Identification of the plasmid-encoded immunity protein for colicin El in the inner membrane of Escherichia coli

A set of plasmids containing portions of the Col El plasmid were transformed into recA⁻ cells. These cells, after UV irradiation, only incorporate labelled amino acids into plasmid-encoded proteins. UV-irradiated cells label a 14.5 kDa band if they are phenotypically immune to colicin E1, and do not contain this band if they are sensitive to colicin E1. We conclude that the 14.5 kDa protein is the colicin E1 immunity protein. When the inner and outer membranes of these cells are fractionated, the labelled band appears in the inner membrane. The immunity protein must be an intrinsic inner membrane protein, confirming the predictions made by hydrophobicity calculations from primary sequence data.MaxicellCol El plasmidImmunity proteinHydrophobicity calculation     

Plasmid-determined immunity of Escherichia coli K-12 to colicin Ia Is mediated by a plasmid-encoded membrane protein.

The colicin Ia structural (cia) and immunity (iia) genes of plasmid pColIa-CA53 have been cloned into the cloning vector pBR322. These two genes are closely linked, and both of them can be isolated on a deoxyribonucleic acid fragment approximately 4,800 base pairs long. An analysis of the polypeptides synthesized in ultraviolet-irradiated cells containing these cloned genes led to the conclusion that the iia gene product is a polypeptide with a molecular weight of approximately 14,500. Insertion of transposon Tn5 into the iia gene led to a concomitant loss of the immune phenotype and the ability to produce this protein. Fractionation of ultraviolet-irradiated cells harboring a plasmid carrying the iia gene showed that the immunity protein is a component of the inner (cytoplasmic) membrane. Furthermore, the mechanism of immunity to colicin Ia appears to operate at the level of the cytoplasmic membrane. This conclusion is based on our finding that membrane vesicles prepared from colicin Ia-immune cells could be depolarized by colicins E1 and Ib but not by colicin Ia.     

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli

The product of the malE—lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Echerichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827–2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.     

Structure and dynamics of the colicin E1 channel

The toxin-like and bactericidal colicin E1 molecule is of interest for problems of toxin action, polypeptide translocation across membranes, voltage-gated channels, and receptor function. Colicin E1 binds to a receptor in the outer membrane and is translocated across the cell envelope to the inner membrane. Import of the colicin channel-forming domain into the inner membrane involves a translocation-competent intermediate state and a membrane potential-dependent movement of one third to one half of the channel peptide into the membrane bilayer. The voltage-gated channel has a conductance sufficiently large to depolarize the Escherichia coli cytoplasmic membrane. Amino acid residues that affect the channel ion selectivity have been identified by site-directed mutagenesis. The colicin E1 channel is one of a few membrane proteins whose secondary structures in the membrane, predominantly alpha-helix, have been determined by physico-chemical techniques. Hypothesis for the identity of the trans-membrane helices, and the mechanism of binding to the membrane, are influenced by the solved crystal structure of the soluble colicin A channel peptide. The protective action of immunity protein is a unique aspect of the colicin problem, and information has been obtained, by genetic techniques, about the probable membrane topography of the imm gene product.     

Delineation of the translocation of colicin E7 across the inner membrane of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The lysis protein of the colicinogenic operon is essential for colicin release and its main function is to activate the outer membrane phospholipase A (OMPLA) for the traverse of colicin across the cell envelope. However, little is known about the involvement of the lysis protein in the translocation of colicin across the inner membrane into the periplasm. The introduction of specific point mutations into the lipobox or sorting signal sequence of the lysE7 gene resulted in the production of various forms of lysis proteins. Our experimental results indicated that cells with wild-type mature LysE7 protein exhibited higher efficiency of colicin E7 translocation across the inner membrane into the periplasm than those with premature LysE7 protein. Moreover, the degree of permeability of the inner membrane induced by the mature LysE7 protein was significantly increased as compared to the unmodified LysE7 precursor. These results suggest that the efficiency of colicin movement into the periplasm is correlated with the increase in inner membrane permeability induced by the LysE7 protein. Thus, we propose that mature LysE7 protein has two critical roles: firstly mediating the translocation of colicin E7 across the inner membrane into the periplasm, and secondly activating the OMPLA to allow colicin release.     

The pore-forming domain of colicin A fused to a signal peptide: a tool for studying pore-formation and inhibition

Pore-forming colicins are plasmid-encoded bacteriocins that kill Escherichia coli and closely related bacteria. They bind to receptors in the outer membrane and are translocated across the cell envelope to the inner membrane where they form voltage-dependent ion-channels. Colicins are composed of three domains, with the C-terminal domain responsible for pore-formation. Isolated C-terminal pore-forming domains produced in the cytoplasm of E. coli are inactive due to the polarity of the transmembrane electrochemical potential, which is the opposite of that required. However, the pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membrane of E. coli from the periplasmic side, forming a functional channel. Sp-pfColA is specifically inhibited by the colicin A immunity protein (Cai). This construct has been used to investigate colicin A channel formation in vivo and to characterise the interaction of pfColA with Cai within the inner membrane. These points will be developed further in this review.     

Mechanism of export of colicin E1 and colicin E3.

The mechanism of export of colicins E1 and E3 was examined. Neither colicin E1, colicin E3, Nor colicin E3 immunity protein appears to be synthesized as a precursor protein with an amino-terminal extension. Instead, the colicins, as well as the colicin E3 immunity protein, appear to leave the cells where they are made, long after their synthesis, by a nonspecific mechanism which results in increased permeability of the producing cells. Induction of ColE3-containing cells with mitomycin C leads to actual lysis of those cells, as some time after synthesis of the colicin E3 and its immunity protein has been completed. Induction of ColE1-containing cells results in increased permeability of the cells, but not in actual lysis, and most of the colicin E1 produced never leaves the producing cells. Intracellular proteins such as elongation factor G can be found outside of colicinogenic cells after mitomycin C induction, along with the colicin. Until substantial increases in permeability occur, most of the colicin remains cell associated, in the soluble cytosol, rather than in a membrane-associated form.     

Channel domain of colicin A modifies the dimeric organization of its immunity protein

Proteins conferring immunity against pore-forming colicins are localized in the Escherichia coli inner membrane. Their protective effects are mediated by direct interaction with the C-terminal domain of their cognate colicins. Cai, the immunity protein protecting E. coli against colicin A, contains four cysteine residues. We report cysteine cross-linking experiments showing that Cai forms homodimers. Cai contains four transmembrane segments (TMSs), and dimerization occurs via the third TMS. Furthermore, we observe the formation of intramolecular disulfide bonds that connect TMS2 with either TMS1 or TMS3. Co-expression of Cai with its target, the colicin A pore-forming domain (pfColA), in the inner membrane prevents the formation of intermolecular and intramolecular disulfide bonds, indicating that pfColA interacts with the dimer of Cai and modifies its conformation. Finally, we show that when Cai is locked by disulfide bonds, it is no longer able to protect cells against exogenous added colicin A.     

Importation of nuclease colicins into E coli cells: endoproteolytic cleavage and its prevention by the immunity protein

A major group of colicins comprises molecules that possess nuclease activity and kill sensitive cells by cleaving RNA or DNA. Recent data open the possibility that the tRNase colicin D, the rRNase colicin E3 and the DNase colicin E7 undergo proteolytic processing, such that only the C-terminal domain of the molecule, carrying the nuclease activity, enters the cytoplasm. The proteases responsible for the proteolytic processing remain unidentified. In the case of colicin D, the characterization of a colicin D-resistant mutant shows that the inner membrane protease LepB is involved in colicin D toxicity, but is not solely responsible for the cleavage of colicin D. The lepB mutant resistant to colicin D remains sensitive to other colicins tested (B, E1, E3 and E2), and the mutant protease retains activity towards its normal substrates. The cleavage of colicin D observed in vitro releases a C-terminal fragment retaining tRNase activity, and occurs in a region of the amino acid sequence that is conserved in other nuclease colicins, suggesting that they may also require a processing step for their cytotoxicity. The immunity proteins of both colicins D and E3 appear to have a dual role, protecting the colicin molecule against proteolytic cleavage and inhibiting the nuclease activity of the colicin. The possibility that processing is an essential step common to cell killing by all nuclease colicins, and that the immunity protein must be removed from the colicin prior to processing, is discussed.     

FtsH-dependent processing of RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm

It has long been suggested that the import of nuclease colicins requires protein processing; however it had never been formally demonstrated. Here we show that two RNase colicins, E3 and D, which appropriate two different translocation machineries to cross the outer membrane (BtuB/Tol and FepA/TonB, respectively), undergo a processing step inside the cell that is essential to their killing action. We have detected the presence of the C-terminal catalytic domains of these colicins in the cytoplasm of target bacteria. The same processed forms were identified in both colicin-sensitive cells and in cells immune to colicin because of the expression of the cognate immunity protein. We demonstrate that the inner membrane protease FtsH is necessary for the processing of colicins D and E3 during their import. We also show that the signal peptidase LepB interacts directly with the central domain of colicin D in vitro and that it is a specific but not a catalytic requirement for in vivo processing of colicin D. The interaction of colicin D with LepB may ensure a stable association with the inner membrane that in turn allows the colicin recognition by FtsH. We have also shown that the outer membrane protease OmpT is responsible for alternative and distinct endoproteolytic cleavages of colicins D and E3 in vitro, presumably reflecting its known role in the bacterial defense against antimicrobial peptides. Even though the OmpT-catalyzed in vitro cleavage also liberates the catalytic domain from colicins D and E3, it is not involved in the processing of nuclease colicins during their import into the cytoplasm.     

The Tip of the Hydrophobic Hairpin of Colicin U Is Dispensable for Colicin U Activity but Is Important for Interaction with the Immunity Protein

The hydrophobic C terminus of pore-forming colicins associates with and inserts into the cytoplasmic membrane and is the target of the respective immunity protein. The hydrophobic region of colicin U of Shigella boydii was mutated to identify determinants responsible for recognition of colicin U by the colicin U immunity protein. Deletion of the tip of the hydrophobic hairpin of colicin U resulted in a fully active colicin that was no longer inactivated by the colicin U immunity protein. Replacement of eight amino acids at the tip of the colicin U hairpin by the corresponding amino acids of the related colicin B resulted in colicin U(575–582ColB), which was inactivated by the colicin U immunity protein to 10% of the level of inactivation of the wild-type colicin U. The colicin B immunity protein inactivated colicin U(575–582ColB) to the same degree. These results indicate that the tip of the hydrophobic hairpin of colicin U and of colicin B mainly determines the interaction with the corresponding immunity proteins and is not required for colicin activity. Comparison of these results with published data suggests that interhelical loops and not membrane helices of pore-forming colicins mainly interact with the cognate immunity proteins and that the loops are located in different regions of the A-type and E1-type colicins. The colicin U immunity protein forms four transmembrane segments in the cytoplasmic membrane, and the N and C termini face the cytoplasm.     

Release of immunity protein requires functional endonuclease colicin import machinery

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. However, it is not known how and at what stage during infection the immunity protein release occurs. Here, we constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein binds the free colicin E2 with a 1:1 stoichiometry and specifically inhibits its DNase activity. The addition of this hybrid complex to susceptible cells reveals that the release of the hybrid immunity protein is a time-dependent process. This process is achieved 20 min after the addition of the complex to the cells. We showed that complex dissociation requires a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicate that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain.     

Immunity protein is not required for the bactericidal activity of colicin E3

Plasmid pLAX3, carrying the colicin E3 gene, was used to direct the in vitro synthesis of a colicin E3* molecule totally devoid of its immunity protein. We established that this molecule is able to kill sensitive Escherichia coli cells in the total absence of immunity protein. Therefore, all of the information required for colicin E3 action is located on the colicin polypeptide itself. Furthermore, our studies indicated that immunity protein protects the C-terminal enzymatic part of native colicin E3 protein against proteolytic degradation before or during its translocation across the cell envelope. These results are discussed in relation to the mode of entry of colicin E3 into bacterial cells.     

Membrane topography of ColE1 gene products: the immunity protein.

The topography of the colicin E1 immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin E1 Imm protein folds in the membrane as three trans-membrane alpha-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to Ser-28, Ile-43 to Phe-62, and Leu-84 to Leu-104. An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin E1 Imm protein for interaction with the translocation apparatus and the colicin E1 ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight-residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side.     

Structure of colicin I receptor bound to the R-domain of colicin Ia: implications for protein import

Colicin Ia is a 69 kDa protein that kills susceptible Escherichia coli cells by binding to a specific receptor in the outer membrane, colicin I receptor (70 kDa), and subsequently translocating its channel forming domain across the periplasmic space, where it inserts into the inner membrane and forms a voltage-dependent ion channel. We determined crystal structures of colicin I receptor alone and in complex with the receptor binding domain of colicin Ia. The receptor undergoes large and unusual conformational changes upon colicin binding, opening at the cell surface and positioning the receptor binding domain of colicin Ia directly above it. We modelled the interaction with full-length colicin Ia to show that the channel forming domain is initially positioned 150 A above the cell surface. Functional data using full-length colicin Ia show that colicin I receptor is necessary for cell surface binding, and suggest that the receptor participates in translocation of colicin Ia across the outer membrane.     

Phospholipase-A-independent damage caused by the colicin A lysis protein during its assembly into the inner and outer membranes of Escherichia coli

The requirement for the activation of phospholipase A by the colicin A lysis protein (Cal) in the efficient release of colicin A by Escherichia coli cells containing colicin A plasmids was studied. In particular, we wished to determine if this activation is the primary effect of Cal or whether it reflects more generalized damage to the envelope caused by the presence of large quantities of this small acylated protein. E. coli tolQ cells, which were shown to be leaky for periplasmic proteins, were transduced to pldA and then transformed with the recombinant colicin A plasmid pKA. Both the pldA and pldA+ strains released large quantities of colicin A following induction, indicating that in these cells phospholipase A activation is not required for colicin release. This release was, however, still dependent on a functioning Cal protein. The assembly and processing of Cal in situ in the cell envelope was studied by combining pulse-chase labelling with isopycnic sucrose density gradient centrifugation of the cell membranes. Precursor Cal and lipid-modified precursor Cal were found in the inner membrane at early times of chase, and gave rise to mature Cal which accumulated in both the inner and outer membrane after further chase. The signal peptide was also visible on these gradients, and its distribution too was restricted to the inner membrane. Gradient centrifugation of envelopes of cells which were overproducing Cal resulted in very poor separation of the membranes. The results of these studies provide evidence that the colicin A lysis protein causes phospholipase A-independent alterations in the integrity of the E. coli envelope.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A pH-induced increase in hydrophobicity as a possible step in the penetration of colicin E3 through bacterial membranes

Exposure to low pH triggers an increase in the hydrophobicity of the colicin E3 molecule. Using a [3H] Triton X-100 binding assay we have shown that the amount of detergent (at supramicellar concentrations) associated with colicin E3 increased dramatically at pH 3.8 and below. Interaction of colicin E3 with asolectin vesicles was monitored by following its cross-linking with two different photoactivatable radioactive phospholipid analogues. At neutral pH colicin E3 was cross-linked with the phospholipid probing the membrane surface whereas at pH 4.5 and below, the bacteriocin reacted with the phospholipid probing the hydrophobic core of the bilayer. With the use of phase partitioning of proteins in Triton X-114 it was shown that at acidic pH whole colicin E3 and its immunity protein segregated in the detergent phase. After trypsin digestion of the colicin-immunity complex, the N-terminal portion of E3 (T1) and the immunity partitioned in the detergent phase at low pH. In contrast, the enzymic domain of the colicin (T2) remained in the aqueous phase and was recovered in a highly active form as a consequence of its dissociation from the immunity protein. These results are discussed in relation to the mechanism of entry of colicin E3 into bacterial cells.     

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli

Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal-like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827-2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.     

Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin E1.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.