Isolation and Identification of Bacillus megaterium Bacteriophages via AFLP Technique (original) (raw)
Isolation and Characterization of a Bacillus megaterium QM B1551 Bacteriophage
Journal of General Virology, 1977
A bacteriophage specific for Bacillus megaterium QM Br55I was isolated. This phage (MP-7) was a lytic phage of typical morphology and distinct stability properties. The DNA was double-stranded, with a mole ~ G + C of 38"9 + 0"7 and a mol. wt. of 42 to 44 x io 6. Bacillus megaterium QM BI55I has been used extensively to study the properties of spores and the mechanism of germination. While there are considerable biochemical data and a variety of mutants available (Hyatt & Levinson, I964; Vary, I972; Setlow, 1975), there is no method to study the genetics of this strain (Vary, I975). Towards this end, we have isolated a bacteriophage that is specific for B. megaterium QM BI55I which may be used (a) to identify this strain, (b) as a source of DNA for transfection studies, and (c) as a potential helper phage in attempts at isolating transducing particles. The properties of this bacteriophage are reported here. Bacteriophage were isolated and purified from the soil by the method of Adams 0959). One of the clear plaques (MP-7) was arbitrarily chosen for further study. Lysates were made from single plaques and assayed for plaque-forming units (p.f.u.) by the overlay method of Adams 0959)-The indicator cells were grown from B. megaterium QM BI55 r by suspending lyophilized spores (approx. io 6 spores) in I ml sterile H~O followed by heat activation at 6o °C for Io min and dilution into 5o ml of supplemented nutrient broth (SNB; Vary, I97z). The culture was incubated on a rotary shaker at 3oo rev/min and 3 o °C until the extinction at 66o nm was about o'5. The characteristics of MP-7 were as follows. The phage produced clear plaques surrounded by a halo as has been reported for other B. megaterium phages (Murphy, I957; Cooney, Jacob & Slepecky, I975). Electron microscopy showed that MP-7 had a hexagonal head of 6o to 7o nm in diam. and a thin tail about ~5 o nm long, not unlike other bacillus lytic and transducing phages. Host range studies showed that MP-7 was specific for B. megaterium strains QM BI55I and ATCC 7o5I and did not infect strains ATCC 9885 or ATCC 19213, B. cereus T, B. licheniformis A5 or B. subtilis I68 at either low (< o.I) or high (> Iooo) multiplicities of infection. With those strains of B. megaterium that were insensitive to MP-7, attempts at phage induction by ultraviolet light irradiation (Yelton & Thorne, I97o) were unsuccessful. In a one-step growth curve by the method of Adams (I959), the latent period was 6o to 65 min, similar to other bacillus phages. However, the eclipse time was 5o min and the burst size was I37O _+ I9o (average + standard deviation from 7 experiments). This large burst size was probably not a result of multiple infections because the multiplicities of infection were < o.I. Some of the other properties of MP-7 (Table I) were its stability to heating at 5o °C but not 6o °C, relatively high stability in organic solvents and sensitivity to salt. The inhibition by salt was a result of inhibition of adsorption similar to that described for Eseherichia eoli T phages and B. subtilis ¢25 phages (Kozloff & Henderson, I955; Doyle et al. I974).
Isolation of bacteriophage from soil sample
2016
Bacteriophage also known as phage, is a virus that attack to a specific bacteria and replicates inside bacteria cell to produce more phages. Bacteriophage can be found in sewage, soil, water and food. This virus kills bacteria by lysis the host cell. There are two cycles of phage which are Lytic Cycle and Lysogenic Cycle. The benefits of phage are they can be used to kill bacteria, as replacement of antibiotic, biological control agent, and phage application in food industry. Phage can also survive in any condition because they have ability to reproduce quickly, high level of specificity and long term survivability. Phages were isolated and partially purified using PEG/ NaCl. The titers of phages were determined by plaque assay methods. The soil sample has different population of bacteriophage. Phage is isolate from soil sample to determine what type of phage in soil. From this study, the characteristic of phage showed that the phage is a type of lytic phage because it has similarit...
A Laboratory Practicum on Screening for Lytic Bacteriophages from Soil Samples
Transactions on Science and Technology, 2018
Bacteriophages are viruses that explicitly infect and lyse bacteria. They are ubiquitous and play vital roles in various biotechnological applications such as phage-based bacterial detection and bacteriophage therapy. This paper describes a simple bacteriophage screening protocol for teaching in secondary and tertiary institutions. This laboratory practicum was designed to enable students explore the presence of bacteriophages from soil samples. The tendency of many bacteriophages to produce plaques that are small or excessively turbid on agar plates limits their proper visualisation and enumeration. One percent of TTC (2,3,5-Triphenyltetrazolium chloride) was incorporated during the pour plate technique to enhance visibility of bacteriophage plaques on Petri dishes. The isolated bacteriophages were then amplified in growth medium containing targeted bacterial hosts. The results showed presence of six bacteriophages with distinctive plaque morphologies. Students were then taken through bacteriophage amplification and storage procedures. A survey was conducted to evaluate student’s learning experience both prior and after laboratory classes. Analysis from the survey showed that only 26% of students attempted all questions from the pre-laboratory bacteriophage practicum questionnaire whereas 74% failed the survey. On the other hand, 53% of the students attempted all questions for the post-laboratory phage practicum questionnaire and 16% failed the survey.
Contribution to the Characterization of Bacillus megaterium
International Journal of Systematic Bacteriology, 1979
A study of the cellular and colonial morphologies, the deoxyribonucleic acid (DNA) base composition, the degree of lysis by lysozyme, and some fundamental biochemical reactions was carried out on 23 strains previously identified as members of Bacillus megaterium. Included among these was NCTC 10342, the neotype strain of B. megaterium. The results showed that, primarily on the basis of their biochemical reactions, two of the strains belong to B. cereus, two belong to B. subtilis, and 19 belong to B. megaterium. The first two strains mentioned above clearly differed from B. megaterium strains with respect to their DNA base composition (33.2 and 34.3 mol% guanine plus cytosine [G+C]), their lysis by lysozyme (5.6 and 6.6%), and the morphology of their macro-and microcolonies. These properties were not helpful in differentiating the other two strains from the B. megaterium strains, for the two strains showed high G+C values (45.9 and 47.4 mol%) and high values of lysis by lysozyme, (92.9 and 96.7%). The 19 remaining strains, confirmed as members of B. megaterium, had G+C contents which varied from 37.3 to 43.0 mol% and lysis percentages which varied from 67.0 to 94.9. For these strains, a significant (P < 0.05) correlation was found between the DNA base composition and lysis by lysozyme. Three biogroups were established on the basis of the biochemical reactions of the 19 strains. These biogroups are similar to the "aggregate" and the "intermediate" strains of B. megaterium established by Gordon et al.
Bacteriophages applications in agriculture
Humboldt kolleg Ibarra 2019, 2019
The bacteriophages life cycle has two stages: a lytic stage where the phages reproduce inside the bacteria and lyse bacteria and a lysogenic stage where the phage is in a stationary stage where do not exist phage reproduction. The understanding of the life cycle of phages is fundamental to understand the advantages of phage offers as biological control applications and how engineered phages work. The bacteriophages are an alternative to fight against the antimicrobial or pesticides because phages offer advantages such as high host specificity, the ability of long term effect, are active against dividing or not dividing bacterial cells, effective elimination of biofilms and are capable vehicles for nucleic acids delivery. Phages have been isolated from water or soil samples in different parts of the world and for specific bacterial pathogens. In the following review, in the main topics in bacteriophages and its applications in agriculture: the bacteriophages life cycle, advantages of...
Review: Indicator bacteriophages in sludge, biosolids, sediments and soils
Environmental Research, 2020
Solid or semisolid matrices polluted with fecal remnants can be highly loaded with pathogens, especially viruses, and play a substantial role in the persistence and dispersion of pathogens in the water cycle. Water quality regulations and guidelines are increasingly including bacteriophages infecting enteric bacteria as indicators of fecal and/or viral pollution. However, more data are needed about viral indicators in contaminated solids to develop effective sanitation strategies for the management of raw and treated sludge, fecal sludge, manures and slurries. Also, the exact role of sediments and soil in the transmission cycle of viral pathogens still needs to be determined. This review aims to provide an update on available data for concentrations of indicator bacteriophages in different solid matrices as well as their resistance to treatments and persistence in solids. The conclusion reached is that there is a need for improved and standardized methodologies for bacteriophage extraction, detection and enumeration in solids. Reports indicate that these contain higher levels of somatic coliphages in comparison with traditional bacterial indicators and F-specific RNA coliphages. Water body sediments and soil have been found to be notable reservoirs of somatic coliphages, which are more persistent in nature and resistant to sludge treatments than Escherichia coli and fecal coliforms and F-specific RNA coliphages. Thus, somatic coliphages show up as excellent complementary indicators for the prediction of pathogenic viruses in solids.
Journal of Visualized Experiments
Bacteriophage isolation from environmental samples has been performed for decades using principles set forth by pioneers in microbiology. The isolation of phages infecting Arthrobacter hosts has been limited, perhaps due to the low success rate of many previous isolation techniques, resulting in an underrepresented group of Arthrobacter phages available for study. The enrichment technique described here, unlike many others, uses a filtered extract free of contaminating bacteria as the base for indicator bacteria growth, Arthrobactersp. KY3901, specifically. By first removing soil bacteria the target phages are not hindered by competition with native soil bacteria present in initial soil samples. This enrichment method has resulted in dozens of unique phages from several different soil types and even produced different types of phages from the same enriched soil sample isolate. The use of this procedure can be expanded to most nutrient rich aerobic media for the isolation of phages in a vast diversity of interesting host bacteria.
Russian Journal of Genetics, 2006
Study of two recently isolated giant bacteriophages Lu11 and OBP that are active on Pseudomonas putida var. Manila and Pseudomonas fluorescens , respectively, demonstrated their similarity in morphotype, genome size, and size of phage particles, with giant bacteriophages of Pseudomonas aeruginosa assigned to the supergroup of φ KZ-like phages of the family Myoviridae. This supergroup was designated in this manner according to the best studied phage φ KZ that belongs to the species of this group widely distributed in nature. Comparison of major polypeptide sizes of mature particles suggests similarity of certain proteins in the phages examined. In OBP particles visualized with an electron microscope, an "inner body" was detected, which points to specific DNA package intrinsic to phages of φ KZ group. In the meantime, phages Lul1 and OBP do not exhibit resemblance among themselves or with any of earlier described φ KZ-like phages in respect to detectable DNA homology. Note that phage Lu11 of P. putida var. Manila exhibits very slight homology with phage Lin68 of the family of P. aeruginosa φ KZ-like phages detected only in blot hybridization. This suggests the possible involvement of these phages in interspecies recombination ("gene shuffling") between phages of various bacterial species. Results of partial sequencing of phage genomes confirmed the phylogenetic relatedness of phage OBP to phages of the φ KZ supergroup, whereas phage Lu11 most probably belongs to a novel species that is not a member of supergroup φ KZ composition. The results of the study are discussed in terms of the evolution of these phages.