[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.

Colicins and T6SS-based competition systems enhance enterotoxigenic E. coli (ETEC) competitiveness.

Diarrheal diseases are still a significant problem for humankind, causing approximately half a million deaths annually. To cause diarrhea, enteric bacterial pathogens must first colonize the gut, which is a niche occupied by the normal bacterial microbiota. Therefore, the ability of pathogenic bacteria to inhibit the growth of other bacteria can facilitate the colonization process. Although enterotoxigenic Escherichia coli (ETEC) is one of the major causative agents of diarrheal diseases, little is known about the competition systems found in and used by ETEC and how they contribute to the ability of ETEC to colonize a host. Here, we collected a set of 94 fully assembled ETEC genomes by performing whole-genome sequencing and mining the NCBI RefSeq database. Using this set, we performed a comprehensive search for delivered bacterial toxins and investigated how these toxins contribute to ETEC competitiveness in vitro. We found that type VI secretion systems (T6SS) were widespread among ETEC (n = 47). In addition, several closely related ETEC strains were found to encode Colicin Ia and T6SS (n = 8). These toxins provide ETEC competitive advantages during in vitro competition against other E. coli, suggesting that the role of T6SS as well as colicins in ETEC biology has until now been underappreciated.

Escherichia coli killing by epidemiologically successful sublineages of Shigella sonnei is mediated by colicins.

BACKGROUND: Shigella sp. are enteric pathogens which causes >125 million cases of shigellosis annually. S. sonnei accounts for about a quarter of those cases and is increasingly prevalent in industrialising nations. Being an enteric pathogen, S. sonnei benefits from outcompeting gut commensals such as Escherichia coli to establish itself and cause disease. There are numerous mechanisms that bacterial pathogens use to outcompete its rivals including molecules called colicins. A Type 6 Secretion System (T6SS) was recently described as contributing to E. coli killing in S. sonnei. METHODS: We used Bulk Phenotyping of Epidemiological Replicates (BPER) which combined bacterial Genome Wide Association Studies (bGWAS) and high throughput phenotyping on a collection of S. sonnei surveillance isolates to identify the genetic features associated with E. coli killing and explore their relationship with epidemiological behaviour. We further explored the presence of colicins and T6SS components in the isolates using genomics, laboratory experimentation, and proteomics. FINDINGS: Our bGWAS analysis returned known and novel colicin and colicin related genes as significantly associated with E. coli killing. In silico analyses identified key colicin clusters responsible for the killing phenotype associated with epidemiologically successful sub-lineages. The killing phenotype was not associated with the presence of a T6SS. Laboratory analyses confirmed the presence of the key colicin clusters and that killing was contact-independent. INTERPRETATION: Colicins are responsible for E. coli killing by S. sonnei, not a T6SS. This phenotype contributes to shaping the observed epidemiology of S. sonnei and may contribute to its increasing prevalence globally. BPER is an epidemiologically relevant approach to phenotypic testing that enables the rapid identification of genetic drivers of phenotypic changes, and assessment of their relevance to epidemiology in natural settings. FUNDING: Biotechnology and Biological Sciences Research Council, Biotechnology and Biological Sciences Research Council Doctoral Training Partnership studentship, Wellcome Trust, Medical Research Council (UK), French National Research Agency.

Phage tRNAs evade tRNA-targeting host defenses through anticodon loop mutations.

Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases.

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.

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.

RtcB2-PrfH Operon Protects E. coli ATCC25922 Strain from Colicin E3 Toxin.

In the bid to survive and thrive in an environmental setting, bacterial species constantly interact and compete for resources and space in the microbial ecosystem. Thus, they have adapted to use various antibiotics and toxins to fight their rivals. Simultaneously, they have evolved an ability to withstand weapons that are directed against them. Several bacteria harbor colicinogenic plasmids which encode toxins that impair the translational apparatus. One of them, colicin E3 ribotoxin, mediates cleavage of the 16S rRNA in the decoding center of the ribosome. In order to thrive upon deployment of such ribotoxins, competing bacteria may have evolved counter-conflict mechanisms to prevent their demise. A recent study demonstrated the role of PrfH and the RtcB2 module in rescuing a damaged ribosome and the subsequent re-ligation of the cleaved 16S rRNA by colicin E3 in vitro. The rtcB2-prfH genes coexist as gene neighbors in an operon that is sporadically spread among different bacteria. In the current study, we report that the RtcB2-PrfH module confers resistance to colicin E3 toxicity in E. coli ATCC25922 cells in vivo. We demonstrated that the viability of E. coli ATCC25922 strain that is devoid of rtcB2 and prfH genes is impaired upon action of colicin E3, in contrast to the parental strain which has intact rtcB2 and prfH genes. Complementation of the rtcB2 and prfH gene knockout with a high copy number-plasmid (encoding either rtcB2 alone or both rtcB2-prfH operon) restored resistance to colicin E3. These results highlight a counter-conflict system that may have evolved to thwart colicin E3 activity.

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.

TisB Protein Protects Escherichia coli Cells Suffering Massive DNA Damage from Environmental Toxic Compounds.

Toxin-antitoxin systems are genetic elements that are widespread in prokaryotes. Although molecular mode of action of many of these toxins has been identified, their biological functions are mostly unknown. We investigated the functional integration of the TisB/IstR toxin-antitoxin system in the Escherichia coli SOS genotoxic stress response network. We showed that the tisB gene is induced in cells exposed to high doses of the genotoxic antibiotic trimethoprim. However, we also found that TisB contributes to trimethoprim-induced lethality. This is a consequence of the TisB-induced drop in the proton motive force (PMF), which results in blocking the thymine import and therefore the functioning of the pyrimidine salvage pathway. Conversely, a TisB-induced PMF drop protects cells by preventing the import of some other toxic compounds, like the aminoglycoside antibiotic gentamicin and colicin M, in the SOS-induced cells. Colicins are cytotoxic molecules produced by Enterobacterales when they are exposed to strong genotoxic stresses in order to compete with other microbiota members. We indeed found that TisB contributes to E. coli's fitness during mouse gut colonization. Based on the results obtained here, we propose that the primary biological role of the TisB toxin is to increase the probability of survival and maintenance in the mammalian gut of their bacterial hosts when they have to simultaneously deal with massive DNA damages and a fierce chemical warfare with other microbiota members. IMPORTANCE The contribution of toxin-antitoxin systems to the persistence of bacteria to antibiotics has been intensively studied. This is also the case with the E. coli TisB/IstR toxin-antitoxin system, but the contribution of TisB to the persistence to antibiotics turned out to be not as straightforward as anticipated. In this study, we show that TisB can decrease, but also increase, cytotoxicity of different antibiotics. This inconsistency has a common origin, i.e., TisB-induced collapse of the PMF, which impacts the import and the action of different antibiotics. By taking into account the natural habitat of TisB bacterial hosts, the facts that this toxin-antitoxin system is integrated into the genotoxic stress response regulon SOS and that both SOS regulon and TisB are required for E. coli to colonize the host intestine, and the phenotypic consequences of the collapse of the PMF, we propose that TisB protects its hosts from cytotoxic molecules produced by competing intestinal bacteria.

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.

Heterologous overexpression and preliminary antimicrobial activity test of salmocin M, a novel colicin M-like bacteriocin against Salmonella sp.

Currently, it is extremely important to identify and describe new alternative compounds with potential antimicrobial properties. Since various natural biological systems are capable of producing active compounds with such properties, many of them have been the subject of intensive study. The aim of this work was to heterologously overexpress, purify and preliminarily investigate the antimicrobial activity of a novel bacteriocin found in Salmonella species. Overexpressed protein shows an amino acid structure homologous to the well-known colicin M and was never expressed previously in the E. coli platform. Purified salmocin M showed an inhibition spectrum against Salmonella and E. coli strains. To determine its potential as an antimicrobial agent for use in medicine or the food industry, preliminary antimicrobial tests against pathogenic bacteria were carried out. Our research demonstrates that bacteriocin can be produced efficiently in bacterial expression systems, which are one of the cheapest and the most popular platforms for recombinant protein production. Moreover, preliminary results of microbiological tests showed its activity against most of the bacterial strains in a dose-dependent manner.

Structural and functional insights into colicin: a new paradigm in drug discovery.

Colicins are agents of allelopathic interactions produced by certain enterobacteria which give them a competitive advantage in the environment. These protein molecules are mostly encoded by plasmids. The colicin operon consists of the activity, immunity and the lysis genes. The activity protein is responsible for the killing activity, the immunity protein protects the producer cell from the lethal action of colicin and the lysis protein facilitates its release. Colicins are primarily composed of three domains, namely the receptor-binding domain, the translocation domain and the cytotoxic domain. The protein molecule binds to its cognate receptor on the target cell via the receptor-binding domain and undergoes translocation into the cell either via the Tol system or the Ton system. After gaining entry into the target cell, there are various mechanisms by which colicins exert their lethality. These comprise DNase activity, RNase activity and pore formation in the target cell membrane or peptidoglycan synthesis inhibition. This review gives a detailed insight into the structural and functional aspect of colicins and their mode of action. This knowledge is of immense significance because colicins are being considered as very useful alternatives to conventional antibiotics in the treatment of multidrug-resistant infections. Besides, they also have a negligible harmful impact on the commensals. Thus, before tapping their therapeutic potential, it is imperative to know their structure and mechanism of action in detail.

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.

Colicin-Mediated Transport of DNA through the Iron Transporter FepA.

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

Design of fusion protein for efficient preparation of cyanovirin-n and rapid enrichment of pseudorabies virus.

OBJECTIVE: Cyanovirin-N (CVN) is a cyanobacterial protein with potent neutralizing activity against enveloped virus. To achieve the economic and functional production of CVN, the CVN N-terminally fused with CL7(A mutant of the Colicin E7 Dnase) was utilized to improve the solubility and stability of CVN fusion protein (CL7-CVN). Additionally, to improve the detection limit of existing PRV diagnostic assays, CL7-CVN was used for Pseudorabies virus (PRV) enrichment from larger sample volumes. RESULTS: CVN fused with CL7 was efficiently expressed at a level of ~ 40% of the total soluble protein in E. coli by optimizing the induction conditions. Also, the stability of CVN fusion protein was enhanced, and 10 mg of CVN with a purity of ~ 99% were obtained from 1 g of cells by one-step affinity purification with the digestion of HRV 3C protease. Moreover, both purified CVN and CL7-CVN could effectively inhibit the infection of PRV to PK15 cells. Considering the bioactivity of CL7-CVN, we explored a strategy for PRV enrichment from larger samples. CONCLUSIONS: CL7 effectively promoted the soluble expression of CVN fusion protein and improved its stability, which was meaningful for its purification and application. The design of CVN fusion protein provides an efficient approach for the economical and functional production of CVN and a new strategy for PRV enrichment.

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.

Structural design principles for specific ultra-high affinity interactions between colicins/pyocins and immunity proteins.

The interactions of the antibiotic proteins colicins/pyocins with immunity proteins is a seminal model system for studying protein-protein interactions and specificity. Yet, a precise and quantitative determination of which structural elements and residues determine their binding affinity and specificity is still lacking. Here, we used comparative structure-based energy calculations to map residues that substantially contribute to interactions across native and engineered complexes of colicins/pyocins and immunity proteins. We show that the immunity protein α1-α2 motif is a unique structurally-dissimilar element that restricts interaction specificity towards all colicins/pyocins, in both engineered and native complexes. This motif combines with a diverse and extensive array of electrostatic/polar interactions that enable the exquisite specificity that characterizes these interactions while achieving ultra-high affinity. Surprisingly, the divergence of these contributing colicin residues is reciprocal to residue conservation in immunity proteins. The structurally-dissimilar immunity protein α1-α2 motif is recognized by divergent colicins similarly, while the conserved immunity protein α3 helix interacts with diverse colicin residues. Electrostatics thus plays a key role in setting interaction specificity across all colicins and immunity proteins. Our analysis and resulting residue-level maps illuminate the molecular basis for these protein-protein interactions, with implications for drug development and rational engineering of these interfaces.

Substrate recognition mechanism of tRNA-targeting ribonuclease, colicin D, and an insight into tRNA cleavage-mediated translation impairment.

Colicin D is a plasmid-encoded bacteriocin that specifically cleaves tRNAArg of sensitive Escherichia coli cells. E. coli has four isoaccepting tRNAArgs; the cleavage occurs at the 3' end of anticodon-loop, leading to translation impairment in the sensitive cells. tRNAs form a common L-shaped structure and have many conserved nucleotides that limit tRNA identity elements. How colicin D selects tRNAArgs from the tRNA pool of sensitive E. coli cells is therefore intriguing. Here, we reveal the recognition mechanism of colicin D via biochemical analyses as well as structural modelling. Colicin D recognizes tRNAArgICG, the most abundant species of E. coli tRNAArgs, at its anticodon-loop and D-arm, and selects it as the most preferred substrate by distinguishing its anticodon-loop sequence from that of others. It has been assumed that translation impairment is caused by a decrease in intact tRNA molecules due to cleavage. However, we found that intracellular levels of intact tRNAArgICG do not determine the viability of sensitive cells after such cleavage; rather, an accumulation of cleaved ones does. Cleaved tRNAArgICG dominant-negatively impairs translation in vitro. Moreover, we revealed that EF-Tu, which is required for the delivery of tRNAs, does not compete with colicin D for binding tRNAArgICG, which is consistent with our structural model. Finally, elevation of cleaved tRNAArgICG level decreases the viability of sensitive cells. These results suggest that cleaved tRNAArgICG transiently occupies ribosomal A-site in an EF-Tu-dependent manner, leading to translation impairment. The strategy should also be applicable to other tRNA-targeting RNases, as they, too, recognize anticodon-loops.Abbreviations: mnm5U: 5-methylaminomethyluridine; mcm5s2U: 5-methoxycarbonylmethyl-2-thiouridine.

Antimicrobial efficacy and prevalence of colicinogenic E. coli in faecal matter of human, cow and sheep.

Colicinogenic Escherichia coli in the human or animal gut prevent pathogen accumulation, thereby protecting against severe gut infections. This ability of commensal human and animal E. coli to restrain Gram-negative pathogens was evaluated invitro. Approximately 13.2% of E. coli isolates inhibited the growth of target pathogens. The greatest inhibition of 36% was observed against E. coli O157:H7 followed by 35%, 27%, 24% and 13% inhibition against E. coli O26:H11, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae, respectively. Detection of 20 colicin determinants in colicinogenic E. coli revealed that >50% isolates exhibited multiple colicin genes. Among all isolates, 70.4% of E. coli had the col E6 gene followed by col Ib (66.4%), E4 (53.6%), E7 (49.9%), J (35.3%) and M (35.2%) determinants. The frequency of col D (8.8%), Ia (27.9%), S4 (12%), E3 (13.2%) and E9 (2.9%) was greater in human samples compared with samples from cows and sheep, whereas col10 (5.8%) and E5 (4.4%) were produced only by cow-derived E. coli. Colicinogenic E. coli belonging to phylogenetic group B2 (52.8%) were more prevalent followed by D1 (16%), B1 (13.2%), A1 (11.6%) and A0 (5.8%). The 16S rRNA sequencing of all colicinogenic E. coli provided 27 non-clinical variants of E. coli that can be further explored for their probiotic properties to minimize risk of gut diseases.