RESUMO
Bacterial resistance to conventional antibiotics stimulated the development of so-called "phage therapies" that rely on cell lysis, which is a process of destroying bacterial cells due to their infections by bacterial viruses. For λ bacteriophages, it is known that the critical role in this process is played by holin proteins that aggregate in cellular membranes before breaking them apart. While multiple experimental studies probed various aspects of cell lysis, the underlying molecular mechanisms remain not well understood. Here we investigate what physicochemical properties of holin proteins are the most relevant for these processes by employing statistical correlation analysis of cell lysis dynamics for different experimentally observed mutant species. Our findings reveal significant correlations between various physicochemical features and cell lysis dynamics. Notably, we uncover a strong inverse correlation between local hydrophobicity and cell lysis times, underscoring the crucial role of hydrophobic interactions in membrane disruption. Stimulated by these observations, a predictive model capable of explicitly estimating cell lysis times for any holin protein mutants based on their mean hydrophobicity values is developed. Our study not only provides important microscopic insights into cell lysis phenomena but also proposes specific routes to optimize medical and biotechnological applications of bacteriophages.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Proteínas Virais , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófago lambda/química , Bacteriólise/efeitos dos fármacos , MutaçãoRESUMO
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.
Assuntos
Imunoprecipitação da Cromatina , DNA Bacteriano , Escherichia coli , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoprecipitação da Cromatina/métodos , Ligação Proteica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Óperon Lac , Reação em Cadeia da Polimerase/métodos , Southern Blotting , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismoRESUMO
Baculoviruses are widely used for their potential as biological pesticide and as platform for the production of recombinant proteins and gene therapy vectors. The Baculovirus Expression Vector System (BEVS) is used for high level of expression of (multiple) proteins in insect cells. Baculovirus recombinants can be quickly constructed by transposition of the gene(s) of interest into a so-called bacmid, which is a baculovirus infectious clone maintained as single-copy, bacterial artificial chromosome in Escherichia coli. A two-step homologous recombineering technique using the lambda-red system in E. coli allows for scarless editing of the bacmid with PCR products based on sequence homology. In the first step, a selection cassette with 50 bp homology arms, typically generated by PCR, is inserted into the designated locus. In the second step, the selection cassette is removed based on a negative selection marker, such as SacB or rpsL. This lambda-red recombineering technique can be used for multiple gene editing purposes, including (large) deletions, insertions, and even single point mutations. Moreover, since there are no remnants of the editing process, successive modifications of the same bacmid are possible. This chapter provides detailed instructions to design and perform two-step homologous recombineering of baculovirus bacmid DNA in E. coli. We present two case studies demonstrating the utility of this technique for creating a deletion mutant of the chitinase and cathepsin genes and for introducing a single point mutation in the baculovirus gene gp41. This scarless genome editing approach can facilitate functional studies of baculovirus genes and improve the production of recombinant proteins using the BEVS.
Assuntos
Baculoviridae , Escherichia coli , Edição de Genes , Vetores Genéticos , Edição de Genes/métodos , Escherichia coli/genética , Baculoviridae/genética , Vetores Genéticos/genética , Cromossomos Artificiais Bacterianos/genética , Genoma Viral , Engenharia Genética/métodos , Bacteriófago lambda/genética , Recombinação HomólogaRESUMO
Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.
Assuntos
Bacteriófago lambda , Cromossomos Bacterianos , Escherichia coli , Lisogenia , Escherichia coli/genética , Escherichia coli/virologia , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Cromossomos Bacterianos/genética , Lisogenia/genética , Integração Viral , Transferência Genética Horizontal , Instabilidade Genômica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Prófagos/genética , Prófagos/fisiologia , Hibridização in Situ Fluorescente , Proteínas Virais Reguladoras e AcessóriasRESUMO
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.
Assuntos
Bacteriófago lambda , Escherichia coli , Escherichia coli/virologia , Escherichia coli/fisiologia , Bacteriófago lambda/fisiologia , Bacteriófago lambda/genética , Lisogenia , Internalização do VírusRESUMO
In numerous malignancies, miRNA-155 is overexpressed and has oncogenic activity because it is one of the most efficient microRNAs for inhibiting apoptosis in human cancer cells. As a result, the highest sensitive detection of the miRNA-155 gene is a technological instrument that can enable early cancer screening. In this study, a miRNA-155 biosensor was created to create a hairpin probe that can bind to the miRNA-155 gene using lambda nucleic acid exonuclease, which can cut the 5' phosphorylated double strand, and by the DNA probe is recognized by the Cas12a enzyme, which then activates Cas12a to catalyze trans-cutting produces strong fluorescence. Research finding, the target concentration's logarithm and corresponding fluorescence intensity have a strong linear connection, and the limit of detection (LOD) of the sensing system was determined to be 8.3 pM. In addition, the biosensor displayed exceptional specificity, low false-positive signal, and high sensitivity in detecting the miRNA-155 gene in serum samples. This study's creation of a biosensor that has high sensitivity, good selectivity, and is simple to operate provides promising opportunities for research into biosensor design and early cancer detection.
Assuntos
Técnicas Biossensoriais , Sistemas CRISPR-Cas , MicroRNAs , MicroRNAs/genética , MicroRNAs/sangue , MicroRNAs/análise , Humanos , Técnicas Biossensoriais/métodos , Sistemas CRISPR-Cas/genética , Proteínas Associadas a CRISPR/metabolismo , Proteínas Associadas a CRISPR/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Limite de Detecção , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/genética , EndodesoxirribonucleasesRESUMO
TMPyP is a porphyrin capable of DNA binding and used in photodynamic therapy and G-quadruplex stabilization. Despite its broad applications, TMPyP's effect on DNA nanomechanics is unknown. Here we investigated, by manipulating λ-phage DNA with optical tweezers combined with microfluidics in equilibrium and perturbation kinetic experiments, how TMPyP influences DNA nanomechanics across wide ranges of TMPyP concentration (5-5120 nM), mechanical force (0-100 pN), NaCl concentration (0.01-1 M) and pulling rate (0.2-20 µm/s). Complex responses were recorded, for the analysis of which we introduced a simple mathematical model. TMPyP binding, which is a highly dynamic process, leads to dsDNA lengthening and softening. dsDNA stability increased at low (<10 nM) TMPyP concentrations, then decreased progressively upon increasing TMPyP concentration. Overstretch cooperativity decreased, due most likely to mechanical roadblocks of ssDNA-bound TMPyP. TMPyP binding increased ssDNA's contour length. The addition of NaCl at high (1 M) concentration competed with the TMPyP-evoked nanomechanical changes. Because the largest amplitude of the changes is induced by the pharmacologically relevant TMPyP concentration range, this porphyrin derivative may be used to tune DNA's structure and properties, hence control the wide array of biomolecular DNA-dependent processes including replication, transcription, condensation and repair.
Assuntos
Pinças Ópticas , Porfirinas , Porfirinas/química , Cinética , DNA/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , Bacteriófago lambda/genética , Nanotecnologia/métodos , DNA Viral/metabolismo , DNA Viral/química , Cloreto de Sódio/química , Cloreto de Sódio/farmacologiaRESUMO
Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Sistemas Toxina-Antitoxina , Sistemas Toxina-Antitoxina/genética , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Bacteriófago lambda/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/fisiologia , Antitoxinas/metabolismo , Antitoxinas/genética , Proteínas da Cauda Viral/metabolismo , Proteínas da Cauda Viral/genéticaRESUMO
Phage-induced lysis of Gram-negative bacterial hosts usually requires a set of phage lysis proteins, a holin, an endopeptidase, and a spanin system, to disrupt each of the three cell envelope layers. Genome annotations and previous studies identified a gene region in the Shewanella oneidensis prophage LambdaSo, which comprises potential holin- and endolysin-encoding genes but lacks an obvious spanin system. By a combination of candidate approaches, mutant screening, characterization, and microscopy, we found that LambdaSo uses a pinholin/signal-anchor-release (SAR) endolysin system to induce proton leakage and degradation of the cell wall. Between the corresponding genes, we found that two extensively nested open-reading frames encode a two-component spanin module Rz/Rz1. Unexpectedly, we identified another factor strictly required for LambdaSo-induced cell lysis, the phage protein Lcc6. Lcc6 is a transmembrane protein of 65 amino acid residues with hitherto unknown function, which acts at the level of holin in the cytoplasmic membrane to allow endolysin release. Thus, LambdaSo-mediated cell lysis requires at least four protein factors (pinholin, SAR endolysin, spanin, and Lcc6). The findings further extend the known repertoire of phage proteins involved in host lysis and phage egress. IMPORTANCE: Lysis of bacteria can have multiple consequences, such as the release of host DNA to foster robust biofilm. Phage-induced lysis of Gram-negative cells requires the disruption of three layers, the outer and inner membranes and the cell wall. In most cases, the lysis systems of phages infecting Gram-negative cells comprise holins to disrupt or depolarize the membrane, thereby releasing or activating endolysins, which then degrade the cell wall. This, in turn, allows the spanins to become active and fuse outer and inner membranes, completing cell envelope disruption and allowing phage egress. Here, we show that the presence of these three components may not be sufficient to allow cell lysis, implicating that also in known phages, further factors may be required.
Assuntos
Bacteriólise , Endopeptidases , Shewanella , Shewanella/virologia , Shewanella/genética , Endopeptidases/metabolismo , Endopeptidases/genética , Proteínas Virais/metabolismo , Proteínas Virais/genética , Bacteriófago lambda/fisiologia , Bacteriófago lambda/genéticaRESUMO
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.
Assuntos
Bacteriófago lambda , Microscopia Crioeletrônica , Shigella sonnei , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Bacteriófago lambda/fisiologia , Shigella sonnei/virologia , Shigella sonnei/metabolismo , Proteínas da Cauda Viral/metabolismo , Proteínas da Cauda Viral/química , Proteínas da Cauda Viral/genética , Porinas/metabolismo , Porinas/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Ligação Proteica , Modelos Moleculares , Conformação Proteica , Receptores ViraisRESUMO
The portal protein of tailed bacteriophage plays essential roles in various aspects of capsid assembly, motor assembly, genome packaging, connector formation, and infection processes. After DNA packaging is complete, additional proteins are assembled onto the portal to form the connector complex, which is crucial as it bridges the mature head and tail. In this study, we report high-resolution cryo-electron microscopy (cryo-EM) structures of the portal vertex from bacteriophage lambda in both its prohead and mature virion states. Comparison of these structures shows that during head maturation, in addition to capsid expansion, the portal protein undergoes conformational changes to establish interactions with the connector proteins. Additionally, the independently assembled tail undergoes morphological alterations at its proximal end, facilitating its connection to the head-tail joining protein and resulting in the formation of a stable portal-connector-tail complex. The B-DNA molecule spirally glides through the tube, interacting with the nozzle blade region of the middle-ring connector protein. These insights elucidate a mechanism for portal maturation and DNA translocation within the phage lambda system. IMPORTANCE: The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.
Assuntos
Bacteriófago lambda , Proteínas do Capsídeo , Capsídeo , Montagem de Vírus , Bacteriófago lambda/fisiologia , Bacteriófago lambda/genética , Capsídeo/metabolismo , Capsídeo/ultraestrutura , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Empacotamento do DNA , DNA Viral/genética , DNA Viral/metabolismo , Modelos Moleculares , Conformação Proteica , Vírion/metabolismo , Vírion/ultraestruturaRESUMO
Redß is a protein from bacteriophage λ that binds to single-stranded DNA (ssDNA) to promote the annealing of complementary strands. Together with λ-exonuclease (λ-exo), Redß is part of a two-component DNA recombination system involved in multiple aspects of genome maintenance. The proteins have been exploited in powerful methods for bacterial genome engineering in which Redß can anneal an electroporated oligonucleotide to a complementary target site at the lagging strand of a replication fork. Successful annealing in vivo requires the interaction of Redß with E. coli single-stranded DNA-binding protein (SSB), which coats the ssDNA at the lagging strand to coordinate access of numerous replication proteins. Previous mutational analysis revealed that the interaction between Redß and SSB involves the C-terminal domain (CTD) of Redß and the C-terminal tail of SSB (SSB-Ct), the site for binding of numerous host proteins. Here, we have determined the x-ray crystal structure of Redß CTD in complex with a peptide corresponding to the last nine residues of SSB (MDFDDDIPF). Formation of the complex is predominantly mediated by hydrophobic interactions between two phenylalanine side chains of SSB (Phe-171 and Phe-177) and an apolar groove on the CTD, combined with electrostatic interactions between the C-terminal carboxylate of SSB and Lys-214 of the CTD. Mutation of any of these residues to alanine significantly disrupts the interaction of full-length Redß and SSB proteins. Structural knowledge of this interaction will help to expand the utility of Redß-mediated recombination to a wider range of bacterial hosts for applications in synthetic biology.
Assuntos
Bacteriófago lambda , DNA de Cadeia Simples , Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Escherichia coli , Proteínas Virais , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sítios de Ligação , Cristalografia por Raios X , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
DNA circuits, as a type of biochemical system, have the capability to synchronize the perception of molecular information with a chemical reaction response and directly process the molecular characteristic information in biological activities, making them a crucial area in molecular digital computing and smart bioanalytical applications. Instead of cascading logic gates, the traditional research approach achieves multiple logic operations which limits the scalability of DNA circuits and increases the development costs. Based on the interface reaction mechanism of Lambda exonuclease, the molecular perceptron proposed in this study, with the need for only adjusting weight and bias parameters to alter the corresponding logic expressions, enhances the versatility of the molecular circuits. We also establish a mathematical model and an improved heuristic algorithm for solving weights and bias parameters for arbitrary logic operations. The simulation and FRET experiment results of a series of logic operations demonstrate the universality of molecular perceptron. We hope the proposed molecular perceptron can introduce a new design paradigm for molecular circuits, fostering innovation and development in biomedical research related to biosensing, targeted therapy, and nanomachines.
Assuntos
Computadores Moleculares , DNA , DNA/química , DNA/metabolismo , Algoritmos , Transferência Ressonante de Energia de Fluorescência , Bacteriófago lambda/genética , Exonucleases/metabolismo , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Técnicas Biossensoriais/métodosRESUMO
RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron-electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.
Assuntos
Bacteriófago lambda , Proteínas de Ligação a DNA , Modelos Moleculares , Proteínas Virais , Bacteriófago lambda/genética , Cristalografia por Raios X , Proteínas Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ligação Proteica , Multimerização Proteica , DNA Viral/genética , DNA Viral/metabolismo , Mutação , Lisogenia , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/metabolismo , DNA/químicaRESUMO
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.
Assuntos
Bacteriófago lambda , Recombinação Genética , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , DNA Viral/genética , DNA Viral/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Sítios de Ligação , Fatores Hospedeiros de Integração/metabolismo , Fatores Hospedeiros de Integração/genéticaRESUMO
The orf63 gene resides in a region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed during infection. In lambda phage and Shiga toxin (Stx) producing phages found in enterohemorrhagic Escherichia coli (EHEC) associated with food poisoning, Orf63 expression reduces the host survival and hastens the period between infection and lysis thereby giving it pro-lytic qualities. The NMR structure of dimeric Orf63 reveals a fold consisting of two helices and one strand that all make extensive intermolecular contacts. Structure-based data mining failed to identify any Orf63 homolog beyond the family of temperate bacteriophages. A machine learning approach was used to design an amphipathic helical ligand that bound a hydrophobic cleft on Orf63 with micromolar affinity. This approach may open a new path towards designing therapeutics that antagonize the contributions of Stx phages in EHEC outbreaks.
Assuntos
Bacteriófago lambda , Escherichia coli Êntero-Hemorrágica , Proteínas Virais , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Escherichia coli Êntero-Hemorrágica/metabolismo , Escherichia coli Êntero-Hemorrágica/virologia , Toxina Shiga/genética , Proteínas Virais/metabolismoRESUMO
The ea22 gene resides in a relatively uncharacterized region of the lambda bacteriophage genome between the exo and xis genes and is among the earliest genes transcribed upon infection. In lambda and Shiga toxin-producing phages found in enterohemorrhagic E. coli (EHEC) associated with food poisoning, Ea22 favors a lysogenic over lytic developmental state. The Ea22 protein may be considered in terms of three domains: a short amino-terminal domain, a coiled-coiled domain, and a carboxy-terminal domain (CTD). While the full-length protein is tetrameric, the CTD is dimeric when expressed individually. Here, we report the NMR solution structure of the Ea22 CTD that is described by a mixed alpha-beta fold with a dimer interface reinforced by salt bridges. A conserved mobile loop may serve as a ligand for an unknown host protein that works with Ea22 to promote bacterial survival and the formation of new lysogens. From sequence and structural comparisons, the CTD distinguishes lambda Ea22 from homologs encoded by Shiga toxin-producing bacteriophages.
Assuntos
Bacteriófagos , Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Humanos , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Lisogenia/genética , Bacteriófagos/genética , Bacteriófagos/metabolismo , Escherichia coli Êntero-Hemorrágica/genética , Toxina Shiga/genética , Infecções por Escherichia coli/microbiologiaRESUMO
In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing. Here, we use cryo-EM to solve the structures of the uS2-deficient 70S ribosome of host E. coli mutant rpsB11 and the wild-type 70S complex with λcI lmRNA and fMet-tRNAfMet. Importantly, the uS2-deficient 70S ribosome also lacks protein bS21. The anti-Shine-Dalgarno (aSD) region is structurally supported by bS21, so that the absence of the latter causes the aSD to divert from the normal mRNA exit pathway, easing the exit of lmRNA. A π-stacking interaction between the monitor base A1493 and A(+4) of lmRNA potentially acts as a recognition signal. Coulomb charge flow, along with peristalsis-like dynamics within the mRNA entrance channel due to the increased 30S head rotation caused by the absence of uS2, are likely to facilitate the propagation of lmRNA through the ribosome. These findings lay the groundwork for future research on the mechanism of translation and the co-evolution of lmRNA and mRNA that includes the emergence of a defined ribosome-binding site of the transcript.
Assuntos
Bacteriófago lambda , Escherichia coli , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro , Proteínas Repressoras , Subunidades Ribossômicas Maiores de Bactérias , Proteínas Virais Reguladoras e Acessórias , Escherichia coli/genética , Escherichia coli/virologia , Proteínas Ribossômicas/metabolismo , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Proteínas Repressoras/genética , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
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.
Assuntos
Bacteriófago lambda , Aptidão Genética , Bacteriófago lambda/genética , Retroalimentação , Mutação , Modelos Genéticos , Evolução BiológicaRESUMO
Bacteriophage lambda has a double-stranded DNA genome and a long, flexible, non-contractile tail encoded by a contiguous block of 11 genes downstream of the head genes. The tail allows host recognition and delivery of viral DNA from the head shell to the cytoplasm of the infected cell. Here, we present a high-resolution structure of the tail complex of bacteriophage lambda determined by cryoelectron microscopy. Most component proteins of the lambda tail were determined at the atomic scale. The structure sheds light on the molecular organization of the extensively studied tail of bacteriophage lambda.