The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

[Characterization of a Taq DNA polymerase fused with a DNA binding domain of Escherichia coli colicin].

Taq DNA polymerase, which was discovered from a thermophilic aquatic bacterium (Thermus aquaticus), is an enzyme that possesses both reverse transcriptase activity and DNA polymerase activity. Colicin E (CE) protein belongs to a class of Escherichia coli toxins that utilize the vitamin receptor BtuB as a transmembrane receptor. Among these toxins, CE2, CE7, CE8, and CE9 are classified as non-specific DNase-type colicins. Taq DNA polymerase consists of a 5'→3' exonuclease domain, a 3'→5' exonuclease domain, and a polymerase domain. Taq DNA polymerase lacking the 5'→3' exonuclease domain (ΔTaq) exhibits higher yield but lower processivity, making it unable to amplify long fragments. In this study, we aimed to enhance the processivity of ΔTaq. To this end, we fused dCE with ΔTaq and observed a significant improvement in the processivity of the resulting dCE-ΔTaq compared to Taq DNA polymerase and dCE-Taq. Furthermore, its reverse transcriptase activity was also higher than that of ΔTaq. The most notable improvement was observed in dCE8-ΔTaq, which not only successfully amplified 8 kb DNA fragments within 1 minute, but also yielded higher results compared to other mutants. In summary, this study successfully enhanced the PCR efficiency and reverse transcription activity of Taq DNA polymerase by fusing ΔTaq DNA polymerase with dCE. This approach provides a novel approach for modifying Taq DNA polymerase and holds potential for the development of improved variants of Taq DNA polymerase.

Cell-specific RNA profiling reveals host genes expressed in Arabidopsis cells haustoriated by downy mildew.

The downy mildew oomycete Hyaloperonospora arabidopsidis, an obligate filamentous pathogen, infects Arabidopsis (Arabidopsis thaliana) by forming structures called haustoria inside host cells. Previous transcriptome analyses have revealed that host genes are specifically induced during infection; however, RNA profiling from whole-infected tissues may fail to capture key transcriptional events occurring exclusively in haustoriated host cells, where the pathogen injects virulence effectors to modulate host immunity. To determine interactions between Arabidopsis and H. arabidopsidis at the cellular level, we devised a translating ribosome affinity purification system using 2 high-affinity binding proteins, colicin E9 and Im9 (immunity protein of colicin E9), applicable to pathogen-responsive promoters, thus enabling haustoriated cell-specific RNA profiling. Among the host genes specifically expressed in H. arabidopsidis-haustoriated cells, we found genes that promote either susceptibility or resistance to the pathogen, providing insights into the Arabidopsis-downy mildew interaction. We propose that our protocol for profiling cell-specific transcripts will apply to several stimulus-specific contexts and other plant-pathogen interactions.

The "Cins" of Our Fathers: Rejuvenated Interest in Colicins to Combat Drug Resistance.

With the growing threat of antibiotic resistance, researchers around the globe are seeking alternatives to stem bacterial pathogenesis. One such alternative is bacteriocins, proteins produced by bacterial species to inhibit the growth and viability of related bacterial species. With their diverse mechanisms, which include pore formation and nuclease activities, and narrow spectrum of activities, which limit their impact to only certain bacterial species, unlike many chemical antibiotics, bacteriocins offer intriguing possibilities to selectively control individual bacterial populations. Within this review, therefore, we highlight current research exploring the application of colicins and microcins, a subset of bacteriocins, with an emphasis on their activities against drug-resistant pathogens, both in in vitro and in vivo settings.

Proteomics analysis reveals that CirA in Aeromonas hydrophila is involved in nutrient uptake.

The colicin I receptor (CirA) is a well-studied outer membrane protein that has been reported to play important roles in antibiotic resistance, virulence, and iron homeostasis, although its exact physiological roles require further investigation. In this study, differentially expressed proteins between the ΔahcirA and wild-type (WT) strains of Aeromonas hydrophila were compared using quantitative proteomics. Bioinformatics analysis revealed that the expression of peptide, histidine, and arginine ATP-binding cassette (ABC) transporter system-related proteins was significantly higher in the ΔahcirA strain. Subsequent growth assays revealed that ΔahcirA grew slower than the WT strain in nutrient-limited medium when supplemented with dipeptide, histidine, and arginine as the carbon source. Far-western blot analysis further confirmed that AhCirA can directly bind to histidine/arginine and dipeptide small-molecule substrates in addition to their periplasmic-binding proteins, AhDppA and AhHisJ, respectively. These results indicate that AhCirA may play an important role in the uptake of amino acids and peptides as a channel-forming porin while also directly interacting with ABC transporters to transport nutrient substances into the plasma membrane. Overall, this study demonstrates that AhCirA is a multifunctional protein in A. hydrophila and extends our understanding of known nutrient transport mechanisms among bacteria.

Colicins of Escherichia coli Lead to Resistance against the Diarrhea-Causing Pathogen Enterotoxigenic E. coli in Pigs.

Gut microbes can affect host adaptation to various environment conditions. Escherichia coli is a common gut species, including pathogenic strains and nonpathogenic strains. This study was conducted to investigate the effects of different E. coli strains in the gut on the health of pigs. In this study, the complete genomes of two E. coli strains isolated from pigs were sequenced. The whole genomes of Y18J and the enterotoxigenic E. coli strain W25K were compared to determine their roles in pig adaptation to disease. Y18J was isolated from feces of healthy piglets and showed strong antimicrobial activity against W25K in vitro. Gene knockout experiments and complementation analysis followed by modeling the microbe-microbe interactions demonstrated that the antagonistic mechanism of Y18J against W25K relied on the bacteriocins colicin B and colicin M. Compared to W25K, Y18J is devoid of exotoxin-coding genes and has more secondary-metabolite-biosynthetic gene clusters. W25K carries more genes involved in genome replication, in accordance with a shorter cell cycle observed during a growth experiment. The analysis of gut metagenomes in different pig breeds showed that colicins B and M were enriched in Laiwu pigs, a Chinese local breed, but were scarce in boars and Duroc pigs. IMPORTANCE This study revealed the heterogeneity of E. coli strains from pigs, including two strains studied by both in silico and wet experiments in detail and 14 strains studied by bioinformatics analysis. E. coli Y18J may improve the adaptability of pigs toward disease resistance through the production of colicins B and M. Our findings could shed light on the pathogenic and harmless roles of E. coli in modern animal husbandry, leading to a better understanding of intestinal-microbe-pathogen interactions in the course of evolution.

Effects of TolC on the pathogenicity of porcine extraintestinal pathogenic Escherichia coli.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a well-known critical pathogenic zoonosis that causes extraintestinal infections in humans and animals by affecting their immune organs. Recently, research on the outer membrane protein of E. coli, tolerant colicin (TolC), a virulent protein in the formation of the ExPEC efflux pump, has been an attractive subject. However, the pathogenic mechanisms remain unclear. This study aimed to explore the role of TolC in the pathogenesis of the ExPEC strain PPECC42; a complementation strain (Cm-TolC) and an isogenic mutant (ΔTolC) were constructed. Loss of TolC drastically impaired the virulence of ExPEC in an experimental mouse model. ΔTolC showed a substantial decrease in the porcine aortic vascular endothelial cell (PAVEC) adherence, invasion, and pro-inflammatory response, in contrast to that of the wild type, with a reduced survival ratio in both the bacterial load and whole blood in mice. ΔTolC also showed decreased expression of necroptosis signals such as receptor-interacting protein kinase 1, phosphorylated mixed-lineage kinase domain-like protein, and mitochondrial proteins such as phosphoglycerate mutase family member 5. Our data suggest that TolC is closely associated with ExPEC pathogenesis. These results provide scientific grounds for exploring the potential of TolC as an effective drug target for controlling ExPEC infection, screening new inhibitors, and developing new drugs. This will allow for further prevention and control of ExPEC infection.

Heterogeneity in the spontaneous induction of the promoter of the ColE9 operon in Escherichia coli.

Spontaneous production of E colicins is known to occur in only a small fraction of colicinogenic population. The current study aimed to determine if the same holds true for the production of colicin E9 in real time, by investigating the induction dynamics of the promoter of the ColE9 operon which results in the expression of the ColE9 activity and functional genes. A novel fluorescent reporter was constructed which carries the fusion of the ColE9 promoter and the gfpmut2 gene in a low copy number plasmid that was compatible with the native ColE9-J plasmid. Using the fluorescent reporter construct in the non colicinogenic E. coli cells, the induction of the ColE9 promoter was investigated. The current study demonstrates that the spontaneous induction of the ColE9 promoter occurs in a heterogenous manner and this heterogeneity is maintained in a bacterial population for several generations suggesting that it is unlikely due to any irreversible mutation in the bacterial culture. Furthermore, the same investigations were repeated using the colicin E9 producing E. coli cells. Flow cytometry analysis revealed that 7.1 ± 0.68% of the colicin E9 producing E. coli cells expressed GFP albeit only 2.45 ± 0.30% was observed from non colicinogenic E. coli cells. The considerable increase in the number of the fluorescent cells was likely due to the DNase activity of colicin E9 produced by their clonemates, resulting the auto-induction, which can be abolished with the inactivation of the DNase activity of the colicin E9.

Colicins and Microcins Produced by Enterobacteriaceae: Characterization, Mode of Action, and Putative Applications.

Enterobacteriaceae are widely present in many environments related to humans, including the human body and the food that they consume, from both plant or animal origin. Hence, they are considered relevant members of the gastrointestinal tract microbiota. On the other hand, these bacteria are also recognized as putative pathogens, able to impair human health and, in food, they are considered indicators for the microbiological quality and hygiene status of a production process. Nevertheless, beneficial properties have also been associated with Enterobacteriaceae, such as the ability to synthesize peptides and proteins, which can have a role in the structure of microbial communities. Among these antimicrobial molecules, those with higher molecular mass are called colicins, while those with lower molecular mass are named microcins. In recent years, some studies show an emphasis on molecules that can help control the development of pathogens. However, not enough data are available on this subject, especially related to microcins. Hence, this review gathers and summarizes current knowledge on colicins and microcins, potential usage in the treatment of pathogen-associated diseases and cancer, as well as putative applications in food biotechnology.

A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Application of a Novel CL7/Im7 Affinity System in Purification of Complex and Pharmaceutical Proteins.

We have developed the CL7/Im7 protein purification system to achieve high-yield, high-purity and high-activity (HHH) products in one step. The system is based on the natural ultrahigh-affinity complex between the two small proteins encoded by colicinogenic plasmids carried by certain E. coli strains, the DNAse domain of colicin E7 (CE7; MW ~ 15 kDa) and its natural endogenous inhibitor, the immunity protein 7 (Im7; MW ~ 10 kDa). CL7 is an engineered variant of CE7, in which the toxic DNA-binding and catalytic activities have been eliminated while retaining the high affinity to Im7. CL7 is used as a protein tag, while Im7 is covalently attached to agarose beads. To make the CL7/Im7 technique easy to use, we have designed a set of the E. coli expression vectors for fusion of a target protein to the protease-cleavable CL7-tag either at the N- or the C-terminus, and also have the options of the dual (CL7/His8) tag. A subset of vectors is dedicated for cloning membrane and multisubunit proteins. The CL7/Im7 system has several notable advatantages over other available affinity purification techniques. First, high concentrations of the small Im7 protein are coupled to the beads resulting in the high column capacities (up to 60 mg/mL). Second, an exceptional stability of Im7 allows for multiple (100+) regeneration cycles with no loss of binding capacities. Third, the CL7-tag improves protein expression levels, solubility and, in some cases, assists folding of the target proteins. Fourth, the on-column proteolytic elution produces purified proteins with few or no extra amino acid residues. Finally, the CL7/Im7 affinity is largely insensitive to high salt concentrations. For many target proteins, loading the bacterial lysates on the Im7 column in high salt is a key to high purity. Altogether, these properties of the CL7/Im7 system allow for a one-step HHH purification of most challenging, biologically and clinically significant proteins.

Reconstruction of the Gram-Negative Bacterial Outer-Membrane Bilayer.

The outer membrane (OM) of gram-negative bacteria is highly asymmetric. The outer leaflet comprises lipopolysaccharides (LPS) and the inner leaflet phospholipids. Here, it is shown that the outer membrane lipid bilayer (OMLB) of Escherichia coli can be reconstructed as a droplet interface bilayer (DIB), which separates two aqueous droplets in oil. The trimeric porin OmpF is inserted into the model OMLB and the translocation of the bacteriocin colicin E9 (colE9) through it is monitored. By contrast with LPS-free bilayers, it is found that colE9 made multiple failed attempts to engage with OmpF in an OMLB before successful translocation occurred. In addition, the observed rate for the second step of colE9 translocation is 3-times smaller than that in LPS-free bilayers, and further, the colE9 dissociates when the membrane potential is reversed. The findings demonstrate the utility of the DIB approach for constructing model OMLBs from physiologically realistic lipids and that the properties of the model OMLBs differ from those of a simple lipid bilayer. The model OMLB offers a credible platform for screening the properties of antibiotics.

Modeling endonuclease colicin-like bacteriocin operons as 'genetic arms' in plasmid-genome conflicts.

Plasmids are acellular propagating entities that depend on bacteria, as molecular parasites, for propagation. A 'tussle' between bacteria and plasmid ensues; bacteria for riddance of the plasmid and plasmid for persistence within a live host. Plasmid-maintenance systems such as endonuclease Colicin-Like Bacteriocins (CLBs) ensure plasmid propagation within the population; (i) the plasmid-cured cells are killed by the CLBs; (ii) damaged cells lyse and release the CLBs that eliminate the competitors, and (iii) the released plasmids invade new bacteria. Surprisingly, endonuclease CLB operons occur on bacterial genomes whose significance is unknown. Here, we study genetics, eco-evolutionary drive, and physiological relevance of genomic endonuclease CLB operons. We investigated plasmidic and genomic endonuclease CLB operons using sequence analyses from an eco-evolutionary perspective. We found 1266 genomic and plasmidic endonuclease CLB operons across 30 bacterial genera. Although 51% of the genomes harbor endonuclease CLB operons, the majority of the genomic endonuclease CLB operons lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the endonuclease CLB operon protects the plasmid-cured host, eliminating the metabolic burden. We show mutual exclusivity of endonuclease CLB operons on genomes and plasmids. We propose an anti-addiction hypothesis for genomic endonuclease CLB operons. Using a stochastic hybrid agent-based model, we show that the endonuclease CLB operons on genomes confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. The conflict between bacterial genome and plasmids allows the emergence of 'genetic arms' such as CLB operons that regulate the ecological interplay of bacterial genomes and plasmids.

Colicin E1 opens its hinge to plug TolC.

The double membrane architecture of Gram-negative bacteria forms a barrier that is impermeable to most extracellular threats. Bacteriocin proteins evolved to exploit the accessible, surface-exposed proteins embedded in the outer membrane to deliver cytotoxic cargo. Colicin E1 is a bacteriocin produced by, and lethal to, Escherichia coli that hijacks the outer membrane proteins (OMPs) TolC and BtuB to enter the cell. Here, we capture the colicin E1 translocation domain inside its membrane receptor, TolC, by high-resolution cryo-electron microscopy to obtain the first reported structure of a bacteriocin bound to TolC. Colicin E1 binds stably to TolC as an open hinge through the TolC pore-an architectural rearrangement from colicin E1's unbound conformation. This binding is stable in live E. coli cells as indicated by single-molecule fluorescence microscopy. Finally, colicin E1 fragments binding to TolC plug the channel, inhibiting its native efflux function as an antibiotic efflux pump, and heightening susceptibility to three antibiotic classes. In addition to demonstrating that these protein fragments are useful starting points for developing novel antibiotic potentiators, this method could be expanded to other colicins to inhibit other OMP functions.

Bacteria are constantly warring with each other for space and resources. As a result, they have developed a range of molecular weapons to poison, damage or disable other cells. For instance, bacteriocins are proteins that can latch onto structures at the surface of enemy bacteria and push toxins through their outer membrane. Bacteria are increasingly resistant to antibiotics, representing a growing concern for modern healthcare. One way that they are able to survive is by using 'efflux pumps' studded through their external membranes to expel harmful drugs before these can cause damage. Budiardjo et al. wanted to test whether bacteriocins could interfere with this defence mechanism by blocking efflux pumps. Bacteriocins are usually formed of binding elements (which recognise specific target proteins) and of a 'killer tail' that can stab the cell. Experiments showed that the binding parts of a bacteriocin could effectively 'plug' efflux pumps in Escherichia coli bacteria: high-resolution molecular microscopy revealed how the bacteriocin fragment binds to the pump, while fluorescent markers showed that it attached to the surface of E. coli and stopped the efflux pumps from working. As a result, lower amounts of antibiotics were necessary to kill the bacteria when bacteriocins were present. The work by Budiardjo et al. could lead to new ways to combat bacteria that will reduce the need for current antibiotics. In the future, bacteriocins could also be harnessed to target other proteins than efflux pumps, allowing scientists to manipulate a range of bacterial processes.

H1 helix of colicin U causes phospholipid membrane permeation.

In light of an increasing number of antibiotic-resistant bacterial strains, it is essential to understand an action imposed by various antimicrobial agents on bacteria at the molecular level. One of the leading mechanisms of killing bacteria is related to the alteration of their plasmatic membrane. We study bio-inspired peptides originating from natural antimicrobial proteins colicins, which can disrupt membranes of bacterial cells. Namely, we focus on the α-helix H1 of colicin U, produced by bacterium Shigella boydii, and compare it with analogous peptides derived from two different colicins. To address the behavior of the peptides in biological membranes, we employ a combination of molecular simulations and experiments. We use molecular dynamics simulations to show that all three peptides are stable in model zwitterionic and negatively charged phospholipid membranes. At the molecular level, their embedment leads to the formation of membrane defects, membrane permeation for water, and, for negatively charged lipids, membrane poration. These effects are caused by the presence of polar moieties in the considered peptides. Importantly, simulations demonstrate that even monomeric H1 peptides can form toroidal pores. At the macroscopic level, we employ experimental co-sedimentation and fluorescence leakage assays. We show that the H1 peptide of colicin U incorporates into phospholipid vesicles and disrupts their membranes, causing leakage, in agreement with the molecular simulations. These insights obtained for model systems seem important for understanding the mechanisms of antimicrobial action of natural bacteriocins and for future exploration of small bio-inspired peptides able to disrupt bacterial membranes.

Topological frustration leading to backtracking in a coupled folding-binding process.

Intrinsically disordered proteins (IDPs) are abundant in all species. Their discovery challenges the traditional "sequence-structure-function" paradigm of protein science because IDPs play important roles in various biological processes without preformed folded structures. Bioinformatic analysis reveals that the intrinsically conformational disorder of IDPs as well as their conformational transition upon binding to their targets is encoded by their amino acid sequences. The rRNase domain of colicin E3 and the immunity protein Im3 are a pair of proteins involved in bacterial survival. While the N-terminal segment and the central segment of E3 make comparable intermolecular contacts with Im3 in the bound state, binding of E3 with Im3 is dominantly triggered by the central segment of E3. In this work, to further investigate the binding mechanism of disordered E3 with Im3, we performed systematic free energy and transition path analysis through coarse-grained molecular dynamics simulations. We observed backtracking of the N-terminal segment of E3 in the binding process, whose occurrence depends on salt concentration. Conformational analysis revealed that initial binding of the N-terminal segment of E3 to Im3 usually leads to misorientation of a central hairpin of E3 on Im3, which generates topological frustration and results in backtracking of the N-terminal segment. Our results not only provide deeper mechanistic insights into the coupled folding-binding process of the E3/Im3 complex, but also suggest that topological frustration could be present in the coupled folding-binding process of IDPs and play an important role in regulating the binding transition pathways.

Reprogramming microbial populations using a programmed lysis system to improve chemical production.

Microbial populations are a promising model for achieving microbial cooperation to produce valuable chemicals. However, regulating the phenotypic structure of microbial populations remains challenging. In this study, a programmed lysis system (PLS) is developed to reprogram microbial cooperation to enhance chemical production. First, a colicin M -based lysis unit is constructed to lyse Escherichia coli. Then, a programmed switch, based on proteases, is designed to regulate the effective lysis unit time. Next, a PLS is constructed for chemical production by combining the lysis unit with a programmed switch. As a result, poly (lactate-co-3-hydroxybutyrate) production is switched from PLH synthesis to PLH release, and the content of free PLH is increased by 283%. Furthermore, butyrate production with E. coli consortia is switched from E. coli BUT003 to E. coli BUT004, thereby increasing butyrate production to 41.61 g/L. These results indicate the applicability of engineered microbial populations for improving the metabolic division of labor to increase the efficiency of microbial cell factories.

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

The Antibiotic Efflux Protein TolC Is a Highly Evolvable Target under Colicin E1 or TLS Phage Selection.

Bacteriophages and bacterial toxins are promising antibacterial agents to treat infections caused by multidrug-resistant (MDR) bacteria. In fact, bacteriophages have recently been successfully used to treat life-threatening infections caused by MDR bacteria (Schooley RT, Biswas B, Gill JJ, Hernandez-Morales A, Lancaster J, Lessor L, Barr JJ, Reed SL, Rohwer F, Benler S, et al. 2017. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 61(10); Chan BK, Turner PE, Kim S, Mojibian HR, Elefteriades JA, Narayan D. 2018. Phage treatment of an aortic graft infected with Pseudomonas aeruginosa. Evol Med Public Health. 2018(1):60-66; Petrovic Fabijan A, Lin RCY, Ho J, Maddocks S, Ben Zakour NL, Iredell JR, Westmead Bacteriophage Therapy Team. 2020. Safety of bacteriophage therapy in severe Staphylococcus aureus infection. Nat Microbiol. 5(3):465-472). One potential problem with using these antibacterial agents is the evolution of resistance against them in the long term. Here, we studied the fitness landscape of the Escherichia coli TolC protein, an outer membrane efflux protein that is exploited by a pore forming toxin called colicin E1 and by TLS phage (Pagie L, Hogeweg P. 1999. Colicin diversity: a result of eco-evolutionary dynamics. J Theor Biol. 196(2):251-261; Andersen C, Hughes C, Koronakis V. 2000. Chunnel vision. Export and efflux through bacterial channel-tunnels. EMBO Rep. 1(4):313-318; Koronakis V, Andersen C, Hughes C. 2001. Channel-tunnels. Curr Opin Struct Biol. 11(4):403-407; Czaran TL, Hoekstra RF, Pagie L. 2002. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci U S A. 99(2):786-790; Cascales E, Buchanan SK, Duché D, Kleanthous C, Lloubès R, Postle K, Riley M, Slatin S, Cavard D. 2007. Colicin biology. Microbiol Mol Biol Rev. 71(1):158-229). By systematically assessing the distribution of fitness effects of ∼9,000 single amino acid replacements in TolC using either positive (antibiotics and bile salts) or negative (colicin E1 and TLS phage) selection pressures, we quantified evolvability of the TolC. We demonstrated that the TolC is highly optimized for the efflux of antibiotics and bile salts. In contrast, under colicin E1 and TLS phage selection, TolC sequence is very sensitive to mutations. Finally, we have identified a large set of mutations in TolC that increase resistance of E. coli against colicin E1 or TLS phage without changing antibiotic susceptibility of bacterial cells. Our findings suggest that TolC is a highly evolvable target under negative selection which may limit the potential clinical use of bacteriophages and bacterial toxins if evolutionary aspects are not taken into account.

Identification of a Dps contamination in Mitomycin-C-induced expression of Colicin Ia.

Colicins are bacterial toxins targeting Gram-negative bacteria, including E. coli and related Enterobacteriaceae strains. Some colicins form ion-gated pores in the inner membrane of attacked bacteria that are lethal to their target. Colicin Ia was the first pore-forming E. coli toxin, for which a high-resolution structure of the monomeric full-length protein was determined. It is so far also the only colicin, for which a low-resolution structure of its membrane-inserted pore was reported by negative-stain electron microscopy. Resolving this structure at the atomic level would allow an understanding of the mechanism of toxin pore formation. Here, we report an observation that we made during an attempt to determine the Colicin Ia pore structure at atomic resolution. Colicin Ia was natively expressed by mitomycin-C induction under a native SOS promotor and purified following published protocols. The visual appearance in the electron microscope of negatively stained preparations and the lattice parameters of 2D crystals obtained from the material were highly similar to those reported earlier resulting from the same purification protocol. However, a higher-resolution structural analysis revealed that the protein is Dps (DNA-binding protein from starved cells), a dodecameric E. coli protein. This finding suggests that the previously reported low-resolution structure of a "Colicin Ia oligomeric pore" actually shows Dps.