the diversification of colicin gene clusters, we examined DNA sequence polymorphism for the colicin gene clusters of 14 colicin E2 (ColE2) plasmids obtained from natural isolates of Escherichia coli. Two types of ColE2 plasmids are revealed, with type II gene clusters generated by recombination between type I ColE2 and ColE7 gene clusters. The levels and patterns of DNA polymorphism are different between the two types. Type I polymorphism is distributed evenly along the gene cluster, while type II accumulates polymorphism at an elevated rate in the 5' end of the colicin gene. These differences may be explained by recombinational origins of type II gene clusters. The pattern of divergence between the ColE2 gene cluster and its close relative ColE9 is not correlated with the pattern of polymorphism within ColE2, suggesting that this gene cluster is not evolving in a neutral fashion. A statistical test confirms significant departures from the predictions of neutrality. These data lend further support to the hypothesis that colicin gene clusters may evolve under the influence of nonneutral forces.      

Colicin E2 production and release by Escherichia coli K12 and other Enterobacteriaceae

Previous work has shown that Escherichia coli K12 ColE2+ cells undergo a form of partial lysis and exhibit increases in lysophosphatidylethanolamine (lysoPE) and free fatty acid content due to activation of phospholipase A when induced to produce and release colicin E2. The increase in lysoPE content was assumed to be essential for efficient colicin release. These same characteristics are also presented by some natural ColE2+ isolates, and by other representatives of the Enterobacteriaceae after transformation with derivatives of a ColE2 plasmid. However, Salmonella typhimurium strains carrying ColE2 plasmids released colicin without partial lysis and without increasing their lysoPE content. A previously undetected minor phospholipid, which appeared in these and other strains only when they were induced to produce colicin, may be an important factor in colicin release. In ColE2+ E. coli K12, production of this new lipid was dependent on phospholipase A activation following expression of the ColE2 lysis gene. Some other ColE2+ strains did not respond to induction of colicin production in the same way as ColE2+ E. coli K12. These strains were less sensitive to inducer (mitomycin C) or unable to produce increased amounts of colicin in response to induction, or unable to degrade colicin once it was released. In general, the results suggest that colicin release occurs by the same or similar processes in the various strains tested, and support the continued use of E. coli K12 as the model strain for studying the mechanisms of colicin release.     

Cloning and characterization of the ColE7 plasmid

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.     

The critical roles of polyamines in regulating ColE7 production and restricting ColE7 uptake of the colicin-producing Escherichia coli

The ColE7 operon is an SOS response regulon, which encodes bacteriocin ColE7 to kill susceptible Escherichia coli and its related enterobacteria under conditions of stress. We have observed for the first time that polyamines confer limited resistance against ColE7 on E. coli cells. Thus, this study aims to investigate the role of polyamines in modulating the protective effect of the E. coli cells against colicin. In the experiments, we surprisingly found that endogenous polyamines are also essential for ColE7 production, and the rate of polyamine synthesis is directly related to the SOS response. Our experimental results further indicated that exogenous polyamines suppress the expression of TolA, BtuB, OmpF, and OmpC proteins that are responsible for ColE7 uptake. Moreover, two-dimensional gel electrophoresis revealed that the production of two periplasmic proteins, PotD and OppA, is increased in E. coli cells under ColE7 exposure. Based on these observations, we propose that endogenous polyamines may play a dual role in the ColE7 system. Polyamines may participate in initiating the expression of the SOS response of the ColE7 operon and simultaneously down-regulate proteins that are essential for colicin uptake, thus conferring a survival advantage on colicin-producing E. coli under stress conditions in the natural environment.     

Obligatory coupling of colicin release and lysis in mitomycin-treated Col+ Escherichia coli

Previous work has shown that Escherichia coli K12 strains carrying the small, high copy number ColE2-P9 plasmid produce large amounts of colicin and then lyse and release colicin when grown in broth culture containing mitomycin C. Strains carrying the larger, low copy number ColIa-CA53 plasmid produced much less colicin and did not lyse or discharge more than 15% of their colicin when grown under the same conditions. Naturally-occurring Col+ strains and E. coli K12 derivatives carrying different Col plasmids could be classified either as ColE2+-like or ColIa+-like according to whether or not they produced large amounts of colicin and lysed and discharged colicin when grown in the presence of mitomycin, and also by the size and presumed copy number of the Col plasmid they carried. Strains carrying multiple copies of the cloned colicin Ia structural gene produced large amounts of colicin but did not lyse or release colicin when grown in the presence of mitomycin. This result rules out the possibility that high level accumulation of colicin is sufficient to cause lysis. Conditions were sought under which colicin Ia could be released from the producing cells. It was found that mitomycin-treated cultures of strains carrying both ColE2 and ColIa plasmids released both colicins when they lysed, although colicin Ia release occurred later than colicin E2 release. It was also noted that colicin Ia-laden cells released their colicin when diluted into fresh culture medium.     

Initial steps of colicin E1 import across the outer membrane of Escherichia coli

Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B(12) receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.     

Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.     

High-resolution crystal structure of a truncated ColE7 translocation domain: implications for colicin transport across membranes

ColE7 is a nuclease-type colicin released from Escherichia coli to kill sensitive bacterial cells by degrading the nucleic acid molecules in their cytoplasm. ColE7 is classified as one of the group A colicins, since the N-terminal translocation domain (T-domain) of the nuclease-type colicins interact with specific membrane-bound or periplasmic Tol proteins during protein import. Here, we show that if the N-terminal tail of ColE7 is deleted, ColE7 (residues 63-576) loses its bactericidal activity against E.coli. Moreover, TolB protein interacts directly with the T-domain of ColE7 (residues 1-316), but not with the N-terminal deleted T-domain (residues 60-316), as detected by co-immunoprecipitation experiments, confirming that the N-terminal tail is required for ColE7 interactions with TolB. The crystal structure of the N-terminal tail deleted ColE7 T-domain was determined by the multi-wavelength anomalous dispersion method at a resolution of 1.7 angstroms. The structure of the ColE7 T-domain superimposes well with the T-domain of ColE3 and TR-domain of ColB, a group A Tol-dependent colicin and a group B TonB-dependent colicin, respectively. The structural resemblance of group A and B colicins implies that the two groups of colicins may share a mechanistic connection during cellular import.     

The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins

Interaction of the TolB box of Group A colicins with the TolB protein in the periplasm of Escherichia coli cells promotes transport of the cytotoxic domain of the colicin across the cell envelope. The crystal structure of a complex between a 107‐residue peptide (TA_1–107) of the translocation domain of colicin A (ColA) and TolB identified the TolB box as a 12‐residue peptide that folded into a distorted hairpin within a central canyon of the β‐propeller domain of TolB. Comparison of this structure with that of the colicin E9 (ColE9) TolB box–TolB complex, together with site‐directed mutagenesis of the ColA TolB box residues, revealed important differences in the interaction of the two TolB boxes with an overlapping binding site on TolB. Substitution of the TolB box residues of ColA with those of ColE9 conferred the ability to competitively recruit TolB from Pal but reduced the biological activity of the mutant ColA. This datum explains (i) the difference in binding affinities of ColA and ColE9 with TolB, and (ii) the inability of ColA, unlike ColE9, to competitively recruit TolB from Pal, allowing an understanding of how these two colicins interact in a different way with a common translocation portal in E. coli cells.