# Colicin-E6 Gene: Structure, Function, and Clinical Significance


## Key Takeaways

- Colicin-E6 is a plasmid-encoded bacteriocin produced by *E. coli*, functioning as a tRNA anticodon nuclease that specifically targets tRNA^Tyr^, leading to protein synthesis arrest and cell death. Its mechanism involves binding to the BtuB receptor, translocation via OmpF and TolB, and subsequent cleavage of tRNA^Tyr^ at the wobble position by its C-terminal RNase domain.
- The gene is located on the ColE6 plasmid and is regulated by the SOS response, with expression induced by DNA damage signals that activate the LexA repressor. The operon also includes immunity (cdi) and lysis (cdl) genes, essential for self-protection and extracellular release.
- Structural analysis reveals a three-domain architecture: an N-terminal translocation (T) domain, a central receptor-binding (R) domain recognizing BtuB, and a C-terminal cytotoxic (C) domain with RNase activity. This modular design facilitates its entry into target cells.
- Clinical significance lies in its potential as a targeted antimicrobial agent against pathogenic *E. coli* strains, sparing commensal flora. Engineered variants are being explored for cancer therapy by fusing the RNase domain to tumor-targeting moieties.
- Naturally occurring mutations can alter Colicin-E6's activity, specificity, and immunity, impacting antimicrobial resistance and gut microbiome dynamics. Detection in clinical microbiology typically involves phenotypic assays (growth inhibition zones) or genotypic methods (PCR for the *cda* gene).

---

## Executive Summary & Key Metadata

Colicin-E6 is a plasmid-encoded bacteriocin produced by *Escherichia coli* strains harboring the ColE6 plasmid. Unlike classical pore-forming colicins, Colicin-E6 is a **tRNA anticodon nuclease** that cleaves a specific transfer RNA (tRNA) within the host cell, leading to protein synthesis arrest and cell death. Its unique substrate specificity—targeting the anticodon loop of tRNA^Tyr^—distinguishes it from the closely related colicins E2, E3, E4, and E5, which target DNA, rRNA, and other tRNAs respectively. The gene product is a three-domain protein comprising an N-terminal translocation (T) domain, a central receptor-binding (R) domain, and a C-terminal cytotoxic (C) domain with intrinsic RNase activity. This manual provides a comprehensive molecular, structural, and clinical reference for Colicin-E6, with emphasis on its biotechnological applications, antimicrobial potential, and emerging role as a scaffold for engineered therapeutics.

| **Attribute** | **Value** |
|---|---|
| **HGNC Symbol** | Colicin-E6 (also *cda* or *colE6* on plasmid) |
| **UniProt Accession** | P17999 |
| **Representative PDB ID** | 1JCH (C-terminal domain); 2Y8V (full-length complex) |
| **Chromosomal Locus** | Not chromosomal; plasmid-borne (ColE6 plasmid, ~6.6 kb) |
| **Primary Molecular Function** | tRNA anticodon nuclease (EC 3.1.27.-); cleaves tRNA^Tyr^ at the wobble position |
| **Disease & Pathology Associations** | None directly; used as antimicrobial agent; implicated in gut microbiome dynamics; engineered variants under investigation for cancer therapy |

---

## 1. Genomic Locus, Chromosomal Organization & Isoforms

### 1.1 Plasmid Context and Genetic Organization

Colicin-E6 is not encoded on the *E. coli* chromosome but on a small, non-conjugative plasmid designated ColE6. The plasmid is approximately 6.6 kilobases (kb) in size and belongs to the ColE1 plasmid family, characterized by a relaxosome-independent, rolling-circle replication mechanism. The genetic organization of the ColE6 plasmid is highly conserved among colicinogenic plasmids, with a core operon structure comprising three genes: **cda** (colicin activity), **cdi** (colicin immunity), and **cdl** (colicin lysis). These genes are arranged in a single transcriptional unit under the control of a promoter that is induced by the SOS response—specifically, by DNA damage signals that activate the LexA repressor [1].

The **cda gene** (colicin D activity) encodes the 522-amino-acid Colicin-E6 precursor protein. The **cdi gene** encodes a small immunity protein (approximately 13 kDa) that binds specifically to the C-terminal domain of Colicin-E6, neutralizing its RNase activity. The **cdl gene** encodes a lysis protein (approximately 7 kDa) that is responsible for the release of colicin into the extracellular milieu by disrupting the bacterial cell envelope [2].

### 1.2 Promoter Architecture and Transcriptional Regulation

The colicin E6 promoter (P_colE6) is a canonical SOS-responsive promoter containing a LexA-binding site (SOS box) located between the −10 and −35 hexamers. Under normal growth conditions, LexA represses transcription. Upon DNA damage (e.g., UV irradiation, mitomycin C treatment), RecA-mediated autocleavage of LexA relieves repression, allowing RNA polymerase to initiate transcription. The promoter exhibits a moderate strength compared to other colicin promoters, with a transcription initiation rate approximately 30% of that observed for colicin E1 [3].

Upstream of the core promoter, a UP element (AT-rich sequence) enhances RNA polymerase binding. Additionally, a binding site for the global regulator **Fis** (factor for inversion stimulation) has been identified at position −80 to −60, which modulates promoter activity in response to growth phase. During exponential growth, Fis binding represses transcription; upon entry into stationary phase, Fis levels decline, leading to derepression and increased colicin production [4].

### 1.3 Transcription Termination and mRNA Stability

The colicin E6 operon terminates at a rho-independent terminator located downstream of the *cdl* gene, forming a stable stem-loop structure followed by a poly(U) tract. The mRNA has a half-life of approximately 4.5 minutes, which is relatively short compared to housekeeping genes, reflecting the need for rapid turnover after the SOS response subsides [5].

### 1.4 Isoforms and Post-Translational Processing

Colicin-E6 is synthesized as a precursor protein with an N-terminal signal peptide of 22 amino acids. This signal peptide directs the protein to the Sec translocon for export to the periplasm. Upon translocation, the signal peptide is cleaved by signal peptidase I, yielding the mature 500-amino-acid protein. No alternative splicing isoforms exist, as the gene lacks introns. However, post-translational modifications include:

- **Disulfide bond formation**: Two cysteine residues (Cys-441 and Cys-467) in the C-terminal domain form a disulfide bond that is essential for structural stability and RNase activity [6].
- **Proteolytic processing**: The T-domain contains a trypsin-sensitive site at Arg-115, which is cleaved during translocation into the target cell, separating the T-domain from the rest of the protein [7].

---

## 2. 3D Protein Domain Architecture & Structural Biology

### 2.1 Overall Architecture

The mature Colicin-E6 protein (500 amino acids, ~55 kDa) adopts a **three-domain architecture** that is characteristic of group A colicins. The domains are connected by flexible linkers and are arranged linearly from the N-terminus to the C-terminus:

1. **N-terminal Translocation (T) domain** (residues 1–135)
2. **Central Receptor-Binding (R) domain** (residues 136–350)
3. **C-terminal Cytotoxic (C) domain** (residues 351–500)

This modular architecture enables the protein to function as a "molecular syringe" that binds to a specific outer membrane receptor, translocates across the cell envelope, and delivers the cytotoxic domain to the cytoplasm [8].

### 2.2 Translocation Domain (Residues 1–135)

The T-domain is intrinsically disordered in solution but adopts a defined structure upon interaction with the translocon machinery (OmpF and TolB). It contains several conserved motifs:

- **TolB-binding motif** (residues 35–55): A β-hairpin that binds to the β-propeller domain of TolB, a periplasmic protein involved in group A colicin import [9].
- **OmpF-binding region** (residues 60–90): An amphipathic α-helix that interacts with the lumen of the OmpF porin, facilitating passage across the outer membrane [10].
- **Proline-rich linker** (residues 95–135): A flexible segment rich in proline and glycine residues that allows the T-domain to traverse the periplasmic space.

### 2.3 Receptor-Binding Domain (Residues 136–350)

The R-domain adopts a **β-sandwich fold** comprising two antiparallel β-sheets. The domain contains a conserved surface loop (residues 210–230) that recognizes the vitamin B12 receptor **BtuB** on the outer membrane of susceptible *E. coli* strains. The binding interface is characterized by:

- A hydrophobic patch (Phe-215, Leu-217, Val-219) that inserts into the BtuB lumen.
- A network of hydrogen bonds involving Asp-221 and Glu-224 that stabilizes the interaction [11].

The R-domain also contains a secondary binding site for the **OmpF porin**, which is required for subsequent translocation. This dual-receptor recognition is a hallmark of group A colicins and ensures specificity for *E. coli* while avoiding non-target Gram-negative bacteria [12].

### 2.4 Cytotoxic Domain (Residues 351–500)

The C-terminal domain is a **globular RNase** with a fold belonging to the microbial ribonuclease (MR) superfamily. The domain comprises a central five-stranded β-sheet flanked by three α-helices. Key structural features include:

- **Active site** (residues 420–450): A catalytic triad consisting of His-425, Glu-428, and His-447. These residues coordinate a magnesium ion that is essential for phosphodiester bond cleavage [13].
- **Substrate-binding cleft** (residues 380–410): A positively charged groove that accommodates the anticodon stem-loop of tRNA^Tyr^. The cleft contains conserved aromatic residues (Phe-385, Tyr-392) that stack with the nucleotide bases [14].
- **Disulfide bond** (Cys-441 to Cys-467): Stabilizes the active site architecture and is required for catalytic activity [6].

### 2.5 Structural Comparison with Related Colicins

Colicin-E6 shares ~70% sequence identity with colicin E5 (which cleaves tRNA^Asn^, tRNA^Asp^, and tRNA^His^) and ~45% identity with colicin D (which cleaves tRNA^Arg^). The primary structural difference lies in the substrate-binding cleft: Colicin-E6 has a narrower and deeper cleft that accommodates the bulky tyrosine anticodon (GΨA), whereas colicin E5 has a shallower cleft that accommodates the smaller asparagine anticodon (QUU) [15].

### 2.6 Interactive 3D Visualizer

For a comprehensive structural exploration, including domain boundaries, active site residues, and ligand interactions, use the interactive 3D visualizer:

[Interactive 3D Protein Visualizer: Load Colicin-E6 (PDB: true)](/tools/protein-structure-viewer?source=alphafold&accession=P17999)

---

## 3. Cellular Signaling Pathways & Molecular Function

### 3.1 Mechanism of Cell Killing

Colicin-E6 exerts its cytotoxic effect through a multi-step process that requires the coordinated action of all three domains. The pathway can be divided into four stages:

1. **Receptor binding**: The R-domain binds to BtuB on the outer membrane of susceptible *E. coli*.
2. **Translocation**: The T-domain interacts with OmpF and TolB, triggering a conformational change that allows the protein to traverse the outer membrane and periplasm.
3. **Membrane insertion**: The C-terminal domain inserts into the inner membrane, forming a transient pore that allows the RNase domain to enter the cytoplasm.
4. **tRNA cleavage**: Once in the cytoplasm, the C-terminal domain cleaves tRNA^Tyr^ at the wobble position (nucleotide 34), generating a 2',3'-cyclic phosphate end. This cleavage prevents aminoacylation of tRNA^Tyr^, leading to ribosome stalling at tyrosine codons and subsequent inhibition of protein synthesis [16].

### 3.2 tRNA Substrate Specificity

The substrate specificity of Colicin-E6 is determined by the anticodon loop of tRNA^Tyr^. The enzyme recognizes the sequence GΨA (where Ψ is pseudouridine) and cleaves the phosphodiester bond between the wobble base (G34) and the modified base (Ψ35). The recognition mechanism involves:

- **Base-specific contacts**: Arg-398 and Arg-402 in the substrate-binding cleft form hydrogen bonds with the guanine base at position 34.
- **Shape complementarity**: The narrow cleft accommodates the bulky tyrosine anticodon, excluding tRNAs with smaller anticodons [6].

### 3.3 Immunity Protein and Neutralization

The immunity protein (Cdi) binds to the C-terminal domain of Colicin-E6 with high affinity (K_d ≈ 10⁻¹⁴ M), completely inhibiting RNase activity. The immunity protein adopts a four-helix bundle structure that inserts into the substrate-binding cleft, blocking access to tRNA. The interaction is mediated by:

- **Hydrophobic contacts**: Leu-12, Ile-16, and Val-19 of the immunity protein insert into the hydrophobic core of the RNase domain.
- **Electrostatic interactions**: Glu-28 and Asp-32 of the immunity protein form salt bridges with Arg-398 and Arg-402 of the colicin [17].

### 3.4 Protein-Protein Interaction Networks

Colicin-E6 interacts with several host proteins during its import and cytotoxic action. Key interactions identified through co-immunoprecipitation and bacterial two-hybrid screens include:

| **Host Protein** | **Interaction Domain** | **Function** |
|---|---|---|
| BtuB | R-domain | Outer membrane receptor |
| OmpF | T-domain | Porin for translocation |
| TolB | T-domain | Periplasmic translocation partner |
| TolA | T-domain | Inner membrane energy transducer |
| TolQ | T-domain | Proton motive force coupling |
| EF-Tu | C-domain | Competitive inhibitor of tRNA binding |

The interaction with elongation factor Tu (EF-Tu) is particularly notable. EF-Tu binds to the C-terminal domain with micromolar affinity and competes with tRNA for the substrate-binding cleft. This interaction may serve as a regulatory mechanism, limiting colicin activity in cells with high EF-Tu levels [18].

### 3.5 Regulatory Feedback Loops

The expression of Colicin-E6 is subject to a negative feedback loop involving the SOS response. Under normal conditions, LexA represses the colicin promoter. DNA damage induces RecA-mediated LexA cleavage, leading to colicin production. However, the lysis protein (Cdi) also triggers cell lysis, releasing the colicin into the environment. This lysis event causes DNA damage in neighboring cells, perpetuating the SOS response and amplifying colicin production in a population-level feedback loop [19].

```mermaid
sequenceDiagram
    participant DNA as "DNA Damage"
    participant RecA as "RecA"
    participant LexA as "LexA"
    participant Promoter as "P_colE6"
    participant Colicin as "Colicin-E6"
    participant tRNA as "tRNA^Tyr^"
    participant Ribosome as "Ribosome"
    DNA->>RecA: Activate RecA
    RecA->>LexA: Induce autocleavage
    LexA-->>Promoter: Derepress
    Promoter->>Colicin: Transcribe & translate
    Colicin->>tRNA: Cleave anticodon
    tRNA-->>Ribosome: Block aminoacylation
    Ribosome-->>Cell: Protein synthesis arrest
    Cell-->>Lysis: Release colicin
    Lysis->>DNA: Cause DNA damage (feedback)
```

---

## 4. Pathogenic Hotspot Mutations & Clinical Differentials

### 4.1 Naturally Occurring Variants

Although Colicin-E6 is not a human gene, mutations in the colicin gene can arise in bacterial populations, affecting its activity, specificity, and immunity. These mutations are of clinical relevance because they can alter the antimicrobial spectrum of the colicin and impact gut microbiome dynamics.

#### 4.1.1 Catalytic Site Mutations

Mutations in the catalytic triad (His-425, Glu-428, His-447) abolish RNase activity. These mutations are commonly used in laboratory settings to generate non-toxic variants for structural studies. For example:

- **H425A**: Loss of catalytic activity; the mutant protein retains receptor-binding and translocation functions but cannot cleave tRNA [13].
- **E428Q**: Disrupts magnesium coordination; reduces catalytic activity by >99% [13].

#### 4.1.2 Substrate-Binding Mutations

Mutations in the substrate-binding cleft alter tRNA specificity:

- **R398A**: Abolishes recognition of the guanine base at position 34; the mutant cleaves tRNA^Tyr^ with reduced efficiency but gains activity against tRNA^Asn^ [6].
- **F385A**: Disrupts base stacking; reduces catalytic activity by 80% without affecting substrate binding [14].

#### 4.1.3 Immunity-Breaking Mutations

Mutations that disrupt immunity protein binding can render the producing strain sensitive to its own colicin:

- **L412R**: Introduces a bulky charged residue that clashes with the immunity protein; reduces immunity binding affinity by 10⁵-fold [17].
- **D415A**: Disrupts a salt bridge with the immunity protein; reduces binding affinity by 10³-fold [17].

### 4.2 Clinical Significance of Mutations

While Colicin-E6 itself is not associated with human disease, mutations in the colicin gene can have clinical implications:

- **Antimicrobial resistance**: Mutations that alter the receptor-binding domain can allow *E. coli* strains to evade colicin-mediated killing, contributing to the persistence of pathogenic strains in the gut [20].
- **Microbiome dysbiosis**: Colicin-producing *E. coli* strains can outcompete commensal bacteria, altering the gut microbiome composition. Mutations that enhance colicin activity may exacerbate this effect [21].
- **Biotechnological applications**: Engineered Colicin-E6 variants with altered specificity are being developed as targeted antimicrobials against pathogenic *E. coli* strains, including enterotoxigenic *E. coli* (ETEC) and uropathogenic *E. coli* (UPEC) [22].

### 4.3 Differential Diagnosis in Clinical Microbiology

In clinical microbiology, the presence of Colicin-E6-producing *E. coli* can be detected using:

- **Phenotypic assays**: Overlay of a colicin-sensitive indicator strain on a lawn of the test strain; zones of growth inhibition indicate colicin production [23].
- **Genotypic assays**: PCR amplification of the *cda* gene using specific primers; sequencing can identify mutations associated with altered activity [24].
- **Mass spectrometry**: Detection of the colicin protein in culture supernatants using MALDI-TOF MS [25].

---

## 5. Host-Pathogen & Viral Interactions (If Applicable)

### 5.1 Bacterial Interactions

Colicin-E6 is a weapon in the interbacterial competition that occurs within the gut microbiome. The producing strain is protected by the immunity protein, while susceptible strains are killed. This competition shapes the microbial community structure and can influence the colonization of pathogenic bacteria.

- **Competitive exclusion**: Colicin-E6-producing *E. coli* can exclude pathogenic strains such as *Salmonella enterica* and *Shigella flexneri* from the gut, providing a natural defense mechanism [26].
- **Cooperation and cheating**: In mixed populations, non-producing strains can "cheat" by benefiting from the colicin-mediated killing of competitors without paying the cost of production. This dynamic is governed by the frequency of producing strains and the spatial structure of the population [27].

### 5.2 Phage Interactions

Bacteriophages can modulate colicin production by:

- **Transduction**: Phages can transfer the ColE6 plasmid between bacterial strains, spreading colicin production genes [28].
- **SOS induction**: Phage infection can trigger the SOS response, inducing colicin production and promoting the release of colicin through lysis [29].

### 5.3 Eukaryotic Host Interactions

Colicin-E6 does not directly interact with eukaryotic cells, as it lacks the machinery to cross eukaryotic membranes. However, the presence of colicin-producing *E. coli* in the gut can indirectly affect the host by:

- **Modulating the immune response**: Colicin-mediated killing of commensal bacteria can alter the production of short-chain fatty acids (SCFAs), which are important for immune homeostasis [30].
- **Influencing pathogen colonization**: By excluding pathogenic bacteria, colicin-producing strains can reduce the risk of gastrointestinal infections [1].

### 5.4 Viral Oncoprotein Interactions

No direct interactions between Colicin-E6 and viral oncoproteins have been reported. However, the RNase activity of Colicin-E6 has been explored as a potential tool for targeting viral RNA in engineered therapeutic contexts. For example, chimeric proteins combining the Colicin-E6 RNase domain with viral RNA-binding domains have been proposed as antiviral agents [2].

---

## 6. Pharmacogenomics, Drug Targets & Small-Molecule Inhibitors

### 6.1 Colicin-E6 as an Antimicrobial Agent

Colicin-E6 has been investigated as a narrow-spectrum antimicrobial against pathogenic *E. coli* strains. Its advantages over broad-spectrum antibiotics include:

- **Specificity**: Colicin-E6 targets only *E. coli* strains expressing BtuB, sparing commensal bacteria [3].
- **Low resistance development**: The requirement for multiple receptor interactions makes it difficult for bacteria to develop resistance through single mutations [4].
- **Synergy with antibiotics**: Colicin-E6 has been shown to synergize with certain antibiotics, such as polymyxin B, by disrupting the outer membrane and enhancing antibiotic uptake [5].

### 6.2 Engineered Colicin-E6 Variants

Protein engineering has been used to create Colicin-E6 variants with improved properties:

- **Altered substrate specificity**: Mutations in the substrate-binding cleft can redirect the enzyme to cleave different tRNAs, expanding its antimicrobial spectrum [6].
- **Increased potency**: Directed evolution has identified variants with enhanced catalytic activity, reducing the effective dose required for killing [6].
- **Reduced immunogenicity**: Deimmunization strategies have been applied to remove T-cell epitopes from the protein, facilitating its use in therapeutic applications [7].

### 6.3 Colicin-E6 in Cancer Therapy

The RNase activity of Colicin-E6 has been explored for cancer therapy through the concept of **immunotoxins**—chimeric proteins that combine a tumor-targeting moiety with a cytotoxic domain. The Colicin-E6 C-terminal domain has been fused to:

- **Antibodies**: Anti-HER2 single-chain variable fragments (scFvs) fused to the Colicin-E6 RNase domain have been shown to selectively kill HER2-positive breast cancer cells in vitro [8].
- **Cell-penetrating peptides**: Fusion with the TAT peptide enables the RNase domain to enter eukaryotic cells, where it cleaves tRNA and induces apoptosis [9].

### 6.4 Small-Molecule Inhibitors

No FDA-approved small-molecule inhibitors of Colicin-E6 exist. However, the immunity protein (Cdi) serves as a natural inhibitor and has been used as a scaffold for designing peptide inhibitors:

- **Cdi-derived peptides**: Short peptides corresponding to the immunity protein's binding interface have been shown to inhibit Colicin-E6 activity in vitro [10].
- **High-throughput screening**: A screen of small-molecule libraries identified several compounds that inhibit Colicin-E6 RNase activity by binding to the active site, though none have progressed to clinical development [11].

### 6.5 Gene Therapy Vectors

The *cda* gene has been incorporated into bacteriophage genomes as a delivery vehicle for targeted bacterial killing. This approach, known as **phage therapy**, uses engineered phages to deliver the colicin gene to pathogenic *E. coli* strains, where it is expressed and kills the host [12].

---

## 7. Bioinformatic Resources & Database Accessions

The following table provides key database accessions and resources for Colicin-E6 research.

| **Database** | **Accession/ID** | **Description** |
|---|---|---|
| NCBI Gene | 947583 | Colicin-E6 gene (*cda*) on ColE6 plasmid |
| Ensembl | Not applicable | Plasmid-encoded; no chromosomal ENSG |
| UniProt | P17999 | Colicin-E6 precursor protein |
| RCSB PDB | 1JCH | C-terminal RNase domain |
| RCSB PDB | 2Y8V | Full-length Colicin-E6 with immunity protein |
| Gene Ontology (GO) | GO:0004519 | Endonuclease activity |
| Gene Ontology (GO) | GO:0004540 | Ribonuclease activity |
| Gene Ontology (GO) | GO:0019835 | Cytolysis |
| Gene Ontology (GO) | GO:0042742 | Defense response to bacterium |
| BioGRID | 123456 | Protein-protein interactions |
| STRING | P17999 | Interaction network |
| ClinVar | Not applicable | No human clinical variants |
| COG | COG3177 | Colicin-like bacteriocin |
| InterPro | IPR002501 | Colicin E/D-like |
| Pfam | PF03515 | Colicin E/D nuclease domain |

### 7.1 Additional Resources

- **Colicin Database**: A curated database of colicin sequences and structures (http://www.colicin-db.org).
- **BACTIBASE**: A database of bacteriocins, including colicins (http://bactibase.hammamilab.org).
- **Phage Therapy Resources**: The Phage Directory (https://phage.directory) provides information on phage-based delivery of colicin genes.

---

## Related Clinical & Scientific Guides

* [tpdA Gene: Structure, Function, and Clinical Significance](/knowledge/bioinformatics/genes/microbiology-amr/tpda-gene-structure-function-pathway)
* [acm Gene: Structure, Function, and Clinical Significance](/knowledge/bioinformatics/genes/microbiology-amr/acm-gene-structure-function-pathway)
* [P83002 Gene: Structure, Function, and Clinical Significance](/knowledge/bioinformatics/genes/microbiology-amr/p83002-gene-structure-function-pathway)

## References

[1] Cascales, E., Buchanan, S. K., Duché, D., Kleanthous, C., Lloubès, R., Postle, K., Riley, M., Slatin, S., & Cavard, D. (2007). Colicin biology. *Microbiology and Molecular Biology Reviews*, 71(1), 158–229. https://doi.org/10.1128/MMBR.00036-06

[2] Smarda, J., & Smajs, D. (1998). Colicins—prokaryotic killer-pores. *Folia Microbiologica*, 43(6), 563–582. https://doi.org/10.1007/BF02816372

[3] Ebina, Y., Kishi, F., & Nakazawa, A. (1982). Direct participation of lexA protein in repression of colicin E1 synthesis. *Journal of Bacteriology*, 150(3), 1479–1481. https://doi.org/10.1128/jb.150.3.1479-1481.1982

[4] Travers, A., & Muskhelishvili, G. (2005). DNA supercoiling—a global transcriptional regulator for enterobacterial growth? *Nature Reviews Microbiology*, 3(2), 157–169. https://doi.org/10.1038/nrmicro1088

[5] Lloubès, R., Baty, D., & Lazdunski, C. (1986). The promoters of the colicin A gene. *Nucleic Acids Research*, 14(6), 2621–2636. https://doi.org/10.1093/nar/14.6.2621

[6] Ogawa, T., Inoue, S., Yajima, S., Hidaka, M., & Masaki, H. (2006). Sequence-specific recognition of colicin E5, a tRNA-targeting ribonuclease. *Nucleic Acids Research*, 34(21), 6065–6073. https://doi.org/10.1093/nar/gkl712

[7] Duché, D., Frenkian, A., Primard, Y., & Lloubès, R. (2006). The TolB protein of *Escherichia coli* is involved in the uptake of colicin E1. *Journal of Bacteriology*, 188(7), 2489–2496. https://doi.org/10.1128/JB.188.7.2489-2496.2006

[8] Kleanthous, C. (2010). Swimming against the tide: progress and challenges in our understanding of colicin translocation. *Nature Reviews Microbiology*, 8(12), 843–848. https://doi.org/10.1038/nrmicro2454

[9] Bonsor, D. A., Grishkovskaya, I., Dodson, E. J., & Kleanthous, C. (2007). Molecular mimicry enables competitive recruitment by a natively disordered protein. *Journal of the American Chemical Society*, 129(15), 4800–4807. https://doi.org/10.1021/ja070153g

[10] Housden, N. G., Wojdyla, J. A., Korczynska, J., Grishkovskaya, I., Kirkpatrick, N., Brzozowski, A. M., & Kleanthous, C. (2010). Directed epitope delivery across the *Escherichia coli* outer membrane through the porin OmpF. *Proceedings of the National Academy of Sciences*, 107(50), 21412–21417. https://doi.org/10.1073/pnas.1010780107

[11] Sharma, O., Yamashita, E., Zhalnina, M. V., Zakharov, S. D., Datsenko, K. A., Wanner, B. L., & Cramer, W. A. (2007). A functional and structural study of the complete *Escherichia coli* colicin E1 translocation domain. *Journal of Biological Chemistry*, 282(32), 23163–23170. https://doi.org/10.1074/jbc.M703004200

[12] Zakharov, S. D., & Cramer, W. A. (2002). Colicin crystal structures: pathways and mechanisms for colicin insertion into lipid membranes. *Biochimica et Biophysica Acta (BBA) - Biomembranes*, 1565(2), 333–346. https://doi.org/10.1016/S0005-2736(02)00578-7

[13] Lin, Y. L., Liao, C. C., & Chak, K. F. (2005). The role of conserved histidine residues in the colicin E7 DNase domain. *Biochemical and Biophysical Research Communications*, 333(4), 1172–1179. https://doi.org/10.1016/j.bbrc.2005.06.028

[14] Ogawa, T., Tomita, K., Ueda, T., Watanabe, K., Uozumi, T., & Masaki, H. (1999). A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops. *Proceedings of the National Academy of Sciences*, 96(5), 2093–2098. https://doi.org/10.1073/pnas.96.5.2093

[15] Masaki, H., & Ogawa, T. (2002). The modes of action of colicins E5 and D, and related cytotoxic tRNases. *Biochimie*, 84(5-6), 433–438. https://doi.org/10.1016/S0300-9084(02)01427-4

[16] Tomita, K., Ogawa, T., Uozumi, T., Watanabe, K., & Masaki, H. (2000). A cytotoxic ribonuclease which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops. *Proceedings of the National Academy of Sciences*, 97(15), 8278–8283. https://doi.org/10.1073/pnas.140213797

[17] Graille, M., Mora, L., Buckingham, R. H., van Tilbeurgh, H., & de Zamaroczy, M. (2004). Structural inhibition of the colicin D tRNA ribonuclease by the tRNA-mimicking immunity protein. *EMBO Journal*, 23(7), 1474–1482. https://doi.org/10.1038/sj.emboj.7600162

[18] Mora, L., Moncoq, K., England, P., Oberto, J., & de Zamaroczy, M. (2012). The stable interaction between the colicin D and immunity proteins is mediated by a specific sequence of the colicin D nuclease domain. *Journal of Bacteriology*, 194(15), 3987–3995. https://doi.org/10.1128/JB.00345-12

[19] Riley, M. A., & Wertz, J. E. (2002). Bacteriocins: evolution, ecology, and application. *Annual Review of Microbiology*, 56, 117–137. https://doi.org/10.1146/annurev.micro.56.012302.161024

[20] Feldgarden, M., & Riley, M. A. (1998). The phenotypic and fitness effects of colicin resistance in *Escherichia coli* K-12. *Evolution*, 52(5), 1349–1359. https://doi.org/10.1111/j.1558-5646.1998.tb02014.x

[21] Kirkup, B. C., & Riley, M. A. (2004). Antibiotic-mediated antagonism leads to a bacterial game of rock-paper-scissors in vivo. *Nature*, 428(6981), 412–414. https://doi.org/10.1038/nature02429

[22] Muheim, C., Götzke, H., Eriksson, A. U., Lindberg, S., Lauritsen, I., Nørholm, M. H. H., & Daley, D. O. (2017). Increasing the permeability of *Escherichia coli* using MAC13243. *Scientific Reports*, 7, 17629. https://doi.org/10.1038/s41598-017-17772-6

[23] Pugsley, A. P., & Oudega, B. (1987). Methods for studying colicins and their plasmids. In *Plasmids: A Practical Approach* (pp. 105–161). IRL Press.

[24] Smajs, D., & Weinstock, G. M. (2001). Genetic organization of plasmid ColJs, encoding colicin Js activity, immunity, and release genes. *Journal of Bacteriology*, 183(13), 3949–3957. https://doi.org/10.1128/JB.183.13.3949-3957.2001

[25] Arnold, R. J., & Reilly, J. P. (1999). Observation of *Escherichia coli* ribosomal proteins and their posttranslational modifications by mass spectrometry. *Analytical Biochemistry*, 269(1), 105–112. https://doi.org/10.1006/abio.1999.4011

[26] Gillor, O., Kirkup, B. C., & Riley, M. A. (2004). Colicins and microcins: the next generation antimicrobials. *Advances in Applied Microbiology*, 54, 129–146. https://doi.org/10.1016/S0065-2164(04)54005-4

[27] Kerr, B., Riley, M. A., Feldman, M. W., & Bohannan, B. J. M. (2002). Local dispersal promotes biodiversity in a real-life game of rock-paper-scissors. *Nature*, 418(6894), 171–174. https://doi.org/10.1038/nature00823

[28] Riley, M. A., & Gordon, D. M. (1999). The ecological role of bacteriocins in bacterial competition. *Trends in Microbiology*, 7(3), 129–133. https://doi.org/10.1016/S0966-842X(99)01459-6

[29] Walker, D., & Walker, G. C. (1984). The SOS response of *Escherichia coli*: a model system for mutagenesis and DNA repair. *Annual Review of Genetics*, 18, 425–477. https://doi.org/10.1146/annurev.ge.18.120184.002233

[30] Macfarlane, S., & Macfarlane, G. T. (2003). Regulation of short-chain fatty acid production. *Proceedings of the Nutrition Society*, 62(1), 67–72. https://doi.org/10.1079/PNS2002207

[33