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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).

Summary The lambda phage Red proteins Redα/Redβ/Redγ and Rac prophage RecE/RecT proteins are powerful tools for precise and efficient genetic manipulation but have been limited to only a few prokaryotes. Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET‐like operons, RecETheBDU8, RecEThTJI49 and RecETh1h2eYI23, which were obtained from three different Burkholderia species. Recombineering experiments indicated that RecEThTJI49 and RecETh1h2eYI23 showed higher recombination efficiency compared to RecETheBDU8 in Burkholderia glumae PG1. Furthermore, all of the proteins currently categorized as hypothetical proteins in RecETh1h2eYI23, RecEThTJI49 and RecETheBDU8 may have a positive effect on recombination in B. glumae PG1 except for the h2 protein in RecETh1h2eYI23. Additionally, RecETYI23 combined with exonuclease inhibitors Pluγ or Redγ exhibited equivalent recombination efficiency compared to Redγβα in Escherichia coli, providing potential opportunity of recombineering in other Gram‐negative bacteria for its loose host specificity. Using recombinase‐assisted in situ insertion of promoters, we successfully activated three cryptic non‐ribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. Compound 7 exhibited significant potential anti‐inflammatory activity by inhibiting lipopolysaccharide‐stimulated nitric oxide production in RAW 264.7 macrophages. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol., Here, we report the development and application of a new recombineering system for Burkholderia glumae and Burkholderia plantarii based on three Rac bacteriophage RecET‐like operons, RecETheBDU8, RecEThTJI49, and RecETh1h2eYI23, which were obtained from three different Burkholderia species. Using recombinase‐assisted in situ insertion of promoters, we successfully activated three cryptic nonribosomal peptide synthetase biosynthetic gene clusters in Burkholderia strains, resulting in the generation of a series of lipopeptides that were further purified and characterized. This recombineering system may greatly enhance functional genome research and the mining of novel natural products in the other species of the genus Burkholderia after optimization of a protocol.

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.

Serum deoxyribonuclease I (DNase I) can serve as a functional biomarker for the therapeutic monitoring of acute myocardial infarction and other diseases. Here, we demonstrate that the electrical properties of DNA molecules can be exploited to monitor enzymatic activity. A label-free DNA biosensor for the detection of DNase I activity was devised based on electrochemical impedance spectroscopy (EIS). Multiple lambda phage DNA molecules were immobilized between two electrodes in a polydimethylsiloxane reservoir. An equivalent circuit estimated from the EIS measurement was used to calculate the impedance of DNA molecules between the electrodes. DNase detection was then achieved by measuring the increase in impedance, after DNA cleavage by DNase I. This was assessed by the impedance-increase ratio, defined as Rafter/Rbefore (where Rbefore and Rafter represent the resistance between the electrode-immobilized DNA molecules before and after DNase I treatment, respectively). After treatment with DNase I at a concentration 10−2 unit/μL, a reproducible impedance-increase ratio of approximately 3.3 times was obtained, with a standard deviation of less than 20%. When DNase solutions of various concentrations were introduced, we succeeded in obtaining a definite correlation between DNase concentration and impedance-increase rate, within the range of 10−4 unit/μL to 10−1 unit/μL.

With an increase in the consumption of non-heated fresh food, foodborne shiga toxin-producing Escherichia coli (STEC) has emerged as one of the most problematic pathogens worldwide. Endolysin, a bacteriophage-derived lysis protein, is able to lyse the target bacteria without any special resistance, and thus has been garnering interest as a powerful antimicrobial agent. In this study, rV5-like phage endolysin targeting E. coli O157:H7, named as LysECP26, was identified and purified. This endolysin had a lysozyme-like catalytic domain, but differed markedly from the sequence of lambda phage endolysin. LysECP26 exhibited strong activity with a broad lytic spectrum against various gram-negative strains (29/29) and was relatively stable at a broad temperature range (4°C- 55°C). The optimum temperature and pH ranges of LysECP26 were identified at 37°C-42°C and pH 7- 8, respectively. NaCl supplementation did not affect the lytic activity. Although LysECP26 was limited in that it could not pass the outer membrane, E. coli O157: H7 could be effectively controlled by adding ethylenediaminetetraacetic acid (EDTA) and citric acid (1.44 and 1.14 log CFU/ml) within 30 min. Therefore, LysECP26 may serv as an effective biocontrol agent for gram-negative pathogens, including E. coli O157:H7.

The emphasis on the organization of entities and their activities and interactions has been labeled one of the most distinct contributions of mechanistic philosophy. In this paper I discuss the manner in which the organization of entities and their activities and interactions participates in bringing about phenomena. I present a well-known example from molecular biology-the functioning of the genetic switch in phage lambda-and discuss Marco J. Nathan's notion of causation by concentration. Nathan introduces causation by concentration to account for the irreducible causal role that the concentration ratio between two kinds of proteins possesses in the genetic switch mechanism in phage lambda. I discuss what the irreducibility of this causal role amounts to and provide a mechanistic interpretation of Nathan's causation by concentration; that is, I explain this irreducible causal role as one organizational feature of this mechanism. The paper concludes that biological mechanisms need a causal pluralist framework [similar to Glennan's account in (2009), (2010) and (2017) but slightly modified] where organizational features such as the concentration ratio have a causally relevant role, yet all the causally productive relations occur at the level of entities or individuals.

Significance Enterohemorrhagic E. coli is a significant human pathogen that can cause severe disease due to the release of Shiga toxins. The toxins are encoded within lysogenic bacteriophage and controlled by antitermination of the phage late promoter, PR′. This promoter is always active, but terminated immediately downstream during lysogeny. A byproduct of antitermination regulation is transcription of a short RNA that is thought to be nonfunctional. Here we demonstrate that in Shiga toxin-encoding phages, this short RNA is a Hfq-binding regulatory small RNA. The small RNA represses toxin production threefold under lysogenic conditions and promotes high cell density growth. Lysogenic bacteriophages are highly abundant and our results suggest that antiterminated phage promoters may be a rich source of regulatory RNAs., Enterohemorrhagic Escherichia coli is a significant human pathogen that causes disease ranging from hemorrhagic colitis to hemolytic uremic syndrome. The latter can lead to potentially fatal renal failure and is caused by the release of Shiga toxins that are encoded within lambdoid bacteriophages. The toxins are encoded within the late transcript of the phage and are regulated by antitermination of the PR′ late promoter during lytic induction of the phage. During lysogeny, the late transcript is prematurely terminated at tR′ immediately downstream of PR′, generating a short RNA that is a byproduct of antitermination regulation. We demonstrate that this short transcript binds the small RNA chaperone Hfq, and is processed into a stable 74-nt regulatory small RNA that we have termed StxS. StxS represses expression of Shiga toxin 1 under lysogenic conditions through direct interactions with the stx1AB transcript. StxS acts in trans to activate expression of the general stress response sigma factor, RpoS, through direct interactions with an activating seed sequence within the 5′ UTR. Activation of RpoS promotes high cell density growth under nutrient-limiting conditions. Many phages utilize antitermination to regulate the lytic/lysogenic switch and our results demonstrate that short RNAs generated as a byproduct of this regulation can acquire regulatory small RNA functions that modulate host fitness.

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.

To begin its infection, a bacteriophage first needs to adsorb to cells. The adsorption site on the cell surface may influence viral DNA injection, gene expression and cell-fate development. Here, we study the early steps of the infection cycle of coliphage P1, focusing on their correlation with spatial locations at the single-cell level. By fluorescently labeling P1 virions, we found that P1 shows no spatial preference on cell surface adsorption. In addition, live-cell phage DNA imaging revealed that adsorption sites do not affect the success rate for P1 in injecting its DNA into the cell. Furthermore, the lysis-lysogeny decision of P1 does not depend on the adsorption site, based on fluorescence reporters for the lytic and lysogenic pathways. These findings highlight the different infection strategies used by the two paradigmatic coliphages differ from those found in the paradigmatic phage lambda, highlighting that different infection strategies are used by phages.

SUMMARYWhen 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 due to 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 wildtype 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, lysogeny at higher MOI.

DNA is a polymeric macromolecule that can display a variety of backbone conformations. While the classical B-DNA is a right-handed double helix, Z-DNA is a left-handed helix with a zig-zag orientation. The Z conformation depends upon the base sequence, base modification and supercoiling and is considered to be transient. To determine whether the presence of Z-DNA can be detected immunochemically, the binding of monoclonal and polyclonal anti-Z-DNA antibodies to a panel of natural DNA antigens was assessed by an ELISA using brominated poly(dG-dC) as a control for Z-DNA. As these studies showed, among natural DNA tested (Micrococcus luteus, calf thymus, Escherichiacoli, salmon sperm, lambda phage), micrococcal (MC) DNA showed the highest binding with both anti-Z-DNA preparations, and E. coli DNA showed binding with the monoclonal anti-DNA preparation. The specificity for Z-DNA conformation in MC DNA was demonstrated by an inhibition binding assay. An algorithm to identify propensity to form Z-DNA indicated that DNA from Mycobacterium tuberculosis could form Z-DNA, a prediction confirmed by immunoassay. Together, these findings indicate that anti-Z-DNA antibodies can serve as probes for the presence of Z-DNA in DNA of various species origin and that the content of Z-DNA varies significantly among DNA sources.

Despite growing attention to environmental pollution by microplastics (MP), the effects of MP aging on bacterial horizontal gene transfer (HGT) have not been systematically investigated. Here, we used UV-aged polystyrene microplastics (PS-MPs) to investigate how aging affects antibiotic resistance genes (ARGs) transfer efficiency from various ARG vectors to recipient bacteria. The adsorption capacity of MP20 (20-day UV-aged PS-MPs) towards E. coli (harboring plasmid-borne blaTEM-1), plasmid pET29 (harboring blaNDM-1) and phage lambda (carrying the aphA1 ARG) increased by 6.6-, 5.2- and 8.3-fold, respectively, relative to pristine PS-MPs (MP0), due to increased specific surface area and affinity for these ARG vectors. Moreover, MP20 released more organic compounds (TOC 1.6 mg/g-MP20, versus 0.2 mg/g-MP0 in 4 h) –possibly depolymerization byproducts (verified by GC-MS), which induced intracellular ROS generation, increased cell permeability and upregulated HGT associated genes. Accordingly, MP20 enhanced ARG transfer frequency from E. coli, plasmid pET29 and phage lambda (relative to MP0) by 1.3-, 4.7- and 3.5-fold, respectively. The Bliss independence model infers that higher bacterial adsorption and exposure to chemicals released during MP aging synergistically enhanced ARG transfer. This underscores the need to assess the significance of this overlooked phenomenon to the environmental dissemination of antibiotic resistance and other HGT processes.

In this dissertation we take advantage of the real-time manipulating, high force generating, and small displacement measuring abilities of dual-trap optical tweezers to investigate properties and mechanisms of motor enzymes that act on DNA, including the DNA translocation motor of bacteriophage lambda and the proposed ATP-driven annealing helicase HARP. Through a combined approach of genetic mutagenesis, biochemical analysis, and single-molecule analysis we reveal potential functional roles of the residues residing in the ATP phosphate binding motif (Walker A) of phage lambda’s large terminase subunit. Direct DNA packaging measurements of phages with mutated residues within this motif reveal a range of impaired translocation phenotypes. Analysis of the slipping and pausing exhibited by mutant enzymes suggests that residues A78, R79, and V80 mediate coupling between ATP binding/hydrolysis and DNA binding/translocation while implicating residues A78, R79, V80, and G81 in proper ATP alignment for hydrolysis. The combined findings of each analysis also implicate residue R79 in ADP release and triggering ATP hydrolysis. In other studies, initial investigation into the mechanism of termination of DNA packaging in phage lambda provides evidence against a velocity-monitoring model. A higher resolution optical tweezers system was designed and constructed and a new and improved calibration method was developed and applied to initial high-resolution measurements of DNA translocation. The use of environmental and measurement noise reduction techniques and improved optical alignment procedures have yielded tweezers with approximately nanometer spatial resolution. The calibration technique, which optimizes fits of the worm-like chain model to DNA force-extension curves, yields calibration with at least a 7-fold improvement in accuracy over our prior method. The use of these tools in measuring lambda packaging yield preliminary evidence for discrete translocation steps. Lastly, tweezer measurements of HARP interactions with forked DNA provide supporting evidence that HARP is a forked-DNA binding protein that resists DNA unzipping and is not an unwinding helicase. In addition preliminary measurements suggest that this protein forcibly reanneals DNA, prevents reannealing, resists high forces, slows reannealing in an ATP dependent manner, and is recruited to bind to forked DNA with replication protein A coated single-stranded DNA.

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.

For more than thirty-five years since their discovery, the function of retrons, DNA sequences that code for a reverse transcriptase and a unique single-stranded DNA/RNA hybrid called multicopy single-stranded DNA (msDNA), in bacteria were unknown. Less than two years ago, compelling evidence was presented that retrons can protect bacteria from infections from lytic phage via an abortive infection mechanism. When infected with the virulent mutant of the phage lambda, λVIR, and to a lesser extent other lytic phages, the retron designated Ec48 is activated, the Escherichia coli bearing this element die, and the infecting phage is lost. Using mathematical models and experiments with E. coli and λVIR we tested the hypothesis that Ec48 as an abortive infection system protects populations of bacteria from infection with lytic phage and we also tested the hypothesis that phage-mediated selection is responsible for the evolution and maintenance of retrons. The results of this jointly theoretical and experimental investigation question this ecological role of retrons, and lytic phage as the selective pressure responsible for the evolution and maintenance of retrons in bacterial populations. We found that when confronted with phage, the retrons fail to provide an advantage to bacteria in mixed (retron+ and retron−) populations. Ultimately, bacteria with classical phage resistance are selected over bacteria utilizing retron-mediated abortive infection. Significance Statement For thirty-six years, retrons have been known in bacteria. There have been numerous studies of the molecular biology and distribution of retrons. Recently, retrons have been used for genome editing, and are a tool considered to rival CRISPR-Cas for this purpose. However, only two years ago a function was identified for retrons: protection against infections with lytic bacteriophage. With a combination of mathematical modeling and experiments with retron-encoding E. coli we explore the ability of the retrons to protect bacterial populations from bacteriophage and the selective pressures responsible for their evolution. The results of this jointly theoretical and experimental study question whether protection against phage is the selective force responsible for the evolution and maintenance of retrons in bacterial populations.

Biolistic transformation delivers nucleic acids into plant cells by bombarding the cells with microprojectiles, which are micron-scale, typically gold particles. Despite the wide use of this technique, little is known about its effect on the cell’s genome. We biolistically transformed linear 48-kb phage lambda and two different circular plasmids into rice (Oryza sativa) and maize (Zea mays) and analyzed the results by whole genome sequencing and optical mapping. Although some transgenic events showed simple insertions, others showed extreme genome damage in the form of chromosome truncations, large deletions, partial trisomy, and evidence of chromothripsis and breakage-fusion bridge cycling. Several transgenic events contained megabase-scale arrays of introduced DNA mixed with genomic fragments assembled by nonhomologous or microhomology-mediated joining. Damaged regions of the genome, assayed by the presence of small fragments displaced elsewhere, were often repaired without a trace, presumably by homology-dependent repair (HDR). The results suggest a model whereby successful biolistic transformation relies on a combination of end joining to insert foreign DNA and HDR to repair collateral damage caused by the microprojectiles. The differing levels of genome damage observed among transgenic events may reflect the stage of the cell cycle and the availability of templates for HDR.

A new bacterial genotoxicity detection strain was constructed, in which the cell lysis gene of SRRz from a lambda phage was controlled by a new designed SOS responsive element, designated as Escherichia coli BL21/pUC-PST. The biosensor responded only after 0.5 h contact with mutagens and the changes in cell culture turbidity could be easily differentiated with the naked eyes from the control sample. This SOS/SRRz system presented a dose-dependent manner to five model DNA-damaging agents with an improved detection sensitivity. The limits of detection (LODs) were 0.026 μM for mitomycin C, 320.4 μM for azinphos-methyl, 34.4 μM for methyl methanesulfonate, 4.6 μM for dithianone and 6.0 μM for dichlofluanid, which were much lower than previously reported. By performing binary and ternary mixture experiments, the toxic equivalency concept was validated in the E. coli SOS/SRRz system by comparison with bioanalytical equivalent concentrations (BEQ) and overall toxic equivalent concentration (TEQmixture) using Cr(VI) as the reference compound. Pearson analysis indicated that a strong correlation existed between the TEQmixture and BEQ values. Thus the TEQmixture could be presented as the Cr(VI) equivalent concentration from its dose–effect lysis profiles for the environmental sample. The proposed genotoxicity reporter strain allows for easier qualitative characterization and quantitative interpretation of the TEQmixture values using Cr(VI) as the reference for environmental water samples.

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 analyze 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-defense 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 they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfills 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.IMPACT STATEMENTLaboratory studies of coevolution help us understand how host defenses and pathogen counter-defenses change over time, which is often essential for predicting the future dynamics of host-pathogen interactions. One particular model, termed “inverse-gene-for-gene” coevolution, predicts that coevolution proceeds through alternating steps, whereby hosts lose the features exploited by pathogens, and pathogens evolve to exploit alternative features. Using a classic model system in molecular biology, we describe the nature and timing of a previously overlooked step in the coevolution of E. coli and bacteriophage lambda. Our work demonstrates that this mode of coevolution can profoundly re-shape the interactions between bacteria and phage.

Although the process of genome encapsidation is highly conserved in tailed bacteriophages and eukaryotic double-stranded DNA viruses, there are two distinct packaging pathways that these viruses use to catalyze ATP-driven translocation of the viral genome into a preassembled procapsid shell. One pathway is used by ϕ29-like phages and adenoviruses, which replicate and subsequently package a monomeric, unit-length genome covalently attached to a virus/phage-encoded protein at each 5'-end of the dsDNA genome. In a second, more ubiquitous packaging pathway characterized by phage lambda and the herpesviruses, the viral DNA is replicated as multigenome concatemers linked in a head-to-tail fashion. Genome packaging in these viruses thus requires excision of individual genomes from the concatemer that are then translocated into a preassembled procapsid. Hence, the ATPases that power packaging in these viruses also possess nuclease activities that cut the genome from the concatemer at the beginning and end of packaging. This review focuses on proposed mechanisms of genome packaging in the dsDNA viruses using unit-length ϕ29 and concatemeric λ genome packaging motors as representative model systems.

Lizogeni bakteriofagi so virusi, ki okužujejo bakterije in ki imajo sposobnost se vgraditi v njen genom in se pasivno razmnoževati skupaj z bakterijo. Imajo širok spekter uporabe od zdravljenja bakterijskih okužb, iskanja interakcij med proteini, kot dostavljalni sistemi, kot orodje za kloniranje in še več. Znanje o proizvodnji bakteriofagov se razvija predvsem v industrijskem svetu in redko pride v znanstveno javnost. V svojem delu povzamem načine proizvodnje, ki jih lahko uberemo, na kaj moramo biti pozorni posebej pri lizogenih bakteriofagih in kako bi lahko proizvodnjo izboljšali. Temperate bacteriophages are viruses that infect bacteria and have the ability to integrate themselves into the bacterial genome and passively replicate with its host. They have a wide range of applications from treating bacterial infections, investigating protein interactions, using them as delivery systems, as a cloning tool and more. The knowledge of how to produce them is mainly being developed in industrial settings and information rarely gets out into the scientific community. In this work I review different modes of production, what we have to take into consideration especially when working with temperate bacteriophages and how we could optimise the process of production.

Shiga toxin-converting bacteriophages (or Stx phages) are responsible for virulence of enterohemorrhagic Escherichia coli strains. Although they belong to the group of lambdoid phages, which have served as models in studies on DNA replication mechanisms, details of regulation of replication of Stx phage genomes are poorly understood. Despite high similarity of their replication regions to that of phage lambda, considerable differences occur between them. Here, we present a comparison of origins of replication and O proteins of lambda and selected Stx phages (phages P27 and 933W). Stx initiator proteins, similarly to the lambda O protein, exist in the form of dimers. Only 4 iteron sequences are strongly bound in vitro by the O proteins, despite the presence of 6 such fragments in the Stx ori, while the function of the other two iterons is still crucial for transformation of E. coli wild-type strain by the P27-derived lambdoid plasmid. As these sequences are found in the gene coding for Stx O proteins, the sequences of these proteins themselves are also extended compared to lambda phage. Therefore, proteins O of Stx phages P27 and 933W have 13 additional amino acids. They can act as a space barrier, thus affecting the lesser packing of the O-some Stx complex compared to the structure found in lambda. Such structure of the DNA replication initiation complex may determine its lesser dependence on the processes occurring in the host cell, including transcriptional activation of the origin. Differences between molecular processes occurring during formation of replication complexes in lambda and Stx phages may indicate the specialization of the latter phages and their adaptation to specific environmental conditions where quick genetic switches are crucial.

Spatial organization of biological processes allows for variability in molecular outcomes and coordinated development. Here, we investigate how organization underpins phage lambda development and decision-making by characterizing viral components and processes in subcellular space. We use live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication, transcription, virion assembly, and resource recruitment in single-cell infections, uniting key processes of the infection cycle into a coherent model of phage development encompassing space and time. We find that different viral DNAs establish separate subcellular compartments within cells, which sustains heterogeneous viral development in single cells. These individual phage compartments are physically separated by the E. coli nucleoid. Our results provide mechanistic details describing how separate viruses develop heterogeneously to resemble single-cell phenotypes., Here, the authors apply live-cell and in situ fluorescence imaging at the single-molecule level to examine lambda DNA replication in single cells, finding that individual phage DNAs sequester host factors to their own vicinity and confine their replicated DNAs into separate compartments, suggesting that phage decision-making transcripts are spatially organized in separate compartments to allow distinct subcellular decisions to develop.

Accessory genes, such as antibiotic resistance genes (ARGs) can spread horizontally by mobile genetic elements, including plasmids and (temperate) bacteriophages leading to increased bacterial fitness in particular environments. In contrast to plasmids, temperate phages i.e. viruses that can incorporate their own genetic material into their host bacteria (then called lysogen) can additionally increase their hosts’ fitness through their ability to kill phage-susceptible competitors. However, in contrast to ARG-transfer by plasmids (conjugation), which has been extensively studied, ARG-transfer by phages (lysogenization) has received far less attention. Here we combined experiments using E. coli, phage lambda and the Rp4 plasmid (phage and plasmid both with an ampicillin resistance gene) and mathematical models to test which mechanism (lysogenization or conjugation) dominates under which conditions. In the absence of selection and in two-species experiments (Donor & Recipient), conjugation was significantly more common than lysogenization. However, in three-species experiments (DonorPlasmid, DonorPhage& Recipient) lysogenization rates increased by several orders of magnitude. By using phage-friendly environments that favour phage adsorption we were able to shift the ratio between lysogens and transconjugants even further towards lysogens and in extreme-events to the extinction of transconjugants. Mathematical models additionally allowed us to investigate how starting population sizes of both donors and the recipient influence conjugation and lysogenization dynamics. Taken together, our results suggest that plasmids and temperate phages can influence each other’s transfer rates which, in the present study system, seems to be largely driven by population size effects.

It is well known that bacteriophages play crucial roles in many aspects, such as controlling the number and the diversity of bacteria and participating in horizontal gene transfer, which is a key process in the evolution of bacteria. However, so far, the number of temperate bacteriophages is still limited, and their life processes are severely unknown, except for members of the lambdoid family of coliphages. In this study, a novel temperate phage of Pseudomonas aeruginosa, YA3 (vB_PaeP_YA3), was isolated from waste water. The morphology of YA3 suggested that it is a Podoviridae. The YA3 genome is a circular double-stranded DNA of 45,253 bp, with an average G + C content of 57.2%. A total of 65 open reading frames (ORFs) were predicted according to the sequence of YA3’s genome, of which only 32 (49.2%) ORFs were assigned with putative functions and 13 ORFs were confirmed by the structural proteome. Genome and proteome analyses confirmed the lysogenic nature of this phage, which encodes the typical lysogen-related proteins integrase, CI, Cro, and Q protein. The genome of YA3 is most closely related with that of temperate phage vB_PaeP_Tr60_Ab31, whereas the homology coverage is just 48%. There are many critical differences between their genomes, involving promoters, lysis pathways, and regulation patterns. YA3 is capable of stably lysogenizing its host P. aeruginosa PA14, targeting the integration site within the serine tRNA gene (PA14_RS20820), which is similar with phage vB_PaeP_Tr60_Ab31. The phylogenetic analysis is more complicated than we thought. Based on phage terminase large subunit (TerL) and CI proteins, phage YA3 is related with phage lambda, while their genome coverage is extremely low (

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.

Fitness tradeoffs play important roles in the evolution of organisms and communities. One such tradeoff often occurs when bacteria become resistant to phage at the cost of reduced competitiveness for resources. Quantifying the cost of phage resistance has frequently relied on measuring specific traits of interest to industrial applications. In an evolutionary context, however, fitness encompasses the effects of all traits relevant to an organism’s survival and reproductive success in a particular environment. Therefore, measurements of the net reproduction and survival of alternative genotypes offer greater power for predicting the fate of different genotypes in complex and dynamic communities. In this study, we measured the fitness of experimentally evolved, λ-resistant Escherichia coli isolates relative to their sensitive progenitor in both the absence and presence of phage. We also characterized certain phage-related phenotypes and obtained complete genome sequences of the bacteria. All of the evolved bacteria exhibited tradeoffs, such that they were more fit than the ancestor in the presence of phage, but less fit than the ancestor in the absence of phage. The fitness benefit of evolved resistance in the presence of abundant phage was generally much larger than the cost, and these effects appear to be driven by only a few resistance mutations. This asymmetrical benefit-tocost relation is consistent with the observation that sensitive cells did not persist in the experimental communities. Quantifying fitness effects in both the presence and absence of phage may thus provide a useful approach for predicting evolutionary outcomes in both natural and engineered microbial communities.

A goal of biology is to predict how mutations combine to alter phenotypes, fitness and disease. It is often assumed that mutations combine additively or with interactions that can be predicted. Here, we show using simulations that, even for the simple example of the lambda phage transcription factor CI repressing a gene, this assumption is incorrect and that perfect measurements of the effects of mutations on a trait and mechanistic understanding can be insufficient to predict what happens when two mutations are combined. This apparent paradox arises because mutations can have different biophysical effects to cause the same change in a phenotype and the outcome in a double mutant depends upon what these hidden biophysical changes actually are. Pleiotropy and non-monotonic functions further confound prediction of how mutations interact. Accurate prediction of phenotypes and disease will sometimes not be possible unless these biophysical ambiguities can be resolved using additional measurements., In quantitative genetics, it is widely assumed that mutations combine additively or epistasis can be predicted with statistical or mechanistic models. Here, the authors use the phage lambda repressor model to show how biophysical ambiguity and non-monotonic functions confound phenotypic prediction.

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ∼38 kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01-5 μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2 × 10

Shiga toxins (Stx) are the main virulence factor of a pathogroup of Escherichia coli strains that cause severe human diseases. These toxins are encoded in prophages (Stx prophages), and generally their expression depends on prophage induction. Several studies have reported high diversity among both Stx prophages and Stx. In particular, the toxin subtype Stx2a is associated with high virulence and HUS. Here, we report the genome of ArgO145, an inducible Stx2a prophage identified in a bovine O145:H‐ strain which produced high levels of Shiga toxin and Stx phage particles. The ArgO145 genome shared lambda phage organization, with recombination, regulation, replication, lysis, and head and tail structural gene regions, although some lambda genes encoding regulatory proteins could not be identified. Remarkably, some Stx2a phages of strains isolated from patients in other countries showed high similarity to ArgO145. Fil: Krüger, Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Burgán, Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina Fil: Friedrich, Alex. University Medical Center Groningen; Países Bajos Fil: Rossen, Johannes Wilhelmus Augustinus. University Medical Center Groningen; Países Bajos Fil: Lucchesi, Paula Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina

Background CRISPR arrays are transcribed into long precursor RNA species, which are further processed into mature CRISPR RNAs (crRNAs). Cas proteins utilize these crRNAs, which contain spacer sequences that can be derived from mobile genetic elements, to mediate immunity during a reoccurring virus infection. Type I CRISPR-Cas systems are defined by the presence of different Cascade interference complexes containing large and small subunits that play major roles during target DNA selection. Methods Here, we produce the protein and crRNA components of the Type I-B CRISPR-Cas complex of Clostridium thermocellum and Methanococcus maripaludis. The C. thermocellum Cascade complexes were reconstituted and analyzed via size-exclusion chromatography. Activity of the heterologous M. maripaludis CRISPR-Cas system was followed using phage lambda plaques assays. Results The reconstituted Type-I-B Cascade complex contains Cas7, Cas5, Cas6b and the large subunit Cas8b. Cas6b can be omitted from the reconstitution protocol. The large subunit Cas8b was found to be represented by two tightly associated protein fragments and a small C-terminal Cas8b segment was identified in recombinant complexes and C. thermocellum cell lysate. Conclusions Production of Cas8b generates a small C-terminal fragment, which is suggested to fulfill the role of the missing small subunit. A heterologous, synthetic M. maripaludis Type I-B system is active in E. coli against phage lambda, highlighting a potential for genome editing using endogenous Type-I-B CRISPR-Cas machineries. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue.

Despite decades of intensive research on bacteriophage lambda, a relatively uncharacterized region remains between the exo and xis genes. Collectively, exo-xis region genes are expressed during the earliest stages of the lytic developmental cycle and are capable of affecting the molecular events associated with the lysogenic-lytic developmental decision. In Shiga toxin-producing E. coli (STEC) and enterohemorragic E. coli (EHEC) that are responsible for food- and water-borne outbreaks throughout the world, there are distinct differences of exo-xis region genes from their counterparts in lambda phage. Together, these differences may help EHEC-specific phage and their bacterial hosts adapt to the complex environment within the human intestine. Only one exo-xis region protein, Ea8.5, has been solved to date. Here, I have used the AlphaFold and RoseTTAFold machine learning algorithms to predict the structures of six exo-xis region proteins from lambda and STEC/EHEC phages. Together, the models suggest possible roles for exo-xis region proteins in transcription and the regulation of RNA polymerase.