RESUMEN
Bacteriophage P1 is the premier transducing phage of E. coli. Despite its prominence in advancing E. coli genetics, modern molecular techniques have not been applied to thoroughly understand P1 structure. Here, we report the proteome of the P1 virion as determined by liquid chromatography tandem mass-spectrometry. Additionally, a library of single-gene knockouts identified the following five previously unknown essential genes: pmgA, pmgB, pmgC, pmgG, and pmgR. In addition, proteolytic processing of the major capsid protein is a known feature of P1 morphogenesis, and we identified the processing site by N-terminal sequencing to be between E120 and S121, producing a 448-residue, 49.3 kDa mature peptide. Furthermore, the P1 defense against restriction (Dar) system consists of six known proteins that are incorporated into the virion during morphogenesis. The largest of these, DarB, is a 250 kDa protein that is believed to translocate into the cell during infection. DarB deletions indicated the presence of an N-terminal packaging signal, and the N-terminal 30 residues of DarB are shown to be sufficient for directing a heterologous reporter protein to the capsid. Taken together, the data expand on essential structural P1 proteins as well as introduces P1 as a nanomachine for cellular delivery.
Asunto(s)
Bacteriófago P1 , Escherichia coli , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , ADN Viral/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin-pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions.
Asunto(s)
Bacteriófago P1 , Proteínas Virales , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Transporte Biológico , Endopeptidasas/metabolismo , Operón , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Cre-loxP gene editing tool enables site-specific editing of DNA without leaving lesions that must be repaired by error-prone cellular processes. Cre recombines two 34-bp loxP DNA sites that feature a pair of palindromic recombinase-binding elements flanking an asymmetric 8-bp spacer region, via assembly of a tetrameric intasome complex and formation of a Holliday junction intermediate. Recombination proceeds by coordinated nucleophilic attack by pairs of catalytic tyrosine residues on specific phosphodiester bonds in the spacer regions of opposing strands. Despite not making base-specific contacts with the asymmetric spacer region of the DNA, Cre exhibits a preference for initial cleavage on one of the strands, suggesting that intrinsic properties of the uncontacted 8-bp spacer region give rise to this preference. Furthermore, little is known about the structural and dynamic features of the loxP spacer that make it a suitable target for Cre. To enable NMR spectroscopic studies of the spacer, we have aimed to identify a fragment of the 34-bp loxP site that retains the structural features of the spacer while minimizing the spectral crowding and line-broadening seen in longer oligonucleotides. Sequence-specific chemical shift differences between spacer oligos of different lengths, and of a mutant that inverts strand cleavage order, reveal how both nearest-neighbor and next-nearest-neighbor effects dominate the chemical environment experienced by the spacer. We have identified a 16-bp oligonucleotide that preserves the structural environment of the spacer, setting the stage for NMR-based structure determination and dynamics investigations.
Asunto(s)
Bacteriófago P1/química , ADN Intergénico/química , Oligonucleótidos/química , Bacteriófago P1/metabolismo , Secuencia de Bases , ADN Intergénico/metabolismo , Integrasas/química , Integrasas/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación de Ácido Nucleico , Oligonucleótidos/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismoRESUMEN
Phage P1 is a temperate phage which makes the lytic or lysogenic decision upon infecting bacteria. During the lytic cycle, progeny phages are produced and the cell lyses, and in the lysogenic cycle, P1 DNA exists as a low-copy-number plasmid and replicates autonomously. Previous studies at the bulk level showed that P1 lysogenization was independent of multiplicity of infection (MOI; the number of phages infecting a cell), whereas lysogenization probability of the paradigmatic phage λ increases with MOI. However, the mechanism underlying the P1 behavior is unclear. In this work, using a fluorescent reporter system, we demonstrated this P1 MOI-independent lysogenic response at the single-cell level. We further observed that the activity of the major repressor of lytic functions (C1) is a determining factor for the final cell fate. Specifically, the repression activity of P1, which arises from a combination of C1, the anti-repressor Coi, and the corepressor Lxc, remains constant for different MOI, which results in the MOI-independent lysogenic response. Additionally, by increasing the distance between phages that infect a single cell, we were able to engineer a λ-like, MOI-dependent lysogenization upon P1 infection. This suggests that the large separation of coinfecting phages attenuates the effective communication between them, allowing them to make decisions independently of each other. Our work establishes a highly quantitative framework to describe P1 lysogeny establishment. This system plays an important role in disseminating antibiotic resistance by P1-like plasmids and provides an alternative to the lifestyle of phage λ. IMPORTANCE Phage P1 has been shown potentially to play an important role in disseminating antibiotic resistance among bacteria during lysogenization, as evidenced by the prevalence of P1 phage-like elements in animal and human pathogens. In contrast to phage λ, a cell fate decision-making paradigm, P1 lysogenization was shown to be independent of MOI. In this work, we built a simple genetic model to elucidate this MOI independency based on the gene-regulatory circuitry of P1. We also proposed that the effective communication between coinfecting phages contributes to the lysis-lysogeny decision-making of P1 and highlighted the significance of spatial organization in the process of cell fate determination in a single-cell environment. Finally, our work provides new insights into different strategies acquired by viruses to interact with their bacterial hosts in different scenarios for their optimal survival.
Asunto(s)
Bacterias/virología , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Regulación Viral de la Expresión Génica , Lisogenia/genética , Interacciones Microbianas , Proteínas Reguladoras y Accesorias Virales/genética , Bacteriófago P1/química , Lisogenia/fisiología , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
The faithful segregation, or "partition," of many low-copy number bacterial plasmids is driven by plasmid-encoded ATPases that are represented by the P1 plasmid ParA protein. ParA binds to the bacterial nucleoid via an ATP-dependent nonspecific DNA (nsDNA)-binding activity, which is essential for partition. ParA also has a site-specific DNA-binding activity to the par operator (parOP), which requires either ATP or ADP, and which is essential for it to act as a transcriptional repressor but is dispensable for partition. Here we examine how DNA binding by ParA contributes to the relative distribution of its plasmid partition and repressor activities, using a ParA with an alanine substitution at Arg351, a residue previously predicted to participate in site-specific DNA binding. In vivo, the parAR351A allele is compromised for partition, but its repressor activity is dramatically improved so that it behaves as a "super-repressor." In vitro, ParAR351A binds and hydrolyzes ATP, and undergoes a specific conformational change required for nsDNA binding, but its nsDNA-binding activity is significantly damaged. This defect in turn significantly reduces the assembly and stability of partition complexes formed by the interaction of ParA with ParB, the centromere-binding protein, and DNA. In contrast, the R351A change shows only a mild defect in site-specific DNA binding. We conclude that the partition defect is due to altered nsDNA binding kinetics and affinity for the bacterial chromosome. Furthermore, the super-repressor phenotype is explained by an increased pool of non-nucleoid bound ParA that is competent to bind parOP and repress transcription.
Asunto(s)
Bacteriófago P1/metabolismo , Cromosomas Bacterianos/metabolismo , ADN Bacteriano/metabolismo , Escherichia coli/metabolismo , Proteínas Virales/metabolismo , Sustitución de Aminoácidos , Bacteriófago P1/química , Bacteriófago P1/genética , Cromosomas Bacterianos/química , Cromosomas Bacterianos/genética , ADN Primasa/química , ADN Primasa/genética , ADN Primasa/metabolismo , ADN Bacteriano/química , ADN Bacteriano/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/virología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación Missense , Regiones Operadoras Genéticas , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Bacterial Type I restriction-modification (R-M) systems present a major barrier to foreign DNA entering the bacterial cell. The temperate phage P1 packages several proteins into the virion that protect the phage DNA from host restriction. Isogenic P1 deletion mutants were used to reconstitute the previously described restriction phenotypes associated with darA and darB. While P1ΔdarA and P1ΔdarB produced the expected phenotypes, deletions of adjacent genes hdf and ddrA also produced darA-like phenotypes and deletion of ulx produced a darB-like phenotype, implicating several new proteins of previously unknown function in the P1 dar antirestriction system. Interestingly, disruption of ddrB decreased P1's sensitivity to EcoB and EcoK restriction. Proteomic analysis of purified virions suggests that packaging of antirestriction components into P1 virions follows a distinct pathway that begins with the incorporation of DarA and Hdf and concludes with DarB and Ulx. Electron microscopy analysis showed that hdf and darA mutants also produce abnormally high proportions of virions with aberrant small heads, which suggests Hdf and DarA play a role in capsid morphogenesis. The P1 antirestriction system is more complex than previously realized and is comprised of multiple proteins including DdrA, DdrB, Hdf, and Ulx in addition to DarA and DarB.
Asunto(s)
Bacteriófago P1/metabolismo , Cápside/fisiología , Proteínas Bacterianas/metabolismo , Bacteriófago P1/genética , Bacteriófagos/genética , Enzimas de Restricción-Modificación del ADN/genética , ADN Viral/metabolismo , Escherichia coli/genética , Morfogénesis , Proteómica , Proteínas Virales/metabolismo , Virión/genéticaRESUMEN
Conditional cooperativity is a common mechanism involved in transcriptional regulation of prokaryotic type II toxin-antitoxin operons and is intricately related to bacterial persistence. It allows the toxin component of a toxin-antitoxin module to act as a co-repressor at low doses of toxin as compared to antitoxin. When toxin level exceeds a certain threshold, however, the toxin becomes a de-repressor. Most antitoxins contain an intrinsically disordered region (IDR) that typically is involved in toxin neutralization and repressor complex formation. To address how the antitoxin IDR is involved in transcription regulation, we studied the phd-doc operon from bacteriophage P1. We provide evidence that the IDR of Phd provides an entropic barrier precluding full operon repression in the absence of Doc. Binding of Doc results in a cooperativity switch and consequent strong operon repression, enabling context-specific modulation of the regulatory process. Variations of this theme are likely to be a common mechanism in the autoregulation of bacterial operons that involve intrinsically disordered regions.
Asunto(s)
Antitoxinas/metabolismo , Entropía , Regulación Alostérica , Antitoxinas/genética , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Operón/genéticaRESUMEN
A step-by-step detailed procedure is presented to progressively truncate genomic DNA inserts from either end in BACs. The bacterial transposon Tn10 carrying a loxP or a lox511 site is inserted at random into BAC DNA inside the bacterial cell. The cells are then infected with bacteriophage P1. The Cre protein expressed by phage P1 generates end-deletions by specifically recombining the inserted loxP (or lox511) with the loxP (or lox511) endogenous to and flanking insert DNA in BACs from the respective end. The Cre protein also helps phage P1 transduce the BAC DNA by packaging it in P1 heads. This packaging by P1 not only recovers the rare BAC clones containing Tn10 insertions efficiently but also selects end-truncated BACs from those containing inversions of portions of their DNA caused by transposition of Tn10 in the opposite orientation. The libraries of end-deleted BACs generated by this procedure are suitable for numerous mapping studies. Because DNA in front of the loxP (or lox511) arrowheads in the Tn10 transposon is retained at the newly created BAC end, exogenous DNA cassettes such as enhancer-traps and iTol2 ends can be efficiently introduced into BAC ends for germline expression in zebrafish or mice. The methodology should facilitate functional mapping studies of long-range cis-acting gene regulatory sequences in these organisms.
Asunto(s)
Bacteriófago P1/genética , Cromosomas Artificiales Bacterianos/genética , Elementos Transponibles de ADN , Elementos de Facilitación Genéticos , Integrasas/genética , Transducción Genética/métodos , Animales , Animales Modificados Genéticamente/genética , Bacteriófago P1/metabolismo , Cromosomas Artificiales Bacterianos/química , Integrasas/metabolismo , Ratones , Plásmidos/química , Plásmidos/genética , Transposasas/genética , Transposasas/metabolismo , Pez Cebra/genéticaRESUMEN
The toxin Doc from the phd/doc toxin-antitoxin module targets the cellular translation machinery and is inhibited by its antitoxin partner Phd. Here we show that Phd also functions as a chaperone, keeping Doc in an active, correctly folded conformation. In the absence of Phd, Doc exists in a relatively expanded state that is prone to dimerization through domain swapping with its active site loop acting as hinge region. The domain-swapped dimer is not capable of arresting protein synthesis in vitro, whereas the Doc monomer is. Upon binding to Phd, Doc becomes more compact and is secured in its monomeric state with a neutralized active site.
Asunto(s)
Bacteriófago P1/genética , Escherichia coli/virología , Regulación Viral de la Expresión Génica , Chaperonas Moleculares/química , Proteínas Virales/química , Bacteriófago P1/química , Bacteriófago P1/metabolismo , Cristalografía por Rayos X , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.
Asunto(s)
Bacteriófago P1/metabolismo , Proteínas de Escherichia coli/metabolismo , Extensión de la Cadena Peptídica de Translación , Factor Tu de Elongación Peptídica/metabolismo , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Antibacterianos/química , Sitios de Unión , Proliferación Celular , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Espectrometría de Masas , Simulación del Acoplamiento Molecular , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Piridonas/química , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Treonina/químicaRESUMEN
Toxin-antitoxin (TA) modules in bacteria are involved in pathogenesis, antibiotic stress response, persister formation and programmed cell death. The toxin Doc, from the phd/doc module, blocks protein synthesis by targeting the translation machinery. Despite a large wealth of biophysical and biochemical data on the regulatory aspects of the operon transcription and role of Doc co-activator and co-repressor, little is still know on the molecular basis of Doc toxicity. Structural information about this toxin is only available for its inhibited state bound to the antitoxin Phd. Here we report the (1)H, (15)N and (13)C backbone and side chain chemical shift assignments of the toxin Doc from of bacteriophage P1 (the model protein from this family of TA modules) in its free state. The BMRB accession number is 18899.
Asunto(s)
Bacteriófago P1/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Virales/química , Secuencia de Aminoácidos , Isótopos de Carbono , Hidrógeno , Datos de Secuencia Molecular , Isótopos de Nitrógeno , Estructura Secundaria de ProteínaRESUMEN
DNA segregation ensures the stable inheritance of genetic material prior to cell division. Many bacterial chromosomes and low-copy plasmids, such as the plasmids P1 and F, employ a three-component system to partition replicated genomes: a partition site on the DNA target, typically called parS, a partition site binding protein, typically called ParB, and a Walker-type ATPase, typically called ParA, which also binds non-specific DNA. In vivo, the ParA family of ATPases forms dynamic patterns over the nucleoid, but how ATP-driven patterning is involved in partition is unknown. We reconstituted and visualized ParA-mediated plasmid partition inside a DNA-carpeted flowcell, which acts as an artificial nucleoid. ParA and ParB transiently bridged plasmid to the DNA carpet. ParB-stimulated ATP hydrolysis by ParA resulted in ParA disassembly from the bridging complex and from the surrounding DNA carpet, which led to plasmid detachment. Our results support a diffusion-ratchet model, where ParB on the plasmid chases and redistributes the ParA gradient on the nucleoid, which in turn mobilizes the plasmid.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Bacteriófago P1/genética , ADN Bacteriano/genética , Factor F/genética , Modelos Biológicos , Proteínas Virales/metabolismo , Bacteriófago P1/metabolismo , División Celular , ADN Bacteriano/metabolismo , Factor F/metabolismo , Hidrólisis , Cinética , Unión Proteica , Multimerización de Proteína , Imagen de Lapso de TiempoRESUMEN
This chapter provides an overview of key tools and methodologies available to practitioners of biocatalysis interested in using microorganisms to carry out biotransformations and describes specific examples of applying genetic modification strategies for strain design. We focus on the use of the polymerase chain reaction (PCR) for gene amplification, plasmid DNA for recombinant gene cloning and expression, and homologous recombination and phage transduction for modifying chromosomal DNA. Specifically we use Escherichia coli as the host organism, and the overproduction of xylitol by reduction of xylose represents the biotransformation of interest.
Asunto(s)
Aldehído Reductasa/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli , Ingeniería Genética/métodos , Proteínas Recombinantes/metabolismo , Xilitol/biosíntesis , Xilosa/metabolismo , Aldehído Reductasa/genética , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Biocatálisis , Candida , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Marcación de Gen/métodos , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Recombinación Genética , Transducción GenéticaRESUMEN
Regulation of the phd/doc toxin-antitoxin operon involves the toxin Doc as co- or derepressor depending on the ratio between Phd and Doc, a phenomenon known as conditional cooperativity. The mechanism underlying this observed behavior is not understood. Here we show that monomeric Doc engages two Phd dimers on two unrelated binding sites. The binding of Doc to the intrinsically disordered C-terminal domain of Phd structures its N-terminal DNA-binding domain, illustrating allosteric coupling between highly disordered and highly unstable domains. This allosteric effect also couples Doc neutralization to the conditional regulation of transcription. In this way, higher levels of Doc tighten repression up to a point where the accumulation of toxin triggers the production of Phd to counteract its action. Our experiments provide the basis for understanding the mechanism of conditional cooperative regulation of transcription typical of toxin-antitoxin modules. This model may be applicable for the regulation of other biological systems.
Asunto(s)
Regulación Alostérica , Regulación de la Expresión Génica , Transcripción Genética , Proteínas Virales/metabolismo , Sitio Alostérico , Bacteriófago P1/metabolismo , ADN/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Regiones Operadoras Genéticas , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Proteínas Virales/química , Difracción de Rayos XRESUMEN
R(21), the lysozyme of coliphage 21, has an N-terminal signal-anchor-release (SAR) domain that directs its secretion in a membrane-tethered, inactive form and then its release and activation in the periplasm. Both genetic and crystallographic studies show that the SAR domain, once extracted from the bilayer, refolds into the body of the enzyme and effects muralytic activation by repositioning one residue of the canonical lysozyme catalytic triad.
Asunto(s)
Bacteriófago P1/metabolismo , Colifagos/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Prokaryotic toxin-antitoxin modules are involved in major physiological events set in motion under stress conditions. The toxin Doc (death on curing) from the phd/doc module on phage P1 hosts the C-terminal domain of its antitoxin partner Phd (prevents host death) through fold complementation. This Phd domain is intrinsically disordered in solution and folds into an alpha-helix upon binding to Doc. The details of the interactions reveal the molecular basis for the inhibitory action of the antitoxin. The complex resembles the Fic (filamentation induced by cAMP) proteins and suggests a possible evolutionary origin for the phd/doc operon. Doc induces growth arrest of Escherichia coli cells in a reversible manner, by targeting the protein synthesis machinery. Moreover, Doc activates the endogenous E. coli RelE mRNA interferase but does not require this or any other known chromosomal toxin-antitoxin locus for its action in vivo.
Asunto(s)
Bacteriófago P1/química , Profagos/química , Pliegue de Proteína , Toxinas Bacterianas/metabolismo , Bacteriófago P1/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/virología , Proteínas de Escherichia coli/metabolismo , Profagos/metabolismo , Estructura Cuaternaria de Proteína/fisiología , Estructura Secundaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Interferencia de ARN/fisiología , Proteínas ViralesRESUMEN
In this study, we demonstrated that the CSKSSDYQC-peptide ligand which was identified from a random phage-peptide library through an in vivo phage display technique with rats could prominently improve the transport efficiency of macromolecules, such as large filamentous phage particles (M13 bacteriophage), across the intestinal mucosal barrier. Synthetic CSKSSDYQC-peptide ligands significantly inhibited the binding of phage P1 encoding CSKSSDYQC-peptide ligands to the intestinal mucosal tissue and immunohistochemical analysis showed that the CSKSSDYQC-peptide ligands could be transported across the intestinal mucosal barrier via goblet cells as their specific gateway. Thus, we inferred that CSKSSDYQC-peptide ligand might have a specific receptor on the goblet cells and transported from intestinal lumen to systemic circulation by transcytosis mechanism. These results suggest that CSKSSDYQC-ligand could be a promising tool for development of an efficient oral delivery system for macromolecular therapeutics in the carrier-drug conjugate strategy.
Asunto(s)
Células Caliciformes/metabolismo , Mucosa Intestinal/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Bacteriófago M13/genética , Bacteriófago M13/metabolismo , Bacteriófago M13/fisiología , Bacteriófago P1/genética , Bacteriófago P1/metabolismo , Bacteriófago P1/fisiología , Transporte Biológico , Células Caliciformes/citología , Inmunohistoquímica , Mucosa Intestinal/citología , Mucosa Intestinal/virología , Intestino Delgado/citología , Intestino Delgado/metabolismo , Intestino Delgado/virología , Masculino , Microscopía Fluorescente , Modelos Teóricos , Biblioteca de Péptidos , Péptidos/química , Unión Proteica , Ratas , Ratas Sprague-DawleyRESUMEN
Transduction frequency with phage P1 had been observed to be very low in Escherichia coli K-12 mutants lacking the operon (ppk1-ppx) responsible for the synthesis of inorganic polyphosphate (poly P). We now find that these mutants, for lack of poly P, are lysogenic for P1 and when infected with phage P1 produce only approximately 1% the number of infective centers compared with the WT host. Both phage adsorption and release were unaffected. The host-encoded P1 late-gene transcriptional activator, SspA, failed to show the transcriptional increase in the mutant, observed in the WT. UV induction of a P1-infected mutant resulted in a 200-fold increase in the production of infectious phage particles. The lysogenized P1 (P1mut) and P1 progeny from the mutant host (Deltappk1-ppx) produced plaques of differing morphologies, whereas P1 progeny from the WT yielded only small, clear plaques. Two discernable variants, one producing small and clear plaques (P1small) and the other large plaques with turbid rims (P1large), had broader host range and produced larger burst sizes in WT compared with P1. Transmission electron microscopy showed P1mut had contractile sheath defects. Thus, the lack of poly P/PPK1 in the mutant host resulted in the formation of defective P1 particles during intracellular growth. A filamentous phage, fd, also failed to produce plaques on a mutant lawn. Although fd adsorbed to the F-pilus, its DNA failed to enter the mutant host.
Asunto(s)
Bacteriófago M13/crecimiento & desarrollo , Bacteriófago M13/metabolismo , Bacteriófago P1/crecimiento & desarrollo , Bacteriófago P1/metabolismo , Lisogenia , Polifosfatos/metabolismo , Bacteriófago M13/genética , Bacteriófago P1/genética , Escherichia coli/virología , Lisogenia/genética , Mutación , Transducción Genética , Replicación Viral/genéticaRESUMEN
The Hot (homolog of theta) protein of bacteriophage P1 can substitute for the Escherichia coli DNA polymerase III theta subunit, as evidenced by its stabilizing effect on certain dnaQ mutants that carry an unstable polymerase III epsilon proofreading subunit (antimutator effect). Here, we show that Hot can also cause an increase in the mutability of various E. coli strains (mutator effect). The hot mutator effect differs from the one caused by the lack of theta. Experiments using chimeric theta/Hot proteins containing various domains of Hot and theta along with a series of point mutants show that both N- and C-terminal parts of each protein are important for stabilizing the epsilon subunit. In contrast, the N-terminal part of Hot appears uniquely responsible for its mutator activity.