Topology and function of the integral membrane protein conferring immunity to colicin A

The topology of the integral membrane protein Cai (colicin A immunity protein), which is required to protect producing cells from the pore-forming colicin A, was analysed using fusions to alkaline phosphatase. The properties of these fusion proteins support the model for Cai topology previously proposed on theoretical grounds. The protein was found to contain four transmembrane sequences and its N- and C-terminal regions were found to be directed towards the cytoplasm. Oligonucleotide-directed mutagenesis and sequence comparisons between Cai, Cbi (colicin B immunity protein), and Cni (colicin N immunity protein) were carried out to determine the functional regions of Cai. The possible roles of the various regions of Cai in its protective function and in its topological organization are discussed.     

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.     

A molecular genetic approach to the functioning of the immunity protein to colicin A

Summary A plasmid (pColAF1), derived from pColA, and lacking the region encoding Cai (colicin A immunity protein) and Cal (colicin A lysis protein) has been constructed. The strains carrying pColAF1 produce normal amounts of colicin A which remains in the cell cytoplasm and does not result in loss of viability. Similar results have also been obtained for transposon insertion mutants lacking Cai. Structure prediction analysis indicates that four peptide regions of Cai might span the cytoplasmic membrane. Since the NH₂-and COOH-terminal regions are charged, this analysis suggests a topology of the 178 residues polypeptide chain in which regions 38 to 70 and 124 to 143 might be exposed at the outer side of the cytoplasmic membrane. With mutants constructed using recombinant DNA techniques, we could demonstrate that the removal of a 30 residue COOH-terminal region, and mutations altering the surface exposed loop comprised of aminoacid residues 124–143 abolish the protecting function of Cai.     

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.     

Colicin A Immunity Protein Interacts with the Hydrophobic Helical Hairpin of the Colicin A Channel Domain in the Escherichia coli Inner Membrane

The colicin A pore-forming domain (pfColA) was fused to a bacterial signal peptide (sp-pfColA). This was inserted into the Escherichia coli inner membrane in functional form and could be coimmunoprecipitated with epitope-tagged immunity protein (EpCai). We constructed a series of fusion proteins in which various numbers of sp-pfColA alpha-helices were fused to alkaline phosphatase (AP). We showed that a fusion protein made up of the hydrophobic alpha-helices 8 and 9 of sp-pfColA fused to AP was specifically coimmunoprecipitated with EpCai produced in the same cells. This is the first biochemical evidence that Cai recognizes and interacts with the colicin A hydrophobic helical hairpin.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

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.     

An alpha-helical hydrophobic hairpin as a specific determinant in protein-protein interaction occurring in Escherichia coli colicin A and B immunity systems.

A collection of chimeric pore-forming domains between colicins A and B was constructed to investigate the specific determinants responsible for recognition by the corresponding immunity proteins. The fusion sites in the hybrid proteins were positioned according to the three-dimensional structure of the soluble form of the colicin A pore-forming domain. The hydrophobic hairpin of colicin pore-forming domains, buried in the core of the soluble structure, was the main determinant recognized by the integral immunity proteins. The immunity protein function may require helix-helix recognition within the lipid bilayer.     

Four plasmid genes are required for colicin V synthesis, export, and immunity.

The colicin V production and immunity genes were isolated from plasmid pColV-K30. A HindIII-to-SalI fragment of 9.4 kilobases was cloned into the compatible vectors pBR322 and pACYC184. Mutants defective in colicin production were generated by Tn5 insertions and by constructing deletions in vitro. Physical analysis of these mutations identified a 4.4-kilobase region of this DNA which contains all the plasmid genes (cva) needed for the production of colicin V. The colicin V immunity determinant (cvi) is in a 700-base-pair fragment located within one end of this region. Complementation tests identified three genes, called cvaA, cvaB, and cvaC, required for colicin production. Analysis of the proteins labeled in minicells harboring various Tn5 insertions allowed us to identify protein products for the cvaA and cvaC genes. Mutations in cvaA and cvaB eliminated colicin activity in culture supernatants, but not within the cells. Mutations in cvaC, however, eliminated all detectable activity. From these results we conclude that the cvaC gene codes for the structural gene for colicin V, while cvaA and cvaB are apparently needed for the normal export of the colicin.     

Hierarchical order of critical residues on the immunity-determining region of the Im7 protein which confer specific immunity to its cognate colicin.

The directed mutagenesis study of the Im7 protein of colicin E7 revealed that three residues, D31, D35, and E39, located in the loop 1 and helix 2 regions of the protein were critical for initiating the complex formation with its cognate colicin E7. Interestingly, the importance of these three critical residues in conferring specific immunity to its own colicin was exhibited in a hierarchical order, respectively. Moreover, we found that existence of the three critical residues was common among the DNase-type Im proteins. Most likely the three residues of the DNase-type immunity proteins are critical for initiating the unique protein-protein interactions with their cognate colicin. In addition, replacement of the helix 2 of Im7 by the corresponding region of Im8 produced a phenotype of the mutant protein very similar to that of Im8. This result suggests that the DNase-type Im proteins indeed share a "homologous-structural framework" and evolution of the Im proteins may be engendered by minor amino acid changes in this specific immunity-determining region without causing structural alteration of the proteins.     

The biology of colicin M

Abstract This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma , has been sequenced and the protein isolated and purified. With a deduced molecular size of 29 453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13 890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

The biology of colicin M

This communication summarizes our present knowledge of colicin M, an unusual member of the colicin group. The gene encoding colicin M, cma, has been sequenced and the protein isolated and purified. With a deduced molecular size of 29,453 Da, colicin M is the smallest of the known colicins. The polypeptide can be divided into functional domains for cell surface receptor binding, uptake into the cell, and killing activity. To kill, the colicin must enter from outside the cell. Colicin M blocks the biosynthesis of both peptidoglycan and O-antigen by inhibiting regeneration of the bactoprenyl-P carrier lipid. Autolysis occurs as a secondary effect following inhibition of peptidoglycan synthesis. Colicin M is the only colicin known to have such a mechanism of action. Immunity to this colicin is mediated by the cmi gene product, a protein of 13,890 Da. This cytoplasmic membrane protein confers immunity by binding to and thus neutralizing the colicin. Cmi shares properties with both immunity proteins of the pore-forming and the cytoplasmically active colicins. Genes for the colicin and immunity protein are found next to each other, but in opposite orientation, on pColM plasmids. The mechanism of colicin M release is not known.     

In vivo and in vitro characterization of overproduced colicin E9 immunity protein.

We report the overproduction of the immunity protein for the DNase colicin E9 and its characterization both in vivo and in vitro. The genes for colicin immunity proteins are normally co-expressed from Col plasmids with their corresponding colicins. In the context of the enzymatic colicins, the two proteins form a complex, thereby protecting the host bacterium from the antibiotic activity of the colicin. This complex is then released into the medium, whereupon the colicin alone translocates (through the appropriate receptor) into sensitive bacterial strains, resulting in bacterial cell death. The immunity protein for colicin E9 (Im9) has been overproduced in a bacterial host in the absence of its colicin, to enable sufficient material to be isolated for structural studies. As a prelude to such studies, the in-vivo and in-vitro properties of overproduced Im9 were analysed. Electrospray mass spectrometry verified the molecular mass of the purified protein and analytical ultracentrifugation indicated that the native protein approximates a symmetric monomer. Fluorescence-enhancement and gel-filtration experiments show that purified Im9 binds to colicin E9 in a 1:1 molar ratio and that this binding neutralizes the DNase activity of the colicin. These results lay the foundations for a full biophysical and structural characterization of the colicin E9 DNase inhibitor protein, Im9.     

Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis.

The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222. Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG). A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222. Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG. The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system.     

Plasmid ColE3 specifies a lysis protein.

Tn5 insertion mutations in plasmid ColE3 were isolated and characterized. Several of the mutants synthesized normal amounts of active colicin E3 but, unlike wild-type colicinogenic cells, did not release measurable amounts of colicin into the culture medium. Cells bearing the mutant plasmids were immune to exogenous colicin E3 at about the same level as wild-type colicinogenic cells. All of these lysis mutants mapped near, but outside of, the structural genes for colicin E3 and immunity protein. Cells carrying the insertion mutations which did not release colicin E3 into the medium were not killed by UV exposure at levels that killed cells bearing wild-type plasmids. The protein specified by the lysis gene was identified in minicells and in mitomycin C-induced cells. A small protein, with a molecular weight between 6,000 and 7,000, was found in cells which released colicin into the medium, but not in mutant cells that did not release colicin. Two mutants with insertions within the structural gene for colicin E3 were also characterized. They produced no colicin activity, but both synthesized a peptide consistent with their map position near the middle of the colicin gene. These two insertion mutants were also phenotypically lysis mutants--they were not killed by UV doses lethal to wild-type colicinogenic cells and they did not synthesize the small putative lysis protein. Therefore, the lysis gene is probably in the same operon as the structural gene for colicin E3.     

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.     

Colicin U, a novel colicin produced by Shigella boydii.

A novel colicin, designated colicin U, was found in two Shigella boydii strains of serovars 1 and 8. Colicin U was active against bacterial strains of the genera Escherichia and Shigella. Plasmid pColU (7.3 kb) of the colicinogenic strain S. boydii M592 (serovar 8) was sequenced, and three colicin genes were identified. The colicin U activity gene, cua, encodes a protein of 619 amino acids (Mr, 66,289); the immunity gene, cui, encodes a protein of 174 amino acids (Mr, 20,688); and the lytic protein gene, cul, encodes a polypeptide of 45 amino acids (Mr, 4,672). Colicin U displays sequence similarities to various colicins. The N-terminal sequence of 130 amino acids has 54% identity to the N-terminal sequence of bacteriocin 28b produced by Serratia marcescens. Furthermore, the N-terminal 36 amino acids have striking sequence identity (83%) to colicin A. Although the C-terminal pore-forming sequence of colicin U shows the highest degree of identity (73%) to the pore-forming C-terminal sequence of colicin B, the immunity protein, which interacts with the same region, displays a higher degree of sequence similarity to the immunity protein of colicin A (45%) than to the immunity protein of colicin B (30.5%). Immunity specificity is probably conferred by a short sequence from residues 571 to residue 599 of colicin U; this sequence is not similar to that of colicin B. We showed that binding of colicin U to sensitive cells is mediated by the OmpA protein, the OmpF porin, and core lipopolysaccharide. Uptake of colicin U was dependent on the TolA, -B, -Q, and -R proteins. pColU is homologous to plasmid pSB41 (4.1 kb) except for the colicin genes on pColU. pSB41 and pColU coexist in S. boydii strains and can be cotransformed into Escherichia coli, and both plasmids are homologous to pColE1.     

Positive selection for colicin diversity in bacteria

To examine the hypothesis that colicin proteins are subject to diversity-enhancing selection, we studied the rates of synonymous, nonsynonymous, and intergenic nucleotide substitution in three pairs of closely related colicin clusters. The results indicate that the immunity gene and the immunity-binding domain of the colicin gene, which interact to provide specific immunity from the lethal action of the colicin toxin, accumulate substitutions at synonymous and nonsynonymous sites several times more rapidly than does the remainder of the colicin cluster. We suggest that this increased level of divergence, centered at the immunity protein, may be the result of the combined action of recombination and positive selection acting to increase colicin diversity in natural populations of Escherichia coli.      

Identification of the plasmid-encoded immunity protein for colicin E1 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.

Maxicell Col El plasmid Immunity protein Hydrophobicity calculation