Structural morphing in the viral portal vertex of bacteriophage lambda.

The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system. IMPORTANCE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

Bacteriophage lambda site-specific recombination.

The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.

Complex effects of the exo-xis region of the Shiga toxin-converting bacteriophage Φ24B genome on the phage development and the Escherichia coli host physiology.

Lambdoid bacteriophages are excellent models in studies on molecular aspects of virus-host interactions. However, some of them carry genes encoding toxins which are responsible for virulence of pathogenic strains of bacteria. Shiga toxin-converting bacteriophages (Stx phages) encode Shiga toxins that cause virulence of enterohemorrhagic Escherichia coli (EHEC), and their effective production depends on Stx prophage induction. The exo-xis region of the lambdoid phage genome consists of genes which are dispensable for the phage multiplication under laboratory conditions; however, they might modulate the virus development. Nevertheless, their exact effects on the phage and host physiology remained unclear. Here, we present results of complex studies on the role of the exo-xis region of bacteriophage Φ24B, one of Stx2b phages. Transcriptomic analyses, together with proteomic and metabolomic studies, provided the basis for understanding the functions of the exo-xis region. Genes from this region promoted lytic development of the phage over lysogenization. Moreover, expression of the host genes coding for DnaK, DnaJ, GrpE, and GroELS chaperones was impaired in the cells infected with the Δexo-xis phage mutant, relative to the wild-type virus, corroborating the conclusion about lytic development promotion by the exo-xis region. Proteomic and metabolomic analyses indicated also modulation of gad and nrf operons, and levels of amino acids and acylcarnitines, respectively. In conclusion, the exo-xis region controls phage propagation and host metabolism by influencing expression of different phage and bacterial genes, directing the virus to the lytic rather than lysogenic developmental mode.

AT-specific DNA visualization revisits the directionality of bacteriophage λ DNA ejection.

In this study, we specifically visualized DNA molecules at their AT base pairs after in vitro phage ejection. Our AT-specific visualization revealed that either end of the DNA molecule could be ejected first with a nearly 50% probability. This observation challenges the generally accepted theory of Last In First Out (LIFO), which states that the end of the phage λ DNA that enters the capsid last during phage packaging is the first to be ejected, and that both ends of the DNA are unable to move within the extremely condensed phage capsid. To support our observations, we conducted computer simulations that revealed that both ends of the DNA molecule are randomized, resulting in the observed near 50% probability. Additionally, we found that the length of the ejected DNA by LIFO was consistently longer than that by First In First Out (FIFO) during in vitro phage ejection. Our simulations attributed this difference in length to the stiffness difference of the remaining DNA within the phage capsid. In conclusion, this study demonstrates that a DNA molecule within an extremely dense phage capsid exhibits a degree of mobility, allowing it to switch ends during ejection.

Characterization of a Primordial Major Capsid-Scaffolding Protein Complex in Icosahedral Virus Shell Assembly.

Capsid assembly pathways are strongly conserved in the complex dsDNA viruses, where major capsid proteins (MCP) self-assemble into icosahedral procapsid shells, chaperoned by a scaffolding protein. Without a scaffold, the capsid proteins aggregate and form aberrant structures. This, coupled with the rapid co-polymerization of MCP and scaffolding proteins, has thwarted characterization of the earliest steps in shell assembly. Here we interrogate the structure and biophysical properties of a soluble, assembly-deficient phage lambda major capsid protein, MCP(W308A). The mutant protein is folded, soluble to high concentrations and binds to the scaffolding protein in an apparent SP2:MCP(W308A)1 stoichiometry but does not assemble beyond this initiating complex. The MCP(W308A) crystal structure was solved to 2.7 Å revealing the canonical HK97 fold in a "pre-assembly" conformation featuring the conserved N-arm and E-loops folded into the body of the protein. Structural, biophysical and computational analyses suggest that MCP(W308A) is thermodynamically trapped in this pre-assembly conformation precluding self-association interactions required for shell assembly. A model is described wherein dynamic interactions between MCP proteins play an essential role in high fidelity viral shell assembly. Scaffold-chaperoned MCP polymerization is a strongly conserved process in all the large dsDNA viruses and our results provide insight into this primordial complex in solution and have broad biological significance in our understanding of virus assembly mechanisms.

Bacteriophage self-counting in the presence of viral replication.

When host cells are in low abundance, temperate bacteriophages opt for dormant (lysogenic) infection. Phage lambda implements this strategy by increasing the frequency of lysogeny at higher multiplicity of infection (MOI). However, it remains unclear how the phage reliably counts infecting viral genomes even as their intracellular number increases because of replication. By combining theoretical modeling with single-cell measurements of viral copy number and gene expression, we find that instead of hindering lambda's decision, replication facilitates it. In a nonreplicating mutant, viral gene expression simply scales with MOI rather than diverging into lytic (virulent) and lysogenic trajectories. A similar pattern is followed during early infection by wild-type phage. However, later in the infection, the modulation of viral replication by the decision genes amplifies the initially modest gene expression differences into divergent trajectories. Replication thus ensures the optimal decision-lysis upon single-phage infection and lysogeny at higher MOI.

Viral recombination systems limit CRISPR-Cas targeting through the generation of escape mutations.

CRISPR-Cas systems provide immunity to bacteria by programing Cas nucleases with RNA guides that recognize and cleave infecting viral genomes. Bacteria and their viruses each encode recombination systems that could repair the cleaved viral DNA. However, it is unknown whether and how these systems can affect CRISPR immunity. Bacteriophage λ uses the Red system (gam-exo-bet) to promote recombination between related phages. Here, we show that λ Red also mediates evasion of CRISPR-Cas targeting. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by phage Exo-Beta, which promotes the generation of mutations within the CRISPR target sequence. Red recombination is strikingly more efficient than the host's RecBCD-RecA in the production of large numbers of phages that escape CRISPR targeting. These results reveal a role for Red-like systems in the protection of bacteriophages against sequence-specific nucleases, which may facilitate their spread across viral genomes.

Bacteriophage λ RexA and RexB functions assist the transition from lysogeny to lytic growth.

The CI and Cro repressors of bacteriophage λ create a bistable switch between lysogenic and lytic growth. In λ lysogens, CI repressor expressed from the PRM promoter blocks expression of the lytic promoters PL and PR to allow stable maintenance of the lysogenic state. When lysogens are induced, CI repressor is inactivated and Cro repressor is expressed from the lytic PR promoter. Cro repressor blocks PRM transcription and CI repressor synthesis to ensure that the lytic state proceeds. RexA and RexB proteins, like CI, are expressed from the PRM promoter in λ lysogens; RexB is also expressed from a second promoter, PLIT , embedded in rexA. Here we show that RexA binds CI repressor and assists the transition from lysogenic to lytic growth, using both intact lysogens and defective prophages with reporter genes under the control of the lytic PL and PR promoters. Once lytic growth begins, if the bistable switch does return to the immune state, RexA expression lessens the probability that it will remain there, thus stabilizing the lytic state and activation of the lytic PL  and PR  promoters. RexB modulates the effect of RexA and may also help establish phage DNA replication as lytic growth ensues.

CroSR391 , an ortholog of the λ Cro repressor, plays a major role in suppressing polVR391 -dependent mutagenesis.

When subcloned into low-copy-number expression vectors, rumAB, encoding polVR391 (RumA'2 B), is best characterized as a potent mutator giving rise to high levels of spontaneous mutagenesis in vivo. This is in dramatic contrast to the poorly mutable phenotype when polVR391 is expressed from the native 88.5 kb R391, suggesting that R391 expresses cis-acting factors that suppress the expression and/or the activity of polVR391 . Indeed, we recently discovered that SetRR391 , an ortholog of λ cI repressor, is a transcriptional repressor of rumAB. Here, we report that CroSR391 , an ortholog of λ Cro, also serves as a potent transcriptional repressor of rumAB. Levels of RumA are dependent upon an interplay between SetRR391 and CroSR391 , with the greatest reduction of RumA protein levels observed in the absence of SetRR391 and the presence of CroSR391 . Under these conditions, CroSR391 completely abolishes the high levels of mutagenesis promoted by polVR391 expressed from low-copy-number plasmids. Furthermore, deletion of croSR391 on the native R391 results in a dramatic increase in mutagenesis, indicating that CroSR391 plays a major role in suppressing polVR391 mutagenesis in vivo. Inactivating mutations in CroSR391 therefore have the distinct possibility of increasing cellular mutagenesis that could lead to the evolution of antibiotic resistance of pathogenic bacteria harboring R391.

Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance.

The evolution of antibiotic-resistant bacteria threatens to become the leading cause of worldwide mortality. This crisis has renewed interest in the practice of phage therapy. Yet, bacteria's capacity to evolve resistance may debilitate this therapy as well. To combat the evolution of phage resistance and improve treatment outcomes, many suggest leveraging phages' ability to counter resistance by evolving phages on target hosts before using them in therapy (phage training). We found that in vitro, λtrn, a phage trained for 28 d, suppressed bacteria ∼1,000-fold for three to eight times longer than its untrained ancestor. Prolonged suppression was due to a delay in the evolution of resistance caused by several factors. Mutations that confer resistance to λtrn are ∼100× less common, and while the target bacterium can evolve complete resistance to the untrained phage in a single step, multiple mutations are required to evolve complete resistance to λtrn. Mutations that confer resistance to λtrn are more costly than mutations for untrained phage resistance. Furthermore, when resistance does evolve, λtrn is better able to suppress these forms of resistance. One way that λtrn improved was through recombination with a gene in a defunct prophage in the host genome, which doubled phage fitness. This transfer of information from the host genome is an unexpected but highly efficient mode of training phage. Lastly, we found that many other independently trained λ phages were able to suppress bacterial populations, supporting the important role training could play during phage therapeutic development.

From Bench to Keyboard and Back Again: A Brief History of Lambda Phage Modeling.

Cellular decision making is the process whereby cells choose one developmental pathway from multiple possible ones, either spontaneously or due to environmental stimuli. Examples in various cell types suggest an almost inexhaustible plethora of underlying molecular mechanisms. In general, cellular decisions rely on the gene regulatory network, which integrates external signals to drive cell fate choice. The search for general principles of such a process benefits from appropriate biological model systems that reveal how and why certain gene regulatory mechanisms drive specific cellular decisions according to ecological context and evolutionary outcomes. In this article, we review the historical and ongoing development of the phage lambda lysis-lysogeny decision as a model system to investigate all aspects of cellular decision making. The unique generality, simplicity, and richness of phage lambda decision making render it a constant source ofmathematical modeling-aided inspiration across all of biology. We discuss the origins and progress of quantitative phage lambda modeling from the 1950s until today, as well as its possible future directions. We provide examples of how modeling enabled methods and theory development, leading to new biological insights by revealing gaps in the theory and pinpointing areas requiring further experimental investigation. Overall, we highlight the utility of theoretical approaches both as predictive tools, to forecast the outcome of novel experiments, and as explanatory tools, to elucidate the natural processes underlying experimental data.

Sustained coevolution of phage Lambda and Escherichia coli involves inner- as well as outer-membrane defences and counter-defences.

Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ's ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.

A snapshot of the λ T4rII exclusion (Rex) phenotype in Escherichia coli.

The lambda (λ) T4rII exclusion (Rex) phenotype is defined as the inability of T4rII to propagate in Escherichia coli lysogenized by bacteriophage λ. The Rex system requires the presence of two lambda immunity genes, rexA and rexB, to exclude T4 (rIIA-rIIB) from plating on a lawn of E. coli λ lysogens. The onset of the Rex phenotype by T4rII infection imparts a harsh cellular environment that prevents T4rII superinfection while killing the majority of the cell population. Since the discovery of this powerful exclusion system in 1955 by Seymour Benzer, few mechanistic models have been proposed to explain the process of Rex activation and the physiological manifestations associated with Rex onset. For the first time, key host proteins have recently been linked to Rex, including σE, σS, TolA, and other membrane proteins. Together with the known Rex system components, the RII proteins of bacteriophage T4 and the Rex proteins from bacteriophage λ, we are closer than ever to solving the mystery that has eluded investigators for over six decades. Here, we review the fundamental Rex components in light of this new knowledge.

Controlled expression of lysis gene E by a mutant of the promoter pL of the thermo-inducible λcI857-pL system.

AIMS: To identify a lambda promoter pL mutant that could extend the thermal stability of the thermo-inducible λcI857-pR/pL system and to evaluate the effects of the modified system for the controlled expression of lysis gene E during the production of bacterial ghosts (BGs). METHODS AND RESULTS: The promoter pL mutant was identified by random mutagenesis and site-directed mutagenesis. The results showed that a T â†’ 35C mutation in the pL promoter was responsible for the phenotype alteration. Under the same induction conditions, the lysis rates of the modified lytic system on Escherichia coli and Salmonella enteritidis were significantly lower than that of the control, while the lysis rates of Escherichia coli with the thermo-inducible lytic system were significantly higher than that of S. enteritidis with the corresponding plasmid (P < 0·05). CONCLUSIONS: Increasing the heat stability of the thermo-inducible lytic systems decreased lysis efficiency during the production of BGs. There exist differences in the lysis efficiency of thermo-inducible lytic systems between different bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings enrich current knowledge about modifications to thermo-inducible systems and provide a reference for the application of these modified systems for the production of BGs and controlled gene expression in bacteria.

A Tale of Good Fortune in the Era of DNA.

Two strains of good fortune in my career were to stumble upon the Watson-Gilbert laboratory at Harvard when I entered graduate school in 1964, and to study gene regulation in bacteriophage λ when I was there. λ was almost entirely a genetic item a few years before, awaiting biochemical incarnation. Throughout my career I was a relentless consumer of the work of previous and current generations of λ geneticists. Empowered by this background, my laboratory made contributions in two areas. The first was regulation of early gene transcription in λ, the study of which began with the discovery of the Rho transcription termination factor, and the regulatory mechanism of transcription antitermination by the λ N protein, subjects of my thesis work. This was developed into a decades-long program during my career at Cornell, studying the mechanism of transcription termination and antitermination. The second area was the classic problem of prophage induction in response to cellular DNA damage, the study of which illuminated basic cellular processes to survive DNA damage.

Engineered Fluorescent E. coli Lysogens Allow Live-Cell Imaging of Functional Prophage Induction Triggered inside Macrophages.

Half of the bacteria in the human gut microbiome are lysogens containing integrated prophages, which may activate in stressful immune environments. Although lysogens are likely to be phagocytosed by macrophages, whether prophage activation occurs or influences the outcome of bacterial infection remains unexplored. To study the dynamics of bacteria-phage interactions in living cells-in particular, the macrophage-triggered induction and lysis of dormant prophages in the phagosome-we adopted a tripartite system where murine macrophages engulf E. coli, which are lysogenic with an engineered bacteriophage λ, containing a fluorescent lysis reporter. Pre-induced prophages are capable of lysing the host bacterium and propagating infection to neighboring bacteria in the same phagosome. A non-canonical pathway, mediated by PhoP, is involved with the native λ phage induction inside phagocytosed E. coli. These findings suggest two possible mechanisms by which induced prophages may function to aid the bactericidal activity of macrophages.

Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.

HORMA Domain Proteins and a Trip13-like ATPase Regulate Bacterial cGAS-like Enzymes to Mediate Bacteriophage Immunity.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

Bacteriophage lambda overcomes a perturbation in its host-viral genetic network through mutualism and evolution of life history traits.

An important driver of evolution in viruses is natural selection to optimize the use of their hosts' genetic network. To learn how viruses respond to this pressure, we disrupted the genetic network of Escherichia coli to inhibit replication of its virus, bacteriophage lambda, and then observed how λ evolved to compensate. We deleted E. coli's dnaJ gene, which lambda uses to initiate DNA replication. Lambda partially restored its ability to reproduce with just two adaptive mutations associated with genes J and S. The location of the mutations was unexpected because they were not in genes that directly interact with DnaJ, rather they affected seemingly unrelated life history traits. A nonsynonymous J mutation increased lambda's adsorption rate and an S regulatory mutation delayed lysis timing. Lambda also recovered some of its reproductive potential through intracellular mutualism. This study offers two important lessons: first, viruses can rapidly adapt to disruptive changes in their host's genetic network. Second, organisms can employ mechanisms thought to operate at the population scale, such as evolution of life history traits and social interactions, in order to overcome hurdles at the molecular level. As life science research progresses and new fields become increasingly specialized, these results remind us of the importance of multiscale and interdisciplinary approaches to understand adaptation.