From DNA to lytic proteins: transcription and translation of the bacteriophage T5 holin/endolysin operon.

The analysis of transcriptional activity of the bacteriophage T5 hol/endo operon conducted in the paper revealed a strong constitutive promoter recognized by E. coli RNA polymerase and a transcription initiation point of the operon. It was also shown that the only translational start codon for holin was a non-canonical TTG. Translation initiation regions (TIRs) of both genes of the operon (hol and endo) were further analyzed using chimeric constructs, in which parts of the hol/endo regulatory regions were fused with the gene of a reporter protein (EGFP). It was found that TIR of hol was 20 times less effective than that of endo. As it turned out, the level of EGFP production was influenced by the composition of the constructs and the type of the hol start codon. Apparently, the translational suppression of holin's accumulation and posttranslational activation of endolysin by Ca2+ are the main factors ensuring the proper timing of the host cell lysis by bacteriophage T5. The approach based on the use of chimeric constructs proposed in the paper can be recommended for studying other native or artificial operons of any complexity: analyzing the impacts of separate DNA regions, as well as their coupled effect, on the processes of transcription and translation of recombinant protein(s).

Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

Bacteriophage defends murine gut from Escherichia coli invasion via mucosal adherence.

Bacteriophage are sophisticated cellular parasites that can not only parasitize bacteria but are increasingly recognized for their direct interactions with mammalian hosts. Phage adherence to mucus is known to mediate enhanced antimicrobial effects in vitro. However, little is known about the therapeutic efficacy of mucus-adherent phages in vivo. Here, using a combination of in vitro gastrointestinal cell lines, a gut-on-a-chip microfluidic model, and an in vivo murine gut model, we demonstrated that a E. coli phage, øPNJ-6, provided enhanced gastrointestinal persistence and antimicrobial effects. øPNJ-6 bound fucose residues, of the gut secreted glycoprotein MUC2, through domain 1 of its Hoc protein, which led to increased intestinal mucus production that was suggestive of a positive feedback loop mediated by the mucus-adherent phage. These findings extend the Bacteriophage Adherence to Mucus model into phage therapy, demonstrating that øPNJ-6 displays enhanced persistence within the murine gut, leading to targeted depletion of intestinal pathogenic bacteria.

Phage predation accelerates the spread of plasmid-encoded antibiotic resistance.

Phage predation is generally assumed to reduce microbial proliferation while not contributing to the spread of antibiotic resistance. However, this assumption does not consider the effect of phage predation on the spatial organization of different microbial populations. Here, we show that phage predation can increase the spread of plasmid-encoded antibiotic resistance during surface-associated microbial growth by reshaping spatial organization. Using two strains of the bacterium Escherichia coli, we demonstrate that phage predation slows the spatial segregation of the strains during growth. This increases the number of cell-cell contacts and the extent of conjugation-mediated plasmid transfer between them. The underlying mechanism is that phage predation shifts the location of fastest growth from the biomass periphery to the interior where cells are densely packed and aligned closer to parallel with each other. This creates straighter interfaces between the strains that are less likely to merge together during growth, consequently slowing the spatial segregation of the strains and enhancing plasmid transfer between them. Our results have implications for the design and application of phage therapy and reveal a mechanism for how microbial functions that are deleterious to human and environmental health can proliferate in the absence of positive selection.

Antibacterial Potential of Non-Tailed Icosahedral Phages Alone and in Combination with Antibiotics.

Non-tailed icosahedral phages belonging to families Fiersviridae (phages MS2 and Qbeta), Tectiviridae (PRD1) and Microviridae (phiX174) have not been considered in detail so far as potential antibacterial agents. The aim of the study was to examine various aspects of the applicability of these phages as antibacterial agents. Antibacterial potential of four phages was investigated via bacterial growth and biofilm formation inhibition, lytic spectra determination, and phage safety examination. The phage phiX174 was combined with different classes of antibiotics to evaluate potential synergistic interactions. In addition, the incidence of phiX174-insensitive mutants was analyzed. The results showed that only phiX174 out of four phages tested against their corresponding hosts inhibited bacterial growth for > 90% at different multiplicity of infection and that only this phage considerably prevented biofilm formation. Although all phages show the absence of potentially undesirable genes, they also have extremely narrow lytic spectra. The synergism was determined between phage phiX174 and ceftazidime, ceftriaxone, ciprofloxacin, macrolides, and chloramphenicol. It was shown that the simultaneous application of agents is more effective than successive treatment, where one agent is applied first. The analysis of the appearance of phiX174 bacteriophage-insensitive mutants showed that mutations occur with a frequency of 10-3. The examined non-tailed phages have a limited potential for use as antibacterial agents, primarily due to a very narrow lytic spectrum and the high frequency of resistant mutants appearance, but Microviridae can be considered in the future as biocontrol agents against susceptible strains of E. coli in combinations with conventional antimicrobial agents.

Non-replicative phage particles delivering CRISPR-Cas9 to target major blaCTX-M variants.

Cluster regularly interspaced short palindromic repeats and CRISPR associated protein 9 (CRISPR-Cas9) is a promising tool for antimicrobial re-sensitization by inactivating antimicrobial resistance (AMR) genes of bacteria. Here, we programmed CRISPR-Cas9 with common spacers to target predominant blaCTX-M variants in group 1 and group 9 and their promoter in an Escherichia coli model. The CRISPR-Cas9 was delivered by non-replicative phagemid particles from a two-step process, including insertion of spacer in CRISPR and construction of phagemid vector. Spacers targeting blaCTX-M promoters and internal sequences of blaCTX-M group 1 (blaCTX-M-15 and -55) and group 9 (blaCTX-M-14, -27, -65, and -90) were cloned into pCRISPR and phagemid pRC319 for spacer evaluation and phagemid particle production. Re-sensitization and plasmid clearance were mediated by the spacers targeting internal sequences of each group, resulting in 3 log10 to 4 log10 reduction of the ratio of resistant cells, but not by those targeting the promoters. The CRISPR-Cas9 delivered by modified ΦRC319 particles were capable of re-sensitizing E. coli K-12 carrying either blaCTX-M group 1 or group 9 in a dose-dependent manner from 0.1 to 100 multiplicity of infection (MOI). In conclusion, CRISPR-Cas9 system programmed with well-designed spacers targeting multiple variants of AMR gene along with a phage-based delivery system could eliminate the widespread blaCTX-M genes for efficacy restoration of available third-generation cephalosporins by reversal of resistance in bacteria.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

Characterization of vB_SalS_PSa2, a T5-like Demerecviridae bacteriophage, and its potential use in food matrices.

This study characterizes a newly isolated Demerecviridae phage, named vB_SalS_PSa2, belonging to the phage T5 group. The main variations between vB_SalS_PSa2 and T5 concern structural proteins related to morphology and host recognition. vB_SalS_PSa2 is infective to 19 out of the 25 tested Salmonella enterica (including the rare "Sendai" and "Equine" serotypes) and Escherichia coli isolates, most of them being multidrug resistant. vB_SalS_PSa2 displayed good thermal stability (4-60 °C) and broad pH stability (4.0-12.0). It also exhibited antibacterial activity against S. enterica sv. Paratyphi A Enb50 at 4 °C in milk during the whole tested period (5 d), and for 3-6 h at both 25 and 37 °C. Furthermore, vB_SalS_PSa2 was able to inhibit biofilm formation and to show degradation activity on mature biofilms of E. coli K12 and S. enterica sv. Paratyphi Enb50 in both LB and milk. Altogether, these results indicate that phage vB_SalS_PSa2 is a valuable candidate for controlling foodborne S. enterica and E. coli pathogens.

In vitro evaluation of two novel Escherichia bacteriophages against multiple drug resistant avian pathogenic Escherichia coli.

BACKGROUND: In recent years, there has been a growing interest in phage therapy as an effective therapeutic tool against colibacillosis caused by avian pathogenic Escherichia coli (APEC) which resulted from the increasing number of multidrug resistant (MDR) APEC strains. METHODS: In the present study, we reported the characterization of a new lytic bacteriophage (Escherichia phage AG- MK-2022. Basu) isolated from poultry slaughterhouse wastewater. In addition, the in vitro bacteriolytic activity of the newly isolated phage (Escherichia phage AG- MK-2022. Basu) and the Escherichia phage VaT-2019a isolate PE17 (GenBank: MK353636.1) were assessed against MDR- APEC strains (n = 100) isolated from broiler chickens with clinical signs of colibacillosis. RESULTS: Escherichia phage AG- MK-2022. Basu belongs to the Myoviridae family and exhibits a broad host range. Furthermore, the phage showed stability under a wide range of temperatures, pH values and different concentrations of NaCl. Genome analysis of the Escherichia phage AG- MK-2022. Basu revealed that the phage possesses no antibiotic resistance genes (ARGs), mobile genetic elements (MGEs), and any E. coli virulence associated genes. In vitro bacterial challenge tests demonstrated that two phages, the Escherichia phage VaT-2019a isolate PE17 and the Escherichia phage AG- MK-2022. Basu exhibited high bactericidal activity against APEC strains and lysed 95% of the tested APEC strains. CONCLUSIONS: The current study findings indicate that both phages could be suggested as safe biocontrol agents and alternatives to antibiotics for controlling MDR-APEC strains isolated from broilers.

Temperate phage-antibiotic synergy across antibiotic classes reveals new mechanism for preventing lysogeny.

A recent demonstration of synergy between a temperate phage and the antibiotic ciprofloxacin suggested a scalable approach to exploiting temperate phages in therapy, termed temperate phage-antibiotic synergy, which specifically interacted with the lysis-lysogeny decision. To determine whether this would hold true across antibiotics, we challenged Escherichia coli with the phage HK97 and a set of 13 antibiotics spanning seven classes. As expected, given the conserved induction pathway, we observed synergy with classes of drugs known to induce an SOS response: a sulfa drug, other quinolones, and mitomycin C. While some ß-lactams exhibited synergy, this appeared to be traditional phage-antibiotic synergy, with no effect on the lysis-lysogeny decision. Curiously, we observed a potent synergy with antibiotics not known to induce the SOS response: protein synthesis inhibitors gentamicin, kanamycin, tetracycline, and azithromycin. The synergy results in an eightfold reduction in the effective minimum inhibitory concentration of gentamicin, complete eradication of the bacteria, and, when administered at sub-optimal doses, drastically decreases the frequency of lysogens emerging from the combined challenge. However, lysogens exhibit no increased sensitivity to the antibiotic; synergy was maintained in the absence of RecA; and the antibiotic reduced the initial frequency of lysogeny rather than selecting against formed lysogens. Our results confirm that SOS-inducing antibiotics broadly result in temperate-phage-specific synergy, but that other antibiotics can interact with temperate phages specifically and result in synergy. This is the first report of a means of chemically blocking entry into lysogeny, providing a new means for manipulating the key lysis-lysogeny decision.IMPORTANCEThe lysis-lysogeny decision is made by most bacterial viruses (bacteriophages, phages), determining whether to kill their host or go dormant within it. With over half of the bacteria containing phages waiting to wake, this is one of the most important behaviors in all of biology. These phages are also considered unusable for therapy because of this behavior. In this paper, we show that many antibiotics bias this behavior to "wake" the dormant phages, forcing them to kill their host, but some also prevent dormancy in the first place. These will be important tools to study this critical decision point and may enable the therapeutic use of these phages.

Centroid of the bacterial growth curves: a metric to assess phage efficiency.

Phage replication can be studied using various approaches, including measuring the optical density (OD) of a bacterial culture in a liquid medium in the presence of phages. A few quantitative methods are available to measure and compare the efficiency of phages by using a single index based on the analysis of OD curves. However, these methods are not always applicable to non-canonical OD curves. Using the concept of center of area (centroid), we developed a metric called Centroid Index (CI), sensitive to the trend of the growth curves (OD distribution) including bacterial regrowth, which is not considered by the methods already available. We also provide a user-friendly software to facilitate the calculation of CI. This method offers an alternative and more precise way to determine phage efficiency by considering the OD variations over time, which may help in the selection of phages for biocontrol applications.

Correlation between the development of phage resistance and the original antibiotic resistance of host bacteria under the co-exposure of antibiotic and bacteriophage.

Bacteriophages (phages) are viruses capable of regulating the proliferation of antibiotic resistant bacteria (ARB). However, phages that directly cause host lethality may quickly select for phage resistant bacteria, and the co-evolutionary trade-offs under varying environmental conditions, including the presence of antibiotics, remains unclear as to their impact on phage and antibiotic resistance. Here, we report the emergence of phage resistance in three distinct E. coli strains with varying resistance to ß-lactam antibiotics, treated with different ampicillin (AMP) concentrations. Hosts exhibiting stronger antibiotic resistance demonstrated a higher propensity to develop and maintain stable phage resistance. When exposed to polyvalent phage KNT-1, the growth of AMP-sensitive E. coli K12 was nearly suppressed within 18 h, while the exponential growth of AMP-resistant E. coli TEM and super-resistant E. coli NDM-1 was delayed by 12 h and 8 h, respectively. The mutation frequency and mutated colony count of E. coli NDM-1 were almost unaffected by co-existing AMP, whereas for E. coli TEM and K12, these metrics significantly decreased with increasing AMP concentration from 8 to 50 µg/mL, becoming unquantifiable at 100 µg/mL. Furthermore, the fitness costs of phage resistance mutation and its impact on initial antibiotic resistance in bacteria were further examined, through analyzing AMP susceptibility, biofilm formation and EPS secretion of the isolated phage resistant mutants. The results indicated that acquiring phage resistance could decrease antibiotic resistance, particularly for hosts lacking strong antibiotic resistance. The ability of mutants to form biofilm contributes to antibiotic resistance, but the correlation is not entirely positive, while the secretion of extracellular polymeric substance (EPS), especially the protein content, plays a crucial role in protecting the bacteria from both antibiotic and phage exposure. This study explores phage resistance development in hosts with different antibiotic resistance and helps to understand the limitations and possible solutions of phage-based technologies.

DNA glycosylases provide antiviral defence in prokaryotes.

Bacteria have adapted to phage predation by evolving a vast assortment of defence systems1. Although anti-phage immunity genes can be identified using bioinformatic tools, the discovery of novel systems is restricted to the available prokaryotic sequence data2. Here, to overcome this limitation, we infected Escherichia coli carrying a soil metagenomic DNA library3 with the lytic coliphage T4 to isolate clones carrying protective genes. Following this approach, we identified Brig1, a DNA glycosylase that excises α-glucosyl-hydroxymethylcytosine nucleobases from the bacteriophage T4 genome to generate abasic sites and inhibit viral replication. Brig1 homologues that provide immunity against T-even phages are present in multiple phage defence loci across distinct clades of bacteria. Our study highlights the benefits of screening unsequenced DNA and reveals prokaryotic DNA glycosylases as important players in the bacteria-phage arms race.

The dsRNA isolated from Escherichia coli infected with the MS2 bacteriophage induces the production of interferons.

Viral infections pose a significant threat to public health, and the production of interferons represents one of the most critical antiviral innate immune responses of the host. Consequently, the screening and identification of compounds or reagents that induce interferon production are of paramount importance. This study commenced with the cultivation of host bacterium 15,597, followed by the infection of Escherichia coli with the MS2 bacteriophage. Utilizing the J2 capture technique, a class of dsRNA mixtures (MS2+15,597) was isolated from the E. coli infected with the MS2 bacteriophage. Subsequent investigations were conducted on the immunostimulatory activity of the MS2+15,597 mixture. The results indicated that the dsRNA mixtures (MS2+15,597) extracted from E. coli infected with the MS2 bacteriophage possess the capability to activate innate immunity, thereby inducing the production of interferon-ß. These dsRNA mixtures can activate the RIG-I and TLR3 pattern recognition receptors, stimulating the expression of interferon stimulatory factors 3/7, which in turn triggers the NF-κB signaling pathway, culminating in the cellular production of interferon-ß to achieve antiviral effects. This study offers novel insights and strategies for the development of broad-spectrum antiviral drugs, potentially providing new modalities for future antiviral therapies.

Involvement of the Cell Division Protein DamX in the Infection Process of Bacteriophage T4.

The molecular mechanism of how the infecting DNA of bacteriophage T4 passes from the capsid through the bacterial cell wall and enters the cytoplasm is essentially unknown. After adsorption, the short tail fibers of the infecting phage extend from the baseplate and trigger the contraction of the tail sheath, leading to a puncturing of the outer membrane by the tail tip needle composed of the proteins gp5.4, gp5 and gp27. To explore the events that occur in the periplasm and at the inner membrane, we constructed T4 phages that have a modified gp27 in their tail tip with a His-tag. Shortly after infection with these phages, cells were chemically cross-linked and solubilized. The cross-linked products were affinity-purified on a nickel column and the co-purified proteins were identified by mass spectrometry, and we found that predominantly the inner membrane proteins DamX, SdhA and PpiD were cross-linked. The same partner proteins were identified when purified gp27 was added to Escherichia coli spheroplasts, suggesting a direct protein-protein interaction.

K1 capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

AIMS: Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. METHODS AND RESULTS: Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. CONCLUSIONS: A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

Bacteriophage LHE83 targeting OmpA as a receptor exhibited synergism with spectinomycin against Escherichia coli.

Understanding the characteristics of bacteriophages is crucial for the optimization of phage therapy. In this study, the biological and genomic characteristics of coliphage LHE83 were determined and its synergistic effects with different types of antibiotics against E. coli E82 were investigated. Phage LHE83 displayed a contractile tail morphology and had a titer of 3.02 × 109 pfu/mL at an optimal MOI of 0.01. Meanwhile, phage LHE83 exhibited good physical and chemical factors tolerance. The 1-step growth analysis revealed a latent period of approx. 10 min with a burst size of 87 pfu/infected cell. Phage LHE83 belongs to the genus Dhakavirus. Its genome consists of 170,464 bp with a 40% GC content, and a total of 268 Open Reading Frames (ORF) were predicted with no detected virulent or resistant genes. ORF 213 was predicted to encode the receptor binding protein (RBP) and confirmed by the antibody-blocking assay. Furthermore, a phage-resistant strain E. coli E82R was generated by co-culturing phage LHE83 with E. coli E82. Genomic analysis revealed that OmpA served as the receptor for phage LHE83, which was further confirmed by phage adsorption assay using E. coli BL21ΔOmpA, E. coli BL21ΔOmpA: OmpA and E. coli BL21:OmpA strains. Additionally, a synergistic effect was observed between phage LHE83 and spectinomycin against the drug-resistant strain E. coli E82. These results provide a theoretical basis for understanding the interactions between phages, antibiotics, and host bacteria, which can assist in the clinical application of phages and antibiotics against drug-resistant bacteria.

The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins.

RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.

How Dedicated Ribosomes Translate a Leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.

Assembly-mediated activation of the SIR2-HerA supramolecular complex for anti-phage defense.

SIR2-HerA, a bacterial two-protein anti-phage defense system, induces bacterial death by depleting NAD+ upon phage infection. Biochemical reconstitution of SIR2, HerA, and the SIR2-HerA complex reveals a dynamic assembly process. Unlike other ATPases, HerA can form various oligomers, ranging from dimers to nonamers. When assembled with SIR2, HerA forms a hexamer and converts SIR2 from a nuclease to an NAD+ hydrolase, representing an unexpected regulatory mechanism mediated by protein assembly. Furthermore, high concentrations of ATP can inhibit NAD+ hydrolysis by the SIR2-HerA complex. Cryo-EM structures of the SIR2-HerA complex reveal a giant supramolecular assembly up to 1 MDa, with SIR2 as a dodecamer and HerA as a hexamer, crucial for anti-phage defense. Unexpectedly, the HerA hexamer resembles a spiral staircase and exhibits helicase activities toward dual-forked DNA. Together, we reveal the supramolecular assembly of SIR2-HerA as a unique mechanism for switching enzymatic activities and bolstering anti-phage defense strategies.