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A major challenge in evolutionary biology is explaining how populations navigate rugged fitness landscapes without getting trapped on local optima. One idea illustrated by adaptive dynamics theory is that as populations adapt, their newly enhanced capacities to exploit resources alter fitness payoffs and restructure the landscape in ways that promote speciation by opening new adaptive pathways. While there have been indirect tests of this theory, to our knowledge none have measured how fitness landscapes deform during adaptation, or test whether these shifts promote diversification. Here, we achieve this by studying bacteriophage [Formula: see text], a virus that readily speciates into co-existing receptor specialists under controlled laboratory conditions. We use a high-throughput gene editing-phenotyping technology to measure [Formula: see text]'s fitness landscape in the presence of different evolved-[Formula: see text] competitors and find that the fitness effects of individual mutations, and their epistatic interactions, depend on the competitor. Using these empirical data, we simulate [Formula: see text]'s evolution on an unchanging landscape and one that recapitulates how the landscape deforms during evolution. [Formula: see text] heterogeneity only evolves in the shifting landscape regime. This study provides a test of adaptive dynamics, and, more broadly, shows how fitness landscapes dynamically change during adaptation, potentiating phenomena like speciation by opening new adaptive pathways.

Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, we present a high-throughput infection method in a microplate reader, where the growth dynamics of the infected culture is measured using the optical density (OD). We find that the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers over nine orders of magnitude and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model allows us to infer the phage growth rate, which is a function of the phage-cell encounter rate, latent period, and burst size. Adding antibiotic selection provides the ability to measure the rate of host lysogenization. Using this method, we found that when E. coli growth slows down, the lytic growth rate of lambda phages decreases, and the propensity for lysogeny increases, demonstrating how host physiology influences the viral developmental program. Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, the authors present a high-throughput method that can be used to estimate the number of phages in a sample, by measuring growth dynamics of the infected culture in a microplate reader. [ABSTRACT FROM AUTHOR]

Bacteriophage infection, a pivotal process in microbiology, initiates with the phage's tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system's biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development. Here, Ge et al use cryo-electron microscopy to resolve the structure of the bacteriophage lambda tail in complex with its LamB receptor from Shigella sonnei and shed light on the conformational changes that the phage tail fiber undergoes in response to binding. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Single-strand annealing (SSA) is initiated when a double strand break (DSB) occurs between two flanking repeated sequences, resulting in a deletion that leaves a single copy of the repeat. We studied budding yeast strains carrying two 200-bp URA3 sequences separated by 2.6 kb of spacer DNA (phage lambda) in which a site-specific DSB can be created by HO or Cas9 endonucleases. Repeat-mediated deletion requires removal of long 3'-ended single-stranded tails (flaps) by Rad1-Rad10 with the assistance of Msh2-Msh3, Saw1 and Slx4. A natural 3% divergence of unequally spaced heterologies between these repeats (designated F and A) causes a significant reduction in the frequency of SSA repair. This decrease is caused by heteroduplex rejection in which mismatches (MMs) in the annealed intermediate are recognized by the MutS (Msh2 and Msh6) components of the MM repair (MMR) pathway coupled to unwinding of the duplex by the Sgs1-Rmi1-Top3 helicase. MutL homologs, Mlh1-Pms1 (MutL), are not required for rejection but play their expected role in mismatch correction. Remarkably, heteroduplex rejection is very low in strains where the divergent repeats were immediately adjacent (Tailless strains) and the DSB was induced by Cas9. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. DNA sequencing analysis of SSA products from the FA Tailed strain showed a gradient of correction favoring the sequence opposite each 3' end of the annealed strand. Mismatches located in the center of the repair intermediate were corrected by Msh2-Msh6 mediated mismatch correction, while correction of MMs at the extremity of the SSA intermediate often appears to use a different mechanism, possibly by 3' nonhomologous tail removal that includes part of the homologous sequence. In contrast, in FA Tailless strains there was a uniform repair of the MMs across the repeat. A distinctive pattern of correction was found in the absence of MSH2, in both Tailed and Tailless strains, different from the spectrum seen in a msh3Δ msh6Δ double mutant. Previous work has shown that SSA is Rad51-independent but dependent on the strand annealing activity of Rad52. However Rad52 becomes dispensable in a Tailless construct where the DSB is induced by Cas9 or in transformation of a plasmid where SSA occurs in the absence of nonhomologous tails. Author summary: Deletions between dispersed, divergent, repeated sequences are frequently found among cancer mutations. We have studied deletion formation by single-strand annealing (SSA) between naturally divergent 200 bp repeats in budding yeast. SSA was initiated by creating a site-specific double-strand break (DSB) in the region between the two repeats, either using HO endonuclease or guide RNA-directed Cas9. In this process, the ends are resected 5' to 3', leaving long 3'-ended single-stranded sequences that can anneal. After annealing any 3'-ended nonhomologous tails are clipped off by the Rad1-Rad10 endonuclease. The presence of nonhomologous tails triggers a Msh2-Msh3-dependent and Sgs1-dependent heteroduplex rejection when there are 7 heterologies within the 200-bp repeats being annealed, whereas a "tailless" construct is not rejected. These results suggest that the presence of nonhomologous tails strongly stimulates heteroduplex rejection in SSA. Mismatch repair within the heteroduplex is Msh2-Msh6-dependent; however, a msh2 deletion proved have a spectrum of mismatch repair different from a msh3 msh6 double mutant, implying that Msh2 may have some additional role when the intermediates have nonhomologous 3' tails. "Tailed" repeats yielded a gradient of repair that favored sequences in the "left" repeat, but "Tailless" SSA yielded a uniform correction of all 7 heterologies in the 200-bp region. A cluster of repeats near the left end of the sequence provokes correction of these markers in a Msh6-independent fashion; this correction is not attributable to 3' to 5' exonuclease activity of DNA polymerase δ and may reflect instances where part of the homologous sequence itself is clipped off by Rad1-Rad10. The presence or absence of nonhomologous tails also affected the requirement for the Rad52 strand annealing protein. Both in the chromosomal SSA assay and in a plasmid assay, Rad52 was largely dispensable when there were no nonhomologous tails after strand annealing. [ABSTRACT FROM AUTHOR]

Siphophages have a long, flexible, and noncontractile tail that connects to the capsid through a neck. The phage tail is essential for host cell recognition and virus–host cell interactions; moreover, it serves as a channel for genome delivery during infection. However, the in situ high-resolution structure of the neck–tail complex of siphophages remains unknown. Here, we present the structure of the siphophage lambda "wild type," the most widely used, laboratory-adapted fiberless mutant. The neck–tail complex comprises a channel formed by stacked 12-fold and hexameric rings and a 3-fold symmetrical tip. The interactions among DNA and a total of 246 tail protein molecules forming the tail and neck have been characterized. Structural comparisons of the tail tips, the most diversified region across the lambda and other long-tailed phages or tail-like machines, suggest that their tail tip contains conserved domains, which facilitate tail assembly, receptor binding, cell adsorption, and DNA retaining/releasing. These domains are distributed in different tail tip proteins in different phages or tail-like machines. The side tail fibers are not required for the phage particle to orient itself vertically to the surface of the host cell during attachment. This study shows that the tail tips, the most diversified region across bacteriophage lambda and other long-tailed phages (or tail-like machines), contain conserved domains, which facilitate tail assembly, receptor binding, cell adsorption, and DNA retention/release. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) causes a wide range of hospital and community-acquired infections worldwide. MRSA is associated with worse clinical outcomes that can lead to multiple organ failure, septic shock, and death, making timely diagnosis of MRSA infections very crucial. In the present work, we develop a method that enables the positive enrichment of bacteria from spiked whole blood using protein coated magnetic beads, followed by their lysis, and detection by a real-time multiplex PCR directly. The assay targeted bacterial 16S rRNA, S. aureus (spa) and methicillin resistance (mecA). In addition, an internal control (lambda phage) was added to determine the assay's true negative. To validate this assay, staphylococcal and non-staphylococcal bacterial strains were used. The three-markers used in this study were detected as expected by monomicrobial and poly-microbial models of the S. aureus and coagulase-negative staphylococci (CoNS). The thermal cycling completed within 30 mins, delivering 100% specificity. The detection LoD of the pre-processing step was ∼ 1 CFU/mL from 2-5mL of whole blood and that of PCR was ∼ 1pg of NA. However, the combined protocol led to a lower detection limit of 100–1000 MRSA CFUs/mL. The main issue with the method developed is in the pre-processing of blood which will be the subject of our future study. [ABSTRACT FROM AUTHOR]

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.

Two novel P. protegens bacteriophages PseuP_222 and Pseu_224 and their host P. protegens CEMTC 4060 were isolated from the same sample (Inya river, Siberia). Both phages have siphovirus morphology and belong to lambdoid phages. Comparative genome analysis revealed a low nucleotide and amino acid sequence similarity of PseuP_222 and PseuP_224 between themselves, and between them and other lambdoid phages. Bioinformatics analysis indicated that PseuP_222 and PseuP_224 are members of a genetically diverse group of phages of environmental Pseudomonas spp.; this group is distant from a large group of P. aeruginosa phages. In phylogenetic trees, the positioning of the terminase large subunits, major capsid proteins, tail tape measure proteins, and CI-like repressors of PseuP_222 and PseuP_224 were remote and changed relative to those of the Escherichia lambda phage and lambdoid phages of Pseudomonas spp. However, the nucleoid-associated protein NdpA/YejK and P5-like structural protein from both phages showed high similarity and were not found in lambda phage and other lambdoid phages of Pseudomonas spp. Substantial divergences of the PseuP_222 and PseuP_224 genomes and proteomes indicated that the evolutionary history of these phages was mostly independent and they probably began to use one host only recently. [ABSTRACT FROM AUTHOR]

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.

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.

Consequently, the phage that "wins" the competition (pink phage in the cartoon) will produce more virions than the other coresident phage (blue phage in the cartoon). Indeed, experiments show that only one temperate phage is predominantly recovered following SOS induction of a polylysogen, presumably the winner of the phage-phage competition [[33]]. Phages are also frequently bacterial parasites, and, consequently, bacteria are under the pervasive threat of infection by phages that can exploit resources for continued propagation and, moreover, that can kill host bacteria in response to particular conditions. Advances in high-throughput culturing, metagenomic sequencing, and genome assembly techniques are revealing new intricacies concerning the nature of phage-host and phage-phage interactions in real-world settings [[18]-[23]]. [Extracted from the article]

Retrons were described in 1984 as DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA). It would not be until 2020 that a function was shown for retrons, when compelling evidence was presented that retrons activate an abortive infection pathway in response to bacteriophage (phage) infection. When infected with the virulent mutant of the phage lambda, λVIR, and to a lesser extent, other phages, a retron designated Ec48 is activated, the Escherichia coli bearing this retron element dies, and the infecting phage is lost. With the aid of a mathematical model, we explore the a priori conditions under which retrons will protect bacterial populations from predation by phage and the conditions under which retron-bearing bacteria will evolve in populations without this element. Using isogenic E. coli with and without Ec48 and λVIR, we estimated the parameters of our model and tested the hypotheses generated from our analysis of its properties. Our models and experiments demonstrate that cells expressing a retron-mediated abortive infection system can protect bacterial populations. Our results demonstrate that retron bearing bacteria only have a competitive advantage under a limited set of conditions. [ABSTRACT FROM AUTHOR]

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution. CRISPR-Cas immune systems provide immunity against viruses using RNA-guided endonucleases like Cas9 and Cas12a. Viruses can evade these Cas endonucleases through the evolution of mutants that block cleavage by creating mismatches between the guiding RNA and the viral DNA. We show that although Cas12a can tolerate some mismatches, the presence and locations of these mismatches strongly influences the outcomes of further viral evolution. [ABSTRACT FROM AUTHOR]

The most significant difference between bacteriophages functionally and ecologically is whether they are purely lytic (virulent) or temperate. Virulent phages can only be transmitted horizontally by infection, most commonly with the death of their hosts. Temperate phages can also be transmitted horizontally, but upon infection of susceptible bacteria, their genomes can be incorporated into that of their host's as a prophage and be transmitted vertically in the course of cell division by their lysogenic hosts. From what we know from studies with the temperate phage Lambda and other temperate phages, in laboratory culture, lysogenic bacteria are protected from killing by the phage coded for by their prophage by immunity; where upon infecting lysogens, the free temperate phage coded by their prophage is lost. Why are lysogens not only resistant but also immune to the phage coded by their prophage since immunity does not confer protection against virulent phages? To address this question, we used a mathematical model and performed experiments with temperate and virulent mutants of the phage Lambda in laboratory culture. Our models predict and experiments confirm that selection would favor the evolution of resistant and immune lysogens, particularly if the environment includes virulent phage that shares the same receptors as the temperate. To explore the validity and generality of this prediction, we examined 10 lysogenic Escherichia coli from natural populations. All 10 were capable of forming immune lysogens, but their original hosts were resistant to the phage coded by their prophage. [ABSTRACT FROM AUTHOR]

Xenotransplantation may compensate the limited number of human allografts for transplantation using pigs as organ donors. Porcine endogenous retroviruses inherit infectious potential if pig cells, tissues, or organs were transplanted to immunosuppressed human recipients. Particularly, ecotropic PERV-C that could recombine with PERV-A to highly replication-competent human-tropic PERV-A/C should be excluded from pig breeds designed for xenotransplantation. Because of their low proviral background, SLAD/D (SLA, swine leukocyte antigen) haplotype pigs are potential candidates as organ donors as they do not bear replication-competent PERV-A and -B, even if they carry PERV-C. In this work, we characterized their PERV-C background isolating a full-length PERV-C proviral clone number 561 from a SLAD/D haplotype pig genome displayed in a bacteriophage lambda library. The provirus truncated in env due to cloning in lambda was complemented by PCR, and the recombinants were functionally characterized, con- firming an increased infectivity in vitro compared to other PERV-C. Recombinant clone PERV-C(561) was chromosomally mapped by its 59-proviral flanking sequences. Full-length PCR using 59-and 39-flanking primers specific to the PERV-C(561) locus verified that this specific SLAD/D haplotype pig harbors at least one full-length PERV-C provirus. The chromosomal location is different from that of the previously described PERV-C(1312) provirus, which was derived from the porcine cell-line MAX-T. The sequence data presented here provide further knowledge about PERV-C infectivity and contribute to targeted knockout in order to generate PERV-C-free founder animals. IMPORTANCE Yucatan SLAD/D haplotype miniature swine are candidates as organ donors for xenotransplantation. A full-length replication-competent PERV-C provirus was characterized. The provirus was chromosomally mapped in the pig genome. In vitro, the virus showed increased infectivity compared to other functional PERV-C isolates. Data may be used for targeted knockout to generate PERV-C free founder animals. [ABSTRACT FROM AUTHOR]

Many viruses employ ATP-powered motors for genome packaging. We combined genetic, biochemical, and single-molecule techniques to confirm the predicted Walker-B ATP-binding motif in the phage λ motor and to investigate the roles of the conserved residues. Most changes of the conserved hydrophobic residues resulted in >107-fold decrease in phage yield, but we identified nine mutants with partial activity. Several were cold-sensitive, suggesting that mobility of the residues is important. Single-molecule measurements showed that the partially active A175L exhibits a small reduction in motor velocity and increase in slipping, consistent with a slowed ATP binding transition, whereas G176S exhibits decreased slipping, consistent with an accelerated transition. All changes to the conserved D178, predicted to coordinate Mg2+•ATP, were lethal except conservative change D178E. Biochemical interrogation of the inactive D178N protein found no folding or assembly defects and near-normal endonuclease activity, but a ∼200-fold reduction in steady-state ATPase activity, a lag in the single-turnover ATPase time course, and no DNA packaging, consistent with a critical role in ATP-coupled DNA translocation. Molecular dynamics simulations of related enzymes suggest that the aspartate plays an important role in enhancing the catalytic activity of the motor by bridging the Walker motifs and precisely contributing its charged group to help polarize the bound nucleotide. Supporting this prediction, single-molecule measurements revealed that change D178E reduces motor velocity without increasing slipping, consistent with a slowed hydrolysis step. Our studies thus illuminate the mechanistic roles of Walker-B residues in ATP binding, hydrolysis, and DNA translocation by this powerful motor.

Localized arrays of proteins cooperatively assemble onto chromosomes to control DNA activity in many contexts. Binding cooperativity is often mediated by specific protein-protein interactions, but cooperativity through DNA structure is becoming increasingly recognized as an additional mechanism. During the site-specific DNA recombination reaction that excises phage λ from the chromosome, the bacterial DNA architectural protein Fis recruits multiple λ-encoded Xis proteins to the attR recombination site. Here, we report X-ray crystal structures of DNA complexes containing Fis + Xis, which show little, if any, contacts between the two proteins. Comparisons with structures of DNA complexes containing only Fis or Xis, together with mutant protein and DNA binding studies, support a mechanism for cooperative protein binding solely by DNA allostery. Fis binding both molds the minor groove to potentiate insertion of the Xis β-hairpin wing motif and bends the DNA to facilitate Xis-DNA contacts within the major groove. The Fis-structured minor groove shape that is optimized for Xis binding requires a precisely positioned pyrimidine-purine base-pair step, whose location has been shown to modulate minor groove widths in Fis-bound complexes to different DNA targets.

Precision genome editing accelerates the discovery of the genetic determinants of phenotype and the engineering of novel behaviors in organisms. Advances in DNA synthesis and recombineering have enabled high-throughput engineering of genetic circuits and biosynthetic pathways via directed mutagenesis of bacterial chromosomes. However, the highest recombination efficiencies have to date been reported in persistent mutator strains, which suffer from reduced genomic fidelity. The absence of inducible transcriptional regulators in these strains also prevents concurrent control of genome engineering tools and engineered functions. Here, we introduce a new recombineering platform strain, BioDesignER, which incorporates (i) a refactored λ-Red recombination system that reduces toxicity and accelerates multi-cycle recombination, (ii) genetic modifications that boost recombination efficiency, and (iii) four independent inducible regulators to control engineered functions. These modifications resulted in single-cycle recombineering efficiencies of up to 25% with a 7-fold increase in recombineering fidelity compared to the widely used recombineering strain EcNR2. To facilitate genome engineering in BioDesignER, we have curated eight context--neutral genomic loci, termed Safe Sites, for stable gene expression and consistent recombination efficiency. BioDesignER is a platform to develop and optimize engineered cellular functions and can serve as a model to implement comparable recombination and regulatory systems in other bacteria.

The model of T4 phage, Lambda phage, and E. coli is often used in research on virus-host interactions. This paper reports for the first time the thermodynamic driving force of biosynthesis, catabolism and metabolism for the three organisms, on the M9 medium. Moreover, the influence of activities of nutrients and metabolic products is analyzed. All three organisms were found to have very similar Gibbs energies of metabolism. Moreover, since they share the same catabolism, their Gibbs energies of catabolism are identical. However, Gibbs energies of biosynthesis differ. The calculated thermodynamic properties have been used to explain the coexistence of both bacteria and phages in a dynamic equilibrium in natural ecosystems. [ABSTRACT FROM AUTHOR]

Site-specific recombinase Int mediates integration of the bacteriophage λ genome into the Escherichia coli chromosome. Integration occurs once the Int tetramer, assisted by the integration host factor IHF, forms the intasome, a higher order structure, within which Int, a heterobivalent protein, interacts with two nonhomologous DNA sequences: the core recombination sites and the accessory arm sites. The binding to these sites is mediated by the catalytic C-terminal domain (CTD) and the regulatory N-terminal domain (NTD) of Int, respectively. Within Int, the NTD can activate or inhibit the recombination activity of the CTD depending on whether the NTD is bound to the arm sites. The CTD alone cannot mediate recombination, and even when the NTD and the CTD are mixed together as individual polypeptides, the NTD cannot trigger recombination in the CTD. In this work, we set to determine what modifications can unlock the recombination activity in the CTD alone and how the CTD can be modified to respond to recombination-triggering signals from the NTD. For this, we performed a series of genetic analyses, which showed that a single mutation that stabilizes the CTD on DNA, E174K, allows the CTD to recombine the core DNA sequences. When the NTD is paired with the CTD (E174K) that also bears a short polypeptide from the C terminus of the NTD, the resulting binary Int can recombine arm-bearing substrates. Our results provide insights into the molecular basis of the regulation of the Int activity and suggest how binary recombinases of the integrase type can be engineered. [ABSTRACT FROM AUTHOR]

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIR is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIR in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility-a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria-their "existence conditions."

A number of transgenic animal models and mutation detection systems have been developed for mutagenicity testing of carcinogens in mammalian cells. Of these, transgenic mice and the Lambda (λ) Select cII Mutation Detection System have been employed for mutagenicity experiments by many research groups worldwide. Here, we describe a detailed protocol for the Lambda Select cII mutation assay, which can be applied to cultured cells of transgenic mice/rats or the corresponding animals treated with a chemical/physical agent of interest. The protocol consists of the following steps: (1) isolation of genomic DNA from the cells or organs/tissues of transgenic animals treated in vitro or in vivo, respectively, with a test compound; (2) recovery of the lambda shuttle vector carrying a mutational reporter gene (i.e., cII transgene) from the genomic DNA; (3) packaging of the rescued vectors into infectious bacteriophages; (4) infecting a host bacteria and culturing under selective conditions to allow propagation of the induced cII mutations; and (5) scoring the cII-mutants and DNA sequence analysis to determine the cII mutant frequency and mutation spectrum, respectively.

Genetic switches frequently include DNA loops secured by proteins. Recent studies of the lambda bacteriophage repressor (CI), showed that this arrangement in which the protein links two sets of three operators separated by approximately 2.3 kbp, optimizes both the stability and dynamics of DNA loops, compared to an arrangement with just two sets of two operators. Because adjacent dimers interact pairwise, we hypothesized that the odd number of operators in each set of the lambda regulatory system might have evolved to allow for semi-specific, pair-wise interactions that add stability to the loop while maintaining it dynamic. More generally, additional CI dimers may bind non-specifically to flanking DNA sequences making the genetic switch more sensitive to CI concentration. Here, we tested this hypothesis using spectroscopic and imaging approaches to study the binding of the lambda repressor (CI) dimer protein to DNA fragments. For fragments with only one operator and a short flanking sequence, fluorescence correlation spectroscopy measurements clearly indicated the presence of two distinct DNA-CI complexes; one is thought to have a non-specifically bound CI dimer on the flanking sequence. Scanning force micrographs of CI bound to DNA with all six operators revealed wild-type or mutant proteins bound at operator positions. The number of bound, wild-type proteins increased with CI concentration and was larger than expected for strictly specific binding to operators. In contrast, a mutant that fails to oligomerize beyond a dimer, D197G, only bound to operators. These data are evidence that CI cooperativity promotes oligomerization that extends from operator sites to influence the thermodynamics and kinetics of CI-mediated looping.

We present hybrid system-based gene regulatory network models for lambda, HK022, and Mu bacteriophages together with dynamics analysis of the modeled networks. The proposed lambda phage model LPH2 is based on an earlier work and incorporates more recent biological assumptions about the underlying gene regulatory mechanism, HK022, and Mu phage models are new. All three models provide accurate representations of experimentally observed lytic and lysogenic behavioral cycles. Importantly, the models also imply that lysis and lysogeny are the only stable behaviors that can occur in the modeled networks. In addition, the models allow to derive switching conditions that irrevocably lead to either lytic or lysogenic behavioral cycle as well as constraints that are required for their biological feasibility. For LPH2 model the feasibility constraints place two mutually independent requirements on comparative order of cro and cI protein binding site affinities. However, HK022 model, while broadly similar, does not require any of these constraints. Biologically very different lysis–lysogeny switching mechanism of Mu phage is also accurately reproduced by its model. In general the results show that hybrid system model (HSM) hybrid system framework can be successfully applied to modeling small (∼ 2 0 gene) regulatory networks and used for comprehensive analysis of model dynamics and stable behavior regions. [ABSTRACT FROM AUTHOR]

Seamless DNA vectors derived from bacterial plasmids are devoid of bacterial genetic elements and represent attractive alternatives for biomedical applications including DNA vaccines. Larger scale production of seamless vectors employs engineered Escherichia coli strains in order to enable tightly regulated expression of site-specific DNA recombinases which precisely delete unwanted sequences from bacterial plasmids. As a novel component of a developing lambda integrase genome editing platform, we describe here strain MG1655-ISC as a means to easily produce different scales of seamless vectors, ranging in size from a few hundred base pairs to more than ten kilo base pairs. Since we employed an engineered lambda integrase that is able to efficiently recombine pairs of DNA crossover sites that differ in sequence, the resulting seamless vectors will be useful for subsequent genome editing in higher eukaryotes to accommodate variations in target site sequences. Future inclusion of single cognate sites for other genome targeting systems could enable modularity. These features, together with the demonstrated simplicity of in vivo seamless vector production, add to their utility in the biomedical space. [ABSTRACT FROM AUTHOR]

After the ejection of viral DNA into the host cytoplasm, the temperate bacteriophage (phage) lambda integrates a cascade of expressions from various regulatory genes, coupled with DNA replication, to commit to a decision between lysis and lysogeny. Higher multiplicity of infection (MOI) greatly shifts the decision toward the lysogenic pathway. However, how the phage separates the MOI from replicated viral DNA during lysis-lysogeny decision-making is unclear. To quantitatively understand the role of viral DNA replication, we constructed a reporter system facilitating the visualization of individual copies of phage DNA throughout the phage life cycle, along with the lysis-lysogeny reporters. We showed that intracellular viral DNA diverges between the lytic and lysogenic pathways from the early phase of the infection cycle, mostly due to the synchronization and success of DNA injection, as well as the competition for replication resources, rather than the replication rate. Strikingly, we observed two distinct replication patterns during lysogenization and surprisingly heterogeneous integration kinetics, which advances our understanding of temperate phage life cycles. We revealed that the weak repression function of Cro is critical for an optimal replication rate and plays a crucial role in establishing stable lysogens., Importance: Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.

ABSTRACT Optimality models have a checkered history in evolutionary biology. While optimality models have been successful in providing valuable insight into the evolution of a wide variety of biological traits, a common objection is that optimality models are overly simplistic and ignore organismal genetics. We revisit evolutionary optimization in the context of a major bacteriophage life history trait, lysis time. Lysis time refers to the period spanning phage infection of a host cell and its lysis, whereupon phage progenies are released. Lysis time, therefore, directly determines phage fecundity assuming progeny assembly does not exhaust host resources prior to lysis. Noting that previous tests of lysis time optimality rely on batch culture, we implemented a quasi-continuous culture system to observe productivity of a panel of isogenic phage λ genotypes differing in lysis time. We report that under our experimental conditions, λ phage productivity is maximized around optimal lysis times ranging from 60 to 100 min, and λ wildtype strain falls within this range. It would appear that natural selection on phage λ lysis time uncovered a set of genetic solutions that optimized progeny production in its ecological milieu relative to alternative genotypes. We discuss this finding in light of recent results that lysis time variation is also minimized in the strains with lysis times closer to the λ wild-type strain. IMPORTANCE Optimality theory presents the idea that natural selection acts on organismal traits to produce genotypes that maximize organismal survival and reproduction. As such, optimality theory is a valuable tool in guiding our understanding of the genetic constraints and tradeoffs organisms experience as their genotypes are selected to produce optimal solutions to biological problems. However, optimality theory is often critiqued as being overly simplistic and ignoring the roles of chance and history in the evolution of organismal traits. We show here that the wild-type genotype of a popular laboratory model organism, the bacteriophage λ, produces a phenotype for a major life history trait, lysis time, that maximizes the reproductive success of bearers of that genotype relative to other possible genotypes. This result demonstrates, as is rarely shown experimentally, that natural selection can achieve optimal solutions to ecological challenges.

The developmental choice made by temperate phages, between cell death (lysis) and viral dormancy (lysogeny), is influenced by the relative abundance of viruses and hosts in the environment. The paradigm for this abundance-driven decision is phage lambda of E. coli , whose propensity to lysogenize increases with the number of viruses coinfecting the same bacterium. It is believed that lambda uses this number to infer whether phages or bacteria outnumber each other. However, this interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). Here, we show this premise to be faulty. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, performed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of phage entry decrease with the number of adsorbed phages. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage attachment, as evidenced by compromised membrane integrity and loss of membrane potential. The dependence of entry dynamics on the surrounding medium results in a strong impact on the infection outcome, while the protracted entry of coinfecting phages increases the heterogeneity in infection outcome at a given MOI. Our findings in lambda, and similar results we obtained for phages T5 and P1, demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection. [Display omitted] • Adsorption and genome entry of individual phages were followed in real time • The efficiency of phage entry decreases at higher multiplicity of infection (MOI) • Phage entry is impeded at high MOI by adsorption-induced perturbation to the cell • MOI-dependent phage entry impacts the choice between lysis and lysogeny Nguyen et al. find that when multiple lambda phages infect the same E. coli cell, they slow or even prevent each other from entering the cell. Similar behavior is observed in other phages. The impeded entry at high multiplicities, caused by phage-induced perturbation to the host cell, impacts the choice between viral reproduction and dormancy. [ABSTRACT FROM AUTHOR]

The COVID‐19 pandemic has necessitated the need to reliably detect the presence of viral genomes in human clinical samples. The most accurate viral tests involve the use of qPCR. Thus, it is important for students to understand the mechanism to detect viral genomes by qPCR including critical qPCR controls and how to properly interpret qPCR data. Herein, we describe a 2‐week undergraduate laboratory to detect a viral genome in a human DNA sample. The strategy follows a SARS‐CoV‐2 qPCR test in numerous ways. Students are provided isolated DNA representing a mock human patient sample, and determine if a viral genome (bacteriophage lambda) is present using qPCR. A battery of control samples and patient pooling strategies are utilized. The laboratory exercise is successful based on high rates of student success in detecting viral genomes, pre‐quiz and post‐quiz assessments focusing on viral qPCR testing, and positive student comments. [ABSTRACT FROM AUTHOR]

Bacteriophage λ is a temperate virus infecting the bacterium Escherichia coli. Temperate phages have the ability to form lysogens where the prophage is maintained in the infected bacterium in a largely quiescent state. The lysogen becomes immune to superinfection by λ by blocking the development of the superinfecting phage. Here we report the λ lysogen not only protected itself from killing by λ phages but also protected λ-sensitive bacteria in mixed culture. This protection required that the lysogen was able to adsorb the superinfecting λ phages. The protection was also sensitive to the growth state of the mixed culture, and the λ lysogen lost efficiency in protecting λ-sensitive bacteria as it stopped growing. A mutant of the λ tail protein, λJ, was not subject to this loss of protection. Adsorption of λ having the wild-type J protein but not the mutant λJ protein to E. coli was inhibited by interference with bacterial energy metabolism. The last observation suggests wild-type λ preferentially infects bacteria with competent energy metabolism. [ABSTRACT FROM AUTHOR]

During the assembly of many viruses, a powerful ATP-driven motor translocates DNA into a preformed procapsid. A Walker-A "P-loop" motif is proposed to coordinate ATP binding and hydrolysis with DNA translocation. We use genetic, biochemical, and biophysical techniques to survey the roles of P-loop residues in bacteriophage lambda motor function. We identify 55 point mutations that reduce virus yield to below detectable levels in a highly sensitive genetic complementation assay and 33 that cause varying reductions in yield. Most changes in the predicted conserved residues K76, R79, G81, and S83 produce no detectable yield. Biochemical analyses show that R79A and S83A mutant proteins fold, assemble, and display genome maturation activity similar to wild-type (WT) but exhibit little ATPase or DNA packaging activity. Kinetic DNA cleavage and ATPase measurements implicate R79 in motor ring assembly on DNA, supporting recent structural models that locate the P-loop at the interface between motor subunits. Single-molecule measurements detect no translocation for K76A and K76R, while G81A and S83A exhibit strong impairments, consistent with their predicted roles in ATP binding. We identify eight residue changes spanning A78-K84 that yield impaired translocation phenotypes and show that Walker-A residues play important roles in determining motor velocity, pausing, and processivity. The efficiency of initiation of packaging correlates strongly with motor velocity. Frequent pausing and slipping caused by changes A78V and R79K suggest that these residues are important for ATP alignment and coupling of ATP binding to DNA gripping. Our findings support recent structural models implicating the P-loop arginine in ATP hydrolysis and mechanochemical coupling.

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

Functional characterization of metagenomic DNA often involves expressing heterologous DNA in genetically tractable microorganisms such as Escherichia coli. Functional expression of heterologous genes can suffer from limitations due to the lack of recognition of foreign promoters or presence of intrinsic terminators on foreign DNA between a vector-based promoter and the transcription start site. Anti-terminator proteins are a possible solution to overcome this limitation. When bacteriophage lambda infects E. coli, it relies on the host transcription machinery to transcribe and express phage DNA. Lambda anti-terminator protein Q (λQ) regulates the expression of late-genes of phage lambda. E. coli RNA polymerase recognizes the PR' promoter on the lambda genome and forms a complex with λQ, to overcome the terminator tR'. Here we show the use of λQ to efficiently transcribe a capsular polysaccharide cluster, cps3, from Lactobacillus plantarum containing intrinsic terminators in Escherichia coli. In addition, we expand the use of anti-terminator λQ in Pseudomonas putida. The results show ~ fivefold higher expression of a fluorescent reporter located ~ 12.5kbp downstream from the promoter, when the transcription is driven by PR' promoter in presence of λQ compared to a lac promoter. These results suggest that λQ could be used in metabolic engineering to enhance expression of heterologous DNA. [ABSTRACT FROM AUTHOR]

Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4). • Tape measure proteins are conserved essential components of long-tailed phages. • Tape measure proteins require Tail Assembly Chaperones (TACs) for their assembly. • TACs and tape measure proteins for the T4-like family of phages have been discovered. • gp26 and gp51 of E. coli phage T4 are TACs. • A new mechanism for generating TACs distinct from translational frameshifting is described. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Genome packaging in many dsDNA phages requires a series of precisely coordinated actions of two phage-coded proteins, namely, large terminase (TerL) and small terminase (TerS) with DNA and ATP, and with each other. Despite the strict functional conservation, TerL and TerS homologs exhibit large sequence variations. We investigated the sequence variability across eight phage types and observed a coevolutionary framework wherein the genealogy of TerL homologs mirrored that of the corresponding TerS homologs. Furthermore, a high purifying selection observed (dN/dS«1) indicated strong structural constraints on both TerL and TerS, and identify coevolving residues in TerL and TerS of phage T4 and lambda. Using the highly coevolving (correlation coefficient of 0.99) TerL and TerS of phage N4, we show that their biochemical features are similar to the phylogenetically divergent phage λ terminases. We also demonstrate using the Surface Plasma Resonance (SPR) technique that phage N4 TerL transiently interacts with TerS. • The large and small terminase proteins of double-stranded DNA phages exhibit strict functional conservation despite high sequence variation. • The large and small terminase proteins are highly coevolving, and their genes are under high purifying selection. • Large terminase and small terminase proteins of N4 phage show strong but transient interaction. [ABSTRACT FROM AUTHOR]

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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. [ABSTRACT FROM AUTHOR]

CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

The stringent response modulators, guanosine tetraphosphate (ppGpp) and protein DksA, bind RNA polymerase (RNAP) and regulate gene expression to adapt bacteria to different environmental conditions. Here, we use Atomic Force Microscopy and in vitro transcription assays to study the effects of these modulators on the conformation and stability of the open promoter complex (RPo) formed at the rrnA P1, rrnB P1, its discriminator (dis) variant and λ pR promoters. In the absence of modulators, RPo formed at these promoters show different extents of DNA wrapping which correlate with the position of UP elements. Addition of the modulators affects both DNA wrapping and RPo stability in a promoter-dependent manner. Overall, the results obtained under different conditions of ppGpp, DksA and initiating nucleotides (iNTPs) indicate that ppGpp allosterically prevents the conformational changes associated with an extended DNA wrapping that leads to RPo stabilization, while DksA interferes directly with nucleotide positioning into the RNAP active site. At the iNTPs-sensitive rRNA promoters ppGpp and DksA display an independent inhibitory effect, while at the iNTPs-insensitive pR promoter DksA reduces the effect of ppGpp in accordance with their antagonistic role.

BackgroundKlebsiella pneumoniae is a leading cause of hospital-acquired urinary tract infections and pneumonia worldwide, and is responsible for many cases of pyogenic liver abscess among diabetic patients in Asia. A defining characteristic of this pathogen is the presence of a thick, exterior capsule that has been reported to play a role in biofilm formation and to protect the organism from threats such antibiotics and host immune challenge.FindingsWe constructed two knockout mutants of K. pneumoniae to investigate how perturbations to capsule biosynthesis alter the cellular phenotype. In the first mutant, we deleted the entire gene cluster responsible for biosynthesis of the extracellular polysaccharide capsule. In the second mutant, we deleted the capsule export subsystem within this cluster. We find that both knockout mutants have lower amounts of capsule but produce greater amounts of biofilm. Moreover, one of the two mutants abolishes fimbriae expression as well.ConclusionsThese results are expected to provide insight into the interaction between capsule biosynthesis, biofilm formation, and fimbriae expression in this organism.