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.
OBJECTIVE: 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 has a double-stranded DNA genome and a long, flexible, non-contractile tail encoded by a contiguous block of 11 genes downstream of the head genes. The tail allows host recognition and delivery of viral DNA from the head shell to the cytoplasm of the infected cell. Here, we present a high-resolution structure of the tail complex of bacteriophage lambda determined by cryoelectron microscopy. Most component proteins of the lambda tail were determined at the atomic scale. The structure sheds light on the molecular organization of the extensively studied tail of bacteriophage lambda.

In physiology and synthetic biology, it can be advantageous to introduce a gene into a naive bacterial host under conditions in which all cells receive the gene and remain fully functional. This cannot be done by the usual chemical transformation and electroporation methods due to low efficiency and cell death, respectively. However, in vivo packaging of plasmids (called cosmids) that contain the 223 bp cos site of phage λ results in phage particles that contain concatemers of the cosmid that can be transduced into all cells of a culture. An historical shortcoming of in vivo packaging of cosmids was inefficient packaging and contamination of the particles containing cosmid DNA with a great excess of infectious λ phage. Manipulation of the packaging phage and the host has eliminated these shortcomings resulting in particles that contain only cosmid DNA. Plasmids have the drawback that they can be difficult to remove from cells. Plasmids with conditional replication provide a means to \"cure\" plasmids from cells. The prevalent conditional replication plasmids are temperature-sensitive plasmids, which are cured at high growth temperature. However, inducible replication plasmids are in some cases more useful, especially since this approach has been applied to plasmids having diverse replication and compatibility properties.

Lambdoid (or Lambda-like) phages are a group of related temperate phages that can infect Escherichia coli and other gut bacteria. A key characteristic of these phages is their mosaic genome structure, which served as the basis for the \'modular genome hypothesis\'. Accordingly, lambdoid phages evolve by transferring genomic regions, each of which constitutes a functional unit. Nevertheless, it is unknown which genes are preferentially transferred together and what drives such co-transfer events. Here we aim to characterize genome modularity by studying co-transfer of genes among 95 distantly related lambdoid (pro-)phages. Based on gene content, we observed that the genomes cluster into 12 groups, which are characterized by a highly similar gene content within the groups and highly divergent gene content across groups. Highly similar proteins can occur in genomes of different groups, indicating that they have been transferred. About 26 % of homologous protein clusters in the four known operons (i.e. the early left, early right, immunity and late operon) engage in gene transfer, which affects all operons to a similar extent. We identified pairs of genes that are frequently co-transferred and observed that these pairs tend to be near one another on the genome. We find that frequently co-transferred genes are involved in related functions and highlight interesting examples involving structural proteins, the cI repressor and Cro regulator, proteins interacting with DNA, and membrane-interacting proteins. We conclude that epistatic effects, where the functioning of one protein depends on the presence of another, play an important role in the evolution of the modular structure of these genomes.

Laboratory-generated hybrids between phage λ and related phages played a seminal role in establishment of the λ model system, which, in turn, served to develop many of the foundational concepts of molecular biology, including gene structure and control. Important λ hybrids with phages 21 and 434 were the earliest of such phages. To understand the biology of these hybrids in full detail, we determined the complete genome sequences of phages 21 and 434. Although both genomes are canonical members of the λ-like phage family, they both carry unsuspected bacterial virulence gene types not previously described in this group of phages. In addition, we determined the sequences of the hybrid phages λ imm21, λ imm434, and λ h434 imm21. These sequences show that the replacements of λ DNA by nonhomologous segments of 21 or 434 DNA occurred through homologous recombination in adjacent sequences that are nearly identical in the parental phages. These five genome sequences correct a number of errors in published sequence fragments of the 21 and 434 genomes, and they point out nine nucleotide differences from Sanger\'s original λ sequence that are likely present in most extant λ strains in laboratory use today. We discuss the historical importance of these hybrid phages in the development of fundamental tenets of molecular biology and in some of the earliest gene cloning vectors. The 434 and 21 genomes reinforce the conclusion that the genomes of essentially all natural λ-like phages are mosaics of sequence modules from a pool of exchangeable segments.

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.

DNA recombination, replication, and repair are intrinsically interconnected processes. From viruses to humans, they are ubiquitous and essential to all life on Earth. Single-strand annealing homologous DNA recombination is a major mechanism for the repair of double-stranded DNA breaks. An exonuclease and an annealase work in tandem, forming a complex known as a two-component recombinase. Redβ annealase and λ-exonuclease from phage lambda form the archetypal two-component recombinase complex. In this short review article, we highlight some of the in vitro studies that have led to our current understanding of the lambda recombinase system. We synthesize insights from more than half a century of research, summarizing the state of our current understanding. From this foundation, we identify the gaps in our knowledge and cast an eye forward to consider what the next 50 years of research may uncover.

During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study-sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection-are broadly applicable to other phage-bacterium systems.IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems.

Critical behaviour pervades scientific disciplines as diverse as geology, economy or sociology. The critical behaviour of cell control systems is an open issue whose role has not yet been fully explored. The control of the expression of lambda phage DNA in the host cell can be classified as a system with critical behaviour. Lambda phage is a virus that infects Escherichia coli. Its core genes maintain one of two states; lysogeny or lysis. Current knowledge of the lambda phage genetic network allows to build a computational model of transcriptional control of the genes involved in the lytic-lysogenic switch and to simulate the temporal changes of their expression. Here, we focused on the computational simulation of these gene expressions to demonstrate critical behaviour of the system.

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