Mature T4 Research Articles

Effect of lactoferrin supplementation on composition, fatty acids composition, lipolysis and sensory characteristics of cheddar cheese

ABSTRACT Lactoferrin is the part of whey proteins, which does not become the part of curd and is lost in the whey. The objective of the current investigation was to develop lactoferrin-supplemented cheddar cheese. Cheddar cheese was supplemented with lactoferrin at 5, 10, 15, 20, and 0 mg/100 g concentrations (T1, T2, T3, T4, and control) and matured for 90 days. The supplementation of lactoferrin at all four concentrations did not affect the compositional attributes of cheddar cheese. In mature cheddar cheese, values of log-colony forming units in T1, T2, T3, T4, and control per mL were 7.82, 7.77, 6.51, 6.21, and 7.95, respectively. Lactoferrin contents of T1, T2, T3, T4, and control were 4.98, 9.89, 14.91, 19.88, and 0.11 mg/100 g cheese. In vitro total antioxidant capacity of mature T1, T2, T3, T4, and control were 32.47%, 47.68%, 65.37%, 78.82%, and 45.79%. High-performance liquid chromatography (HPLC) analysis revealed that concentrations of citric acid, lactic acid, acetic acid, and propionic acid in mature T4 were 2389, 16,468, 126, and 141 mg/kg. Analysis of cheese samples on gas chromatography–mass spectrometry (GC-MS) showed that fatty acids composition was not influenced by the supplementation of lactoferrin. Peroxide value of matured T1, T2, T3, T4, and control was 0.31, 0.30, 0.28, 0.35, and 0.28 (meqO2/kg) with no variation in color, flavor and texture score. The results of the current investigation proved that lactoferrin can be used for the supplementation of cheddar cheese.

Gated Ethidium- and Bleomycin-Loading in Phage T4 That Is Subsequently Purified Leak-Free

Chemotherapy-inhibiting tumor cell evolution to drug-resistance is potentially suppressed by using a drug delivery vehicle (DDV) that has gating. Gating would be used to increase tumor-selectivity of delivery of DDV packaged drug. Tumor-selectivity increase would make possible increase in tumor-delivered drug dose, which would suppress opportunities to evolve drug resistance. Currently used DDVs do not have gating but gating is a natural feature of some bacteriophages (phages). Phage T4, which has recently been found highly persistent in murine blood, is a potential gated DDV. Thus, here, we proceed towards a T4-DDV by developing (1) improved procedure for generating high concentrations and amounts of phage T4, (2) elevated temperature-driven gate-opening and ethidium- and bleomycin-loading, and (3) purification of loaded T4 by rate zonal centrifugation. We test for loading by native agarose gel electrophoresis (AGE) with fluorescence detection. We observe loading in both phage T4 and T4 (tail-free) heads. The loaded particles have an openable, closed gate. Stored, mature T4 phages and phage heads do not release ethidium during at least a month at 4 °C and 6 days at 37 and 42 °C. Tumor-specific T4 phage delivery is projected via both the EPR effect and high T4 persistence.

Structural changes in bacteriophage T7 upon receptor-induced genome ejection

Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.

Efeito da poda apical nos atributos morfofisiológicos do porta-enxerto clonal de seringueira GT1

Os porta-enxertos de seringueira requerem um período em viveiro para atingir o estádio de enxertia, sendo necessária a aplicação de práticas para otimizar o tempo de formação das mudas. O objetivo do trabalho foi avaliar o efeito da poda apical nos atributos morfofisiológicos dos porta-enxertos de seringueira GT1. O experimento foi realizado no período de maio de 2014 a maio de 2015 e os tratamentos consistiram em T1: sem poda apical; T2: poda apical realizada uma vez após o terceiro lançamento foliar maduro; T3: poda apical realizada mensalmente após o terceiro lançamento foliar maduro; T4: poda apical realizada uma vez após o quarto lançamento foliar maduro; T5: poda apical realizada mensalmente após o quarto lançamento foliar maduro; T6: poda apical realizada uma vez após o quinto lançamento foliar maduro e T7: poda apical realizada mensalmente após o quinto lançamento foliar maduro. As mensurações foram realizadas mensalmente, medindo-se o diâmetro e a altura das plantas, de 90 até 360 DAT (dias após o transplantio). Aos 360 DAT foram avaliados: massa seca de raiz, caule, folhas e massa seca total; número de folíolos; comprimento da raiz pivotante; volume do sistema radicular; área foliar e teores de elementos minerais nas folhas. A poda apical influenciou no diâmetro do caule, da altura, da massa seca de caule, da massa seca foliar e da área foliar das plantas. Plantas submetidas às podas apicais apresentaram a massa seca de raiz, a massa seca total, o comprimento de raiz, o volume de raiz e o número de folíolos semelhantes às não despontadas. Os tratamentos com poda apical realizada mensalmente apresentaram maior concentração de elementos minerais nas folhas. A poda apical realizada mensalmente após o quarto lançamento foliar maduro possibilitou o desenvolvimento em diâmetro dos porta-enxertos de seringueira em período semelhante às plantas não despontadas, porém aliado a uma menor altura, que facilita os tratos culturais.

Seed targeted RNAi-mediated silencing of GmMIPS1 limits phytate accumulation and improves mineral bioavailability in soybean

Phytic acid (PA), the major phosphorus reserve in soybean seeds (60–80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.

Forced phage uncorking: viral DNA ejection triggered by a mechanically sensitive switch.

The foremost event of bacteriophage infection is the ejection of genomic material into the host bacterium after virus binding to surface receptor sites. How ejection is triggered is yet unknown. Here we show, in single mature T7 phage particles, that tapping the capsid wall with an oscillating atomic-force-microscope cantilever triggers rapid DNA ejection via the tail complex. The triggering rate increases exponentially as a function of force, following transition-state theory, across an activation barrier of 23 kcal mol-1 at 1.2 nm along the reaction coordinate. The conformation of the ejected DNA molecule revealed that it had been exposed to a propulsive force. This force, arising from intra-capsid pressure, assists in initiating the ejection process and the transfer of DNA across spatial dimensions beyond that of the virion. Chemical immobilization of the tail fibers also resulted in enhanced DNA ejection, suggesting that the triggering process might involve a conformational switch that can be mechanically activated either by external forces or via the tail-fiber complex.

Molecular Rearrangements Involved in the Capsid Shell Maturation of Bacteriophage T7

Maturation of dsDNA bacteriophages involves assembling the virus prohead from a limited set of structural components followed by rearrangements required for the stability that is necessary for infecting a host under challenging environmental conditions. Here, we determine the mature capsid structure of T7 at 1 nm resolution by cryo-electron microscopy and compare it with the prohead to reveal the molecular basis of T7 shell maturation. The mature capsid presents an expanded and thinner shell, with a drastic rearrangement of the major protein monomers that increases in their interacting surfaces, in turn resulting in a new bonding lattice. The rearrangements include tilting, in-plane rotation, and radial expansion of the subunits, as well as a relative bending of the A- and P-domains of each subunit. The unique features of this shell transformation, which does not employ the accessory proteins, inserted domains, or molecular interactions observed in other phages, suggest a simple capsid assembling strategy that may have appeared early in the evolution of these viruses.

Mechanistic Coupling of Bacteriophage T4 DNA Packaging to Components of the Replication-dependent Late Transcription Machinery

Regulation of the terminal stage of viral DNA development, DNA packaging, is poorly understood. A new phage T4 in vitro DNA packaging assay employed purified proheads, terminase (gp17 + gp16), and ATP to encapsidate DNA resistant to nuclease. Mature phage T4 DNA and linearized plasmid DNAs containing or lacking a cloned T4 gene were packaged with high (approximately 10%) efficiency. Supercoiled, relaxed covalently closed, and nicked circular plasmid DNAs were packaged inefficiently, if at all, by these components. However, efficient packaging is achieved for nicked circular plasmid DNA, but not covalently closed plasmid DNA, upon addition to packaging mixtures of the purified T4 late transcription-replication machinery proteins: gp45 (sliding clamp), gp44/gp62 (clamp loader complex), gp55 (late sigma-factor), and gp33 (transcriptional co-activator). The small terminase subunit (gp16) is inhibitory for packaging linear DNAs, but enhances the transcription-replication protein packaging of nicked plasmid DNA. Taken together with genetic and biochemical evidence of a requirement for gp55 for concatemer packaging to assemble active wild-type phage particles (1), the plasmid packaging results show that initiation of phage T4 packaging on "endless" concatemeric DNA in vivo by terminase depends upon interaction with the DNA loaded gp45 coupled late transcription-replication machinery. The results suggest a close mechanistic connection in vivo between DNA packaging and developmentally concurrent replication-dependent late transcription.

Maturation of phage T7 involves structural modification of both shell and inner core components

The double-stranded DNA bacteriophages are good model systems to understand basic biological processes such as the macromolecular interactions that take place during the virus assembly and maturation, or the behavior of molecular motors that function during the DNA packaging process. Using cryoelectron microscopy and single-particle methodology, we have determined the structures of two phage T7 assemblies produced during its morphogenetic process, the DNA-free prohead and the mature virion. The first structure reveals a complex assembly in the interior of the capsid, which involves the scaffolding, and the core complex, which plays an important role in DNA packaging and is located in one of the phage vertices. The reconstruction of the mature virion reveals important changes in the shell, now much larger and thinner, the disappearance of the scaffolding structure, and important rearrangements of the core complex, which now protrudes the shell and interacts with the tail. Some of these changes must originate by the pressure exerted by the DNA in the interior of the head.

The tail structure of bacteriophage T4 and its mechanism of contraction

Bacteriophage T4 and related viruses have a contractile tail that serves as an efficient mechanical device for infecting bacteria. A three-dimensional cryo-EM reconstruction of the mature T4 tail assembly at 15-A resolution shows the hexagonal dome-shaped baseplate, the extended contractile sheath, the long tail fibers attached to the baseplate and the collar formed by six whiskers that interact with the long tail fibers. Comparison with the structure of the contracted tail shows that tail contraction is associated with a substantial rearrangement of the domains within the sheath protein and results in shortening of the sheath to about one-third of its original length. During contraction, the tail tube extends beneath the baseplate by about one-half of its total length and rotates by 345 degrees , allowing it to cross the host's periplasmic space.

Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry

Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor.

Molecular architecture of the prolate head of bacteriophage T4

The head of bacteriophage T4 is a prolate icosahedron with one unique portal vertex to which the phage tail is attached. The three-dimensional structure of mature bacteriophage T4 head has been determined to 22-A resolution by using cryo-electron microscopy. The T4 capsid has a hexagonal surface lattice characterized by the triangulation numbers T(end) = 13 laevo for the icosahedral caps and T(mid) = 20 for the midsection. Hexamers of the major capsid protein gene product (gp)23* and pentamers of the vertex protein gp24*, as well as the outer surface proteins highly antigenic outer capsid protein (hoc) and small outer capsid protein (soc), are clearly evident in the reconstruction. The size and shape of the gp23* hexamers are similar to the major capsid protein organization of bacteriophage HK97. The binding sites and shape of the hoc and soc proteins have been established by analysis of the soc(-) and hoc(-)soc(-) T4 structures.

Role of His-16 in Turnover of T4 Pyrimidine Dimer Glycosylase

Previously, the histidine residue at position 16 in the mature T4 pyrimidine dimer glycosylase (T4-PDG) protein has been suggested to be involved in general (non-target) DNA binding. This interpretation is likely correct, but, in and of itself, cannot account for the most dramatic phenotype of mutants at this position: their inability to restore ultraviolet light resistance to a DNA repair-deficient Escherichia coli strain. Accordingly, this residue has been mutated to serine, glutamic, aspartic acid, lysine, cysteine, and alanine. The mutant proteins were expressed, purified, and their abilities to carry out several functions of T4-PDG were assessed. The mutant proteins were able to perform most functions tested in vitro, albeit at reduced rates compared with the wild type protein. The most likely explanation for the biochemical phenotypes of the mutants is that the histidine residue is required for rapid turnover of the enzyme. This role is interpreted and discussed in the context of a reaction mechanism able to account for the complete spectrum of products generated by T4-PDG during a single turnover cycle.

Molecular Mechanisms In Bacteriophage T7 Procapsid Assembly, Maturation, And Dna Containment

Bacteriophage T7 is a double-stranded DNA bacteriophage that has attracted particular interest in studies of gene expression and regulation and of morphogenesis, as well as in biotechnological applications of expression vectors and phage display. We report here studies of T7 capsid assembly by cryoelectron microscopy and image analysis. T7 follows the canonical pathway of first forming a procapsid that converts into the mature capsid, but with some novel variations. The procapsid is a round particle with an icosahedral triangulation number of 7 levo, composed of regular pentamers and elongated hexamers. A singular vertex in the procapsid is occupied by the connector/portal protein, which forms 12-fold and 13-fold rings when overexpressed, of which the 12-mer appears to be the assembly-competent form. This vertex is the site of two symmetry mismatches: between the connector and the surrounding five gp 10 hexamers; and between the connector and the 8-fold cylindrical core mounted on its inner surface. The scaffolding protein, gp9, which is required for assembly, forms nubbin-like protrusions underlying the hexamers but not the pentamers, with no contacts between neighboring gp9 monomers. We propose that gp9 facilitates assembly by binding to gp10 hexamers, locking them into a morphogenically correct conformation. gp9 is expelled as the procapsid matures into the larger, thinner walled, polyhedral capsid. Several lines of evidence implicate the connector vertex as the site at which the maturation transformation is initiated: in vivo, maturation appears to be triggered by DNA packaging whereby the signal may involve interaction of the connector with DNA. In the mature T7 head, the DNA is organized as a tightly wound coaxial spool, with the DNA coiled around the core in at least four and perhaps as many as six concentric shells.

The Chaperonins of Synechocystis PCC 6803 Differ in Heat Inducibility and Chaperone Activity

The chaperonins GroEL and Cpn60 were isolated from the cyanobacterium Synechocystis PCC 6803 and characterized. In cells grown under optimal conditions their ratio was about one to one. However, the amount of GroEL increased considerably more than that of Cpn60 in response to heat stress. The labile chaperonin oligomer required stabilization by MgATP or glycerol during isolation. Use of the E. coli mutant strain, groEL44 revealed that the functional properties of the two cyanobacterial chaperonins are strikingly different. Overexpression of cyanobacterial GroEL in the E. coli mutant strain allowed growth at elevated temperature, the formation of mature bacteriophage T4, and active Rubisco enzyme assembly. In contrast, Cpn60 partially complemented the temperature-sensitive phenotype, the Rubisco assembly defect and did not promote the growth of the bacteriophage T4. The difference in chaperone activity of the two cyanobacterial chaperonins very probably reflects the unique chaperonin properties required during the life of Synechocystis PCC 6803.

Bacteriophage T4 self-assembly: localization of gp3 and its role in determining tail length.

Gene 3 of bacteriophage T4 participates at a late stage in the T4 tail assembly pathway, but the hypothetical protein product, gp3, has never been identified in extracts of infected cells or in any tail assembly intermediate. In order to overcome this difficulty, we expressed gp3 in a high-efficiency plasmid expression vector and subsequently purified it for further analysis. The N-terminal sequence of the purified protein showed that the initial methionine had been removed. Variant C-terminal amino acid sequences were resolved by determining the cysteine content of the protein. The molecular mass of 20.6 kDa for the pure protein was confirmed by Western blotting, using a specific anti-gp3 serum for which the purified protein was the immunogen. We also demonstrated, for the first time, the physical presence of gp3 in the mature T4 phage particle and localized it to the tail tube. By finding a nonleaky, nonpermissive host for a gene 3 mutant, we could clearly demonstrate a new phenotype: the slow, aberrant elongation of the tail tube in the absence of gp3.

Polymorphism of bacteriophage T7

For viruses made of nucleic acid and protein, the structure of the protein outer shell has, in the past, been found to be uniquely determined by the viral genome. However, here, non-denaturing agarose gel electrophoresis of bacteriophage T7 reveals two states of the mature T7 capsid; the conditions of growth are found to alter the population by T7 of these two electrophoretically defined states. Both states have been previously observed for a genetically altered T7 and they are observed here for wild-type T7. The average electrical surface charge density of a bacteriophage particle (δ) determines its state; the δ of particles in both states is negative. For a given condition of growth, the population of these two states is influenced by the extent to which the major T7 outer shell protein, p10A, is accompanied by its minor readthrough variant, p10B. Comparison of the two electrophoretic states reveals the following. (1) No difference in radius is present in the outer shell (±2%). (2) As the pH of electrophoresis is either increased or decreased from neutrality, the state becomes more highly populated for which δ is greater in magnitude (state 1). By changing the pH, some T7 particles are made to change state. (3) Particles in state 1 adsorb less quickly to host cells than do the particles in the alternative state (state 2). This latter observation suggests the hypothesis that state 1 evolved to reduce the probability of re-initiating an infection when conditions are not favorable for growth. This hypothesis is supported by the observation that, as conditions of growth become apparently more unfavorable, progeny increasingly populate state 1.

Specific Single-Stranded Breaks in Mature Bacteriophage T7 DNA

Both rate zonal centrifugation and gel electrophoresis have revealed that the mature double-stranded DNA of bacteriophage T5 has single-stranded breaks (nicks) at specific sites. Neither of those procedures has previously revealed site-specific nicks in the double-stranded DNA of other bacteriophages, including T7. In the present study, denaturing gel electrophoresis, followed by specific DNA detection, reveals that a small fraction of mature T7 DNA molecules, like most T5 DNA molecules, has site-specific nicks. The procedure of specific detection is to probe with an oligonucleotide specific for one of the ends of T7 DNA. If position 0.0 is the left genetic end and position 100.0 is the right genetic end of T7 DNA, the nicks on the 5′ left-oriented strand are at 11.3, 12.4, 65.7, 79.2, and 86.0; the nicks on the 5′ right-oriented strand are at 23.3 and 26.5 (±0.5). The positions of the three rightmost nicks are indistinguishable from those of double-stranded breaks that produce previously demonstrated shorter than mature length DNAs packaged in vivo. We propose that the T7 nicks are produced by premature activity of the T7 terminase during DNA packaging.

Specific, Nonproductive Cleavage of Packaged Bacteriophage T7 DNA in Vivo

The morphogenesis of bacteriophage T7 includes assembly of a procapsid that subsequently both packages DNA and changes in structure. The DNA packaged by T7 is concatemeric and is cleaved to mature length during packaging. In the present study, packaged DNA obtained from T7-infected cells was analyzed after release from DNase-treated capsids. After fractionation by agarose gel electrophoresis, in-gel probing with oligonucleotides reveals that some of this DNA is shorter than mature T7 DNA; most of this short DNA has the T7 right end, but not the left end. Some of this short, packaged DNA is the product of left-to-right injection of DNA at the beginning of a T7 infection. However, subsequently produced short, packaged DNA has characteristics of a DNA that was produced during DNA packaging (incompletely packaged DNA or ipDNA). In contrast to results previously obtained in vitro, the profile of right-end-containing ipDNA is sometimes dominated by discrete bands. Some of the band-forming right-end-containing ipDNA appears with the kinetics of an abortive end product of packaging; cleavage in vivo appears to have arrested DNA packaging in this case. Other band-forming right-end-containing ipDNA appears with kinetics that have some characteristics expected of a precursor to the mature DNA; cleavage appears to have occurred after arrest of packaging in this case. The finding here of both left-to-right injection and right-to-left packaging is the most direct demonstration of polarity for these events in vivo.

Purification and Characterization of Giant Empty Proheads from Packaging-Defective 23ptg Mutants of Bacteriophage T4

We constructed packaging-defective gene 23ptg mutants of bacteriophage T4 that produced giant proheads of varying lengths. The giant proheads constituted up to 20% of the total proheads. A purification protocol was developed to prepare large quantities of highly pure giant proheads. The purified giant proheads appeared to package both mature T4 DNA and concatemeric AD10 plasmid DNA in vitro.