Growth Of T7 Research Articles

Bactericidal activity of some plant essential oils against Ralstonia solanacearum infection

Potato plants and their tubers in Egypt are affected by one of the most renowned soil-borne pathogen, Ralstonia solanacearum, that caused brown rot in potato tubers and wilt in plants. There is no efficient therapeutic bactericide so; control of bacterial wilt is very rough.The study investigated three different concentrations of seven essential plant oils under in vitro and in vivo conditions as a result of their effects on Ralstonia solanacearum growth and their possibility use as potato seed pieces dressing for controlling bacterial wilt disease incidence. In vitro, anise oil at the three tested different concentrations (0.04, 0.07, and 0.14% vol/vol) was the most effective one inhibiting the growth of T4 and W9 isolates of Ralstonia solanacearum then pursued by thyme, lemongrass, and clove oils. On the other hand, rocket oil at the tested concentration was the least effective one followed by fennel oil. However, wheat germ oil was not completely effective. In vivo, experiment revealed that anise oil at the three concentrations significantly reduced disease incidence and severity in sponta and hermes potato cultivars and their effect was associated with increase of peroxidase, polyphenoloxidase, phenols and the foliar fresh weight of treated plants as well as the weight of tubers/plant followed by thyme and lemongrass oils compared to the infected untreated control.Morphological differences in bacterial cell structure have been observed using a transmission electron microscope (TEM). Anise oil at higher concentration caused of cell wall rupture and degraded cellular components.

Fe3+ enhanced degradation of oxytetracycline in water by pseudomonas

The application and fate of antibiotics are closely related to human health and the ecological balance, which has gradually aroused the widespread global concerns. Long-term antibiotic residues can easily induce antibiotic resistance and antibiotic resistance genes (ARGs) in the environment. Although many studies have investigated the metabolic pathways of biosynthesis or degradation of oxytetracycline (OTC) and its influencing factors under laboratory or controlled conditions, the understanding of OTC degradation pathways and influencing factors in the environment is still poor. In the present study, the role of Pseudomonas (T4) in OTC biodegradation were investigated with different carbon sources, metal ions, substrate concentrations, temperatures, and pH values, as well as the temporal changes in the relative abundance of OTC ARGs. It was found that OTC could be degraded by T4 as a sole carbon source. Comparison with Cu2+, the addition of Fe3+ could significantly promote the growth of T4, and then increased the OTC degradation percentage to 65.3%. The initial concentration of OTC, temperature, and pH had significant impacts on OTC degradation. At the initial OTC concentration of 50 mg L−1, the percentage degradation of OTC by T4 could reach 81.0% at the presence of Fe3+, and at 40 °C and pH = 7. Common tetracycline ARGs were not found during the OTC degradation by T4 in the present study. The eight main putative OTC degradation byproducts were identified by ultra-high definition accurate-mass quadrupole time-of-flight tandem mass spectrometry (QTOF/MS). Six different reaction types and seven possible degradation pathways were proposed, including enol-ketone conversion, hydroxylation, dehydration, deamination, demethylation and decarbonylation. Under optimal conditions, the OTC degradation percentages by T4 could reach to 88.2%, 91.6% and 92.0% in pond water, fish wastewater and industrial wastewater, respectively. These results demonstrate the high effectiveness of T4 at the presence of Fe3+ for the enhanced biodegradation of OTC in water environment, without resulting in the occurrence of ARGs. This has important implications for the removal of OTC from aquatic environments by the technology proposed from this study.

Hydrophobic Residue in Escherichia coli Thioredoxin Critical for the Processivity of T7 DNA Polymerase.

Bacteriophage T7 uses the thioredoxin of its host, Escherichia coli, to enhance the processivity of its DNA polymerase, a requirement for the growth of phage T7. The evolutionarily conserved structure and high degree of homology of amino acid sequence of the thioredoxin family imply that homologues from other organisms might also interact with T7 DNA polymerase to support the phage growth. Despite the structural resemblance, human thioredoxin, whose X-ray crystallographic structure overlaps with that of the E. coli protein, cannot support T7 phage growth. It does not form a complex with T7 DNA polymerase as determined by surface plasmon resonance and thus does not increase the processivity. Homologous scanning analysis using this nonfunctional homologue reveals that the 60 N-terminal and the 12 C-terminal amino acid residues of E. coli thioredoxin can be substituted for its human counterpart without significantly affecting phage growth. Comparison of chimeric thioredoxins, followed by site-directed mutagenesis, identifies leucine 95 as a critical element. This residue may contribute to hydrophobic interactions with the thioredoxin-binding loop of the polymerase; levels of DNA binding and thus nucleotide polymerization are significantly decreased in the absence of this residue. The results suggest that the specific interactions at the interface of thioredoxin and DNA polymerase, rather than the overall structure, are important in the interactions that promote high processivity.

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.

Bacteriophage T7 DNA Translocation during Infection is Bolstered by the Host ATP Synthase Complex

Translocation of bacteriophage T7 dsDNA into its host cell is an integral step in infection initiation that is energy dependent and is catalyzed principally by transcription per host and viral RNA polymerases. Here we show utilizing high-throughput cryo-electron tomography, a hexameric ring complex exists below the T7 trans-envelope channel in the host-cell cytoplasm during infection. Using sub-tomogram averaging and extensive classification we isolated T7 in the brief stage of genome translocation in coordination with the ring. This host-cell nanomachine is ∼30nm in diameter, ∼8nm in height with a ∼10nm channel. The complex was not observed in ATP synthase-deficient Escherichia coli mutants, suggesting that the complex functions in an energy-dependent manner. T7 grows in these mutant strains, and thus DNA ejection is not dependent on the complex. The ring is not host-specific; it has also been observed in an infected rough strain of Salmonella typhimurium, which supports the growth of T7. These results, only obtainable by in situ structural analysis, provide details on the essential process of DNA translocation and reveal the ability of T7 to recruit host-cell machinery to aid in the initiation of infection.

Coupling dTTP Hydrolysis with DNA Unwinding by the DNA Helicase of Bacteriophage T7

The DNA helicase encoded by gene 4 of bacteriophage T7 assembles on single-stranded DNA as a hexamer of six identical subunits with the DNA passing through the center of the toroid. The helicase couples the hydrolysis of dTTP to unidirectional translocation on single-stranded DNA and the unwinding of duplex DNA. Phe(523), positioned in a β-hairpin loop at the subunit interface, plays a key role in coupling the hydrolysis of dTTP to DNA unwinding. Replacement of Phe(523) with alanine or valine abolishes the ability of the helicase to unwind DNA or allow T7 polymerase to mediate strand-displacement synthesis on duplex DNA. In vivo complementation studies reveal a requirement for a hydrophobic residue with long side chains at this position. In a crystal structure of T7 helicase, when a nucleotide is bound at a subunit interface, Phe(523) is buried within the interface. However, in the unbound state, it is more exposed on the outer surface of the helicase. This structural difference suggests that the β-hairpin bearing the Phe(523) may undergo a conformational change during nucleotide hydrolysis. We postulate that upon hydrolysis of dTTP, Phe(523) moves from within the subunit interface to a more exposed position where it contacts the displaced complementary strand and facilitates unwinding.

Gene 1.7 of Bacteriophage T7 Confers Sensitivity of Phage Growth to Dideoxythymidine

Bacteriophage T7 has long been used as a model for studying DNA replication. One of the interesting features of this system is the ability of T7 DNA polymerase to efficiently incorporate dideoxynucleotides into DNA, resulting in chain termination. We show that dideoxythymidine (ddT) present in the media at levels not toxic to the host Escherichia coli results in inhibition of phage T7 growth. We have isolated 100 T7 phage that are resistant to ddT. Surprisingly, all contain a mutation in T7 gene 1.7, a nonessential gene of unknown function. When T7 gene 1.7 is expressed from a plasmid, suppressor T7 phage that are resistant to ddT still arise; analysis of 36 of these mutants reveals that all have mutations in gene 5, that encodes the T7 DNA polymerase. The strongest suppressors change tyrosine 526 of gene 5 to phenylalanine. This mutation is known to dramatically increase the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. These results suggest that in the presence of the T7 gene 1.7 protein, ddT is inhibitory to phage T7 because it acts as a chain terminator for DNA synthesis. In support of this, whereas DNA synthesis in E. coli infected with wild‐type T7 is inhibited by ddT, no inhibition is observed in the absence of gene 1.7. Overexpression of gene 1.7 from a plasmid renders E. coli cells sensitive to ddT in the absence of T7 phage, indicating that gene 1.7 protein does not require other T7 proteins to confer sensitivity to ddT. We propose that gene 1.7 protein plays a critical role in the conversion of ddT to ddTTP.

Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage growth on hosts overexpressing udk

Overexpression of udk, an Escherichia coli gene encoding a uridine/cytidine kinase, interferes with T7 bacteriophage growth. We show here that inhibition of T7 phage growth by udk overexpression can be overcome by inhibition of host RNA polymerase. Overexpression of gene 2, whose product inhibits host RNA polymerase, restores T7 phage growth on hosts overexpressing udk. In addition, rifampicin, an inhibitor of host RNA polymerase, restores the burst size of T7 phage on udk-overexpressing hosts to normal. In agreement with these findings, suppressor mutants that overcome the inhibition arising from udk overexpression gain the ability to grow on hosts that are resistant to inhibition of RNA polymerase by gene 2 protein, and suppressor mutants that overcome a lack of gene 2 protein gain the ability to grow on hosts that overexpress udk. Mutations that eliminate or weaken strong promoters for host RNA polymerase in T7 DNA, and mutations in T7 gene 3.5 that affect its interaction with T7 RNA polymerase, also reduce the interference with T7 growth by host RNA polymerase. We propose a general model for the requirement of host RNA polymerase inhibition.

Chi hotspot activity in Escherichia coli without RecBCD exonuclease activity: implications for the mechanism of recombination.

The major pathway of genetic recombination and DNA break repair in Escherichia coli requires RecBCD enzyme, a complex nuclease and DNA helicase regulated by Chi sites (5'-GCTGGTGG-3'). During its unwinding of DNA containing Chi, purified RecBCD enzyme has two alternative nucleolytic reactions, depending on the reaction conditions: simple nicking of the Chi-containing strand at Chi or switching of nucleolytic degradation from the Chi-containing strand to its complement at Chi. We describe a set of recC mutants with a novel intracellular phenotype: retention of Chi hotspot activity in genetic crosses but loss of detectable nucleolytic degradation as judged by the growth of mutant T4 and lambda phages and by assay of cell-free extracts. We conclude that RecBCD enzyme's nucleolytic degradation of DNA is not necessary for intracellular Chi hotspot activity and that nicking of DNA by RecBCD enzyme at Chi is sufficient. We discuss the bearing of these results on current models of RecBCD pathway recombination.

Genomewide screens for Escherichia coli genes affecting growth of T7 bacteriophage.

Use of bacteriophages as a therapy for bacterial infection has been attempted over the last century. Such an endeavor requires the elucidation of basic aspects of the host-virus interactions and the resistance mechanisms of the host. Two recently developed bacterial collections now enable a genomewide search of the genetic interactions between Escherichia coli and bacteriophages. We have screened >85% of the E. coli genes for their ability to inhibit growth of T7 phage and >90% of the host genes for their ability to be used by the virus. In addition to identifying all of the known interactions, several other interactions have been identified. E. coli CMP kinase is essential for T7 growth, whereas overexpression of the E. coli uridine/cytidine kinase inhibits T7 growth. Mutations in any one of nine genes that encode enzymes for the synthesis of the E. coli lipopolysaccharide receptor for T7 adsorption leads to T7 resistance. Selection of T7 phage that can recognize these altered receptors has enabled the construction of phage to which the host is 100-fold less resistant.

Evolutionary design on a budget: robustness and optimality of bacteriophage T7

Exploring how biological systems have been 'designed' by evolution to achieve robust behaviours is now a subject of increasing research effort. Yet, it still remains unclear how environmental factors may contribute to this process. This issue is addressed by employing a detailed computer model for the intracellular growth of phage T7. More than 150 000 in silico T7 mutants were generated and the rates and efficiencies of their growth in two host environments, namely, a realistic environment that offered finite host resources for the synthesis of phage functions and a hypothetical environment where the phage was supplied infinite host resources, were evaluated. Results revealed two key properties of phage T7. First, T7 growth was overall robust with respect to perturbations in its parameters, but fragile with respect to changes in the ordering of its genetic elements. Secondly, the wild-type T7 had close to optimal fitness in the finite environment. Furthermore, a strong correlation was found between fitness and growth efficiency in the finite environment. The results underscore the potential importance of the environment in shaping robust design of a biological system. In particular, the strong correlation between fitness and growth efficiency suggests that T7 may have evolved to maximise its growth rate by minimising waste of finite resources.

Acidic Residues in the Nucleotide-binding Site of the Bacteriophage T7 DNA Primase

DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.

Utilization of Fruit Processing Wastes in the Diet of Labeo rohita Fingerling

A feeding trial was conducted for 60 days to study the utilization of fruits processing wastes as feed ingredient in the diet of Labeo rohita fingerlings. One hundred and sixty fingerlings (av. body weight, 1.65 g±0.03) were equally distributed in four experimental groups having 4 replicates each. Four different experimental diets were prepared by replacing wheat flour and rice bran with either orange (T2) (Cirtus qurantium), pineapple (T3) (Ananas spp. and Pseudananas spp.) or sweet lime (T4) (Citrus sinensis) wastes to the basal diet along with the control (T1, without any fruit wastes) keeping the CP level at around 40%. The water quality parameters like DO, CO2, pH, total alkalinity, total hardness, ammonia and water temperature were recorded within the optimum range. The diet containing 25% pineapple wastes (T3) showed significantly higher growth in terms of SGR (1.50), FCR (2.09) and PER (1.19) than the other groups. However, growth of T4 and T2 groups were not significantly different than the control group (T1). Protease activity (17.17 unit/mg protein), protein digestibility (91.57%) and carbohydrate digestibility (41.62%) were not significantly different among the different groups. Survival of the fingerlings were not significantly different among the experimental groups. It concludes that waste of orange, pineapple and sweet lime can be used at 25% level as a substitute of wheat flour and rice bran in the diet of Labeo rohita. (Asian-Aust. J. Anim. Sci. 2003. Vol 16, No. 11 : 1661-1665)

Effects of Escherichia coli physiology on growth of phage T7 in vivo and in silico.

Phage development depends not only upon phage functions but also on the physiological state of the host, characterized by levels and activities of host cellular functions. We established Escherichia coli at different physiological states by continuous culture under different dilution rates and then measured its production of phage T7 during a single cycle of infection. We found that the intracellular eclipse time decreased and the rise rate increased as the growth rate of the host increased. To develop mechanistic insight, we extended a computer simulation for the growth of phage T7 to account for the physiology of its host. Literature data were used to establish mathematical correlations between host resources and the host growth rate; host resources included the amount of genomic DNA, pool sizes and elongation rates of RNA polymerases and ribosomes, pool sizes of amino acids and nucleoside triphosphates, and the cell volume. The in silico (simulated) dependence of the phage intracellular rise rate on the host growth rate gave quantitatively good agreement with our in vivo results, increasing fivefold for a 2.4-fold increase in host doublings per hour, and the simulated dependence of eclipse time on growth rate agreed qualitatively, deviating by a fixed delay. When the simulation was used to numerically uncouple host resources from the host growth rate, phage growth was found to be most sensitive to the host translation machinery, specifically, the level and elongation rate of the ribosomes. Finally, the simulation was used to follow how bottlenecks to phage growth shift in response to variations in host or phage functions.

Dependence of epistasis on environment and mutation severity as revealed by in silico mutagenesis of phage t7.

Understanding how interactions among deleterious mutations affect fitness may shed light on a variety of fundamental biological phenomena, including the evolution of sex, the buffering of genetic variations, and the topography of fitness landscapes. It remains an open question under what conditions and to what extent such interactions may be synergistic or antagonistic. To address this question, we employed a computer model for the intracellular growth of bacteriophage T7. We created in silico 90,000 mutants of phage T7, each carrying from 1 to 30 mutations, and evaluated the fitness of each by simulating its growth cycle. The simulations sought to account for the severity of single deleterious mutations on T7 growth, as well as the effect of the resource environment on our fitness measures. We found that mildly deleterious mutations interacted synergistically in poor-resource environments but antagonistically in rich-resource environments. However, severely deleterious mutations always interacted antagonistically, irrespective of environment. These results suggest that synergistic epistasis may be difficult to experimentally distinguish from nonepistasis because its effects appear to be most pronounced when the effects of mutations on fitness are most challenging to measure. Our approach demonstrates how computer simulations of developmental processes can be used to quantitatively study genetic interactions at the population level.

Simulating the growth of viruses.

To explore how the genome of an organism defines its growth, we have developed a computer simulation for the intracellular growth of phage T7 on its E. coli host. Our simulation, which incorporates 30 years of genetic, biochemical, physiological, and biophysical data, is used here to study how the intracellular resources of the host, determined by the specific growth rate of the host, contribute toward phage development. It is also used to probe how changes in the linear organization of genetic elements on the T7 genome can affect T7 development. Further, we show how time-series trajectories of T7 mRNA and protein levels generated by the simulation may be used as raw data to test data-mining strategies, specifically, to identify partners in protein-protein interactions. Finally, we suggest how generalization of this work can lead to a knowledge-driven simulation for the growth of any virus.

In Vivo and in Vitro Function of GroEL Mutants with Impaired Allosteric Properties

Escherichia coli cells that produce only plasmid-encoded wild-type or mutant GroEL were generated by bacteriophage P1 transduction. Effects of mutations that affect the allosteric properties of GroEL were characterized in vivo. Cells containing only GroEL(R197A), which has reduced intra-ring positive cooperativity and inter-ring negative cooperativity in ATP binding, grow poorly upon a temperature shift from 25 to 42 degrees C. This strain supports the growth of phages T4 and T5 but not phage lambda and produces light at 28 degrees C when transformed with a second plasmid containing the lux operon. In contrast, cells containing only GroEL(R13G, A126V) which lacks negative cooperativity between rings but has intact intra-ring positive cooperativity grow normally and support phage growth but do not produce light at 28 degrees C. In vitro refolding of luciferase in the presence of this mutant is found to be less efficient compared with wild-type GroEL or other mutants tested. Our results show that allostery in GroEL is important in vivo in a manner that depends on the physiological conditions and is protein substrate specific.

Patterns of Regulation from mRNA and Protein Time Series

The rapid advance of genome sequencing projects challenges biologists to assign physiological roles to thousands of unknown gene products. We suggest here that regulatory functions and protein–protein interactions involving specific products may be inferred from the trajectories over time of their mRNA and free protein levels within the cell. The level of a protein in the cytoplasm is governed not only by the level of its mRNA and the rate of translation, but also by the protein's folding efficiency, its biochemical modification, its complexation with other components, its degradation, and its transport from the cytoplasmic space. All these co- and post translational events cause the concentration of the protein to deviate from the level that would result if we only accounted for translation of its mRNA. The dynamics of such deviations can create patterns that reflect regulatory functions. Moreover, correlations among deviations highlight protein pairs involved in potential protein–protein interactions. We explore and illustrate these ideas here using a genetically structured simulation for the intracellular growth of bacteriophage T7.

Toward antiviral strategies that resist viral escape.

We studied the effect on viral growth of drugs targeting different virus functions using a computer simulation for the intracellular growth of bacteriophage T7. We found that drugs targeting components of negative-feedback loops gain effectiveness against mutant viruses that attenuate the drug-target interaction. The greater inhibition of such mutants than of the wild type suggests a drug design strategy that would hinder the development of drug resistance.

Recombination and recombination-dependent DNA replication in bacteriophage T4.

General recombination is essential for growth of phage T4, because origin initiation of DNA replication is inactivated during development, and recombination-dependent initiation is necessary for continuing DNA replication. The requirement of recombination for T4 growth has apparently been a driving force to acquire and maintain multiple recombination mechanisms. This requirement makes this phage an excellent model to analyze several recombination mechanisms that appear redundant under optimal growth conditions but become essential under other conditions, or at different stages of the developmental program. The most important substrate for wild-type T4 recombination is single-stranded DNA generated by incomplete replication of natural or artificial chromosomal ends, or by nucleolytic degradation from induced breaks, or nicks. Recombination circumvents the further erosion of such ends. There are multiple proteins and multiple pathways to initiate formation of recombinants (by single-strand annealing or by strand invasion) and to convert recombinational intermediates into final recombinants ("cut and paste" or "cut and package"), or to initiate extensive DNA replication by "join-copy" or "join-cut-copy" mechanisms. Most T4 recombination is asymmetrical, favoring the initiation of replication. In wild-type T4 these pathways are integrated with physiological changes of other DNA transactions: mainly replication, transcription, and packaging. DNA replication and packaging enzymes participate in recombination, and recombination intermediates supply substrates for replication and packaging. The replicative recombination pathways are also important for transmission of intron DNA to intronless genomes ("homing"), and are implicated in horizontal transfer of foreign genes during evolution of the T-even phages. When horizontal transfer involves heteroduplex formation and repair, it is intrinsically mutagenic and contributes to generation of species barriers between phages.