Isolation Characterization Of Bacillus Thuringiensis Strains Biology Essay

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ABSTRACT:

Bacillus thuringiensis var israelensis (serotype: H14) a sporigen soil bacterium characterized by production of parasporal crystals during its sporulation composed of protein molecules of different weights ranging between 27-140KDa known as delta endotoxin or insecticidal crystal (cry) proteins against mosquito larvae. This endotoxin of Bti is effective against three orders of insect pests namely Lepidoptera, Diptera, and Coleoptera.. This study aimed to find native strains of Bacillus thuringiensis var israelensis (serotype: H14) a sporigen bacterium from different soils. Five Bt strains were isolated from soil samples collected from five different agriculture related habitats of Kancheepuram District in Tamilnadu, India. The isolates were screened for Bti by culturing in NYSM conventional medium (Li and Youstin, 1975) and these isolates were evaluated in terms of their novel activities according to the following criteria: Cellular morphology was determined from Gram-stained cultures grown for 24 h on NYSM agar at 37 °C using oil immersion microscopy. Isolates appearing as slender rods with a sub terminal, oval endospore in a swollen sporangium were retained. Strains on NYSM agar were stored at room temperature, subcultured in LB broth and subjected to separation and purification of crystals by sucrose gelatin gradient method (Priest et al.1988). These purified crystal proteins were subjected to quantitative and qualitative molecular characterization by ELISA and SDS-PAGE.

INTRODUCTION:

Bacillus thuringiensis scientific classification (TAXONOMY):

Kingdom: Eubacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales

Family: Bacillaceae

Genus: Bacillus

Species: thuringiensis

Figure 1: Bacillus thuringiensis  Figure 2: Proposed mode of action

Plates supplied by Neil Crickmore Diagrams provided by Neil Crickmore

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming soil bacterial species of genome size of 2.4 to 5.7 million base pairs which during its sporulation produces insecticidal parasporal crystal (cry) proteins used as highly specific insecticides in agriculture and forestry because they are specifically toxic to particular orders and species of insects, like Lepidoptera, Diptera, and Coleoptera which causes deadly diseases. Amongst vector borne diseases, malaria Anopheles mosquito occupies a predominant position since it is probably the leading cause of death in the world despite intense national and international efforts to control it (Pickett, 1990; Smyth, 1994).These genes are also used to engineer insect-resistant transgenic crops, which are widely cultivated .It also produces antibiotic compounds that are of antifungal activity. Cry proteins are also used as commercial insecticides. Since the genetic diversity and toxic potential of Bt strains differ from region to region, Bt strains have been collected and characterized all over the world from various habitats, including soil, stored-product dusts, insects, deciduous and coniferous sources.

Bacillus thuringiensis var. israelensis (Bti):

Bacillus thuringiensis israelensis (Serotype H-14) is a subspecies of the common insecticidal bacterium Bt. Bti-based products is one of the most efficient and the safest methods to control some larval mosquitoes, black flies' midges' populations. Their very specific and target-oriented mode of action of Bti makes it very safe for human health and non-target organisms. Bt strain was discovered with high toxicity to mosquito larvae (Goldberg and Margalit 1977) which was later identified and designated Bt var. israelensis, serotype H14 (de Barjac 1978).

OCCURRENCE

The occurrence of bacillus thuringiensis is identified in the following natural sources:

Soil sediments

Plants

Animal feces

Insects

Water

CLASSIFICATION OF Bt:

Bt are broadly classified into many significant varieties:

Bt subspecies  kurstaki - controls various types of lepidopterous insects. (Most commonly used)

Bt subspecies  israelensis - effective against mosquitoes, blackflies' and some midges. 

Bt subspecies tenebrionis - effective against certain beetle (chrysomelids) species and the boll weevil. 

Bt subspecies Japonensis - effective against many species of scarabid beetles. 

Bt subspecies aizawai - used against wax moth larvae in honeycombs.

Bt TOXIN AND THEIR CLASSIFICATION:

A class of crystalline pore forming proteins produced by strains of Bacillus thuringiensis, and engineered into crop plants to give resistance against insect pests. Their mechanism involves the lysis of midgut epithelial cells by inserting into the target cell membrane and forming pores. There are two types of toxins produced from Bt strains:

Cry(crystal) toxins

Cyt(cytolytic) toxins

Domain 1 = responsible for inserting into the gut membrane and creating a pore where ions can pass freely

Domain 2 = responsible for binding to the receptors on the epithelial lining of the midgut

Domain 3 = responsible to protect the endotoxin from cleavage by gut proteases, or may be involved in ion channel formation, receptor binding, and insect specificity

Cry PROTIN STRUCTURE:

GENETICS OF Bti:

Complete Sequence and Organization of pBtoxis, the Toxin-Coding Plasmid of Bacillus thuringiensis subsp.israelensis:

Figure 4: Circular representation of pBtoxis. The inner circle represents GC bias [(G - C)/(G + C)], with positive values in khaki and negative values in purple; the second circle represents G+C content; and the outer two circles represent predicted genes on the reverse and forward strands (selected CDSs are numbered for reference). Color coding for the genes is as follows: gray, toxin and peptide antibiotic; pink, transposon related; orange, conserved hypothetical; red, DNA metabolism; blue, regulatory; bright green, surface associated; pale green unknown; yellow, miscellaneous metabolic genes. The outer scale is marked in kilo bases. (Taken from Applied and Environmental Microbiology, October 2002, p. 5082-5095, Vol. 68, No. 10)

Tabe2: Some significant cry genes are listed below:

Gene

Crystal shape

Protein size(kDa)

Insect activity

cry I [several subgroups:

A(a), A(b), A(c), B, C, D, E, F, G]

bipyramidal

130-138

lepidoptera larvae

cry II [subgroups A, B, C]

cuboidal

69-71

lepidoptera and diptera

cry III [subgroups A, B, C]

flat/irregular

73-74

coleoptera

cry IV [subgroups A, B, C, D]

bipyramidal

73-134

diptera

cry V-IX

various

35-129

various

MODE OF ACTION OF CRY PROTEIN:

Bti bacterium produces a protein crystal which is toxic only to mosquito and black fly larvae during the spore-forming stage of its life cycle. When the insects feed, these microscopic crystals are ingested by insect larvae. The crystals are dissolved thus converted into toxic protein molecules which destroy the walls of the insect's stomach in the alkaline environment of the susceptible insect's digestive system. The insect stops feeding within hours and dies within days.

Figure 3:mode of action of bt toxin from http://web.utk.edu/jurat/

Bt BASED BIOPESTICIDES :

According to the United States Environmental Protection Agency (EPA), "biopesticides" are naturally occurring substances (biochemical pesticides) that control pests, microorganisms that control pests (microbial pesticides), and pesticidal substances produced by plants containing added genetic material, plant-incorporated protectants.

The strains of Bt characterized so far affect members of three insect orders:

Lepidoptera (butterfl ies and moths)

Diptera (mosquitoes and biting fl ies)

Coleoptera (beetles)

Commercially available Environmental Protection Agency-registered Bt products include:

B.t. aizawai (Lepidoptera)-used for wax moth larvae in honeycombs.

B.t. israelensis (Diptera)-frequently used for mosquitoes.

B.t. kurstaki (Lepidoptera)- frequently used for gypsy moth, spruce budworm, and many vegetable pests as it will kill many leaf-feeding larvae on vegetables, shrubs, fruit trees, and conifers.

B.t. san diego and tenebrionis (Coleoptera)-frequently used for elm leaf beetle, Colorado potato beetle.

Bt.japonensis and kumamotoensis (Coleoptera)-used on several turf beetle species.

Bt.gallerie (Coleoptera)-used on Japanese beetles.

COMMERCIAL PRODUCTION:

TABLE 4 : Some Bti products for mosquito and blackfly control (possibly not all currently available) (modified from Becker and Margalit 1993).

Product

Formulation

Company

Teknar TC

Powder

Novartis (sold by Triology)

Teknar HP-D

Fluid

Novartis (sold by Triology)

Teknar G

Granules

Novartis (sold by Triology)

VectoBac TP

Powder

Abbott Laboratories

VectoBac 12 AS

Fluid

Abbott Laboratories

VectoBac G

Granules

Abbott Laboratories

VectoBac CG

Abbott Laboratories

Bactimos WP

Powder

Abbott Laboratories

Bactimos G

Granules

Abbott Laboratories

Bactimos

Briquettes/pellets

Abbott Laboratories

Bactimos PP

Abbott Laboratories

Cybate (Australian label)

Fluid

Cyanamid

Skeetal FC

Fluid

Entotec/Novo

BMC WP

Powder

Reuter

Duplex

methoprene + Bti

Zoecon - PPM

GMOs and Bt:

A GMO (genetically modified organism) is defined as 'an organism whose genetic map has been modified in a different way from what happens in nature by cross breeding or natural genetic combination' (directive CEE 90/220 and French law 92/654).

Table 5: Various Genetically Modified crops produced by using Bt :

SL.NO.

CROP

GENE/EVENT

1

Potato

RB gene

2

Cabbage

cry1Ac

cry1Ba & cry1ac3

3

Okra

cry1Ac

4

Corn

cry1Ac + cp4epsp4

5

Brinjal

cry1Ac

cry1Aa & cry1Aabc

6

Cauliflower

cry1Ac

cry1Ba & cry1ac3

7

Tomato

unedited NAD9

8

Rice

cry1Ab, cry1C & bar

9

Groundnut

chitinase gene

GMO REGULATIONS IN INDIA

Some of the regulatory guidelines followed for Genetically Modified Organisms in India are:

Environment Protection Act 1986 (EPA).

Indian biosafety regulatory framework comprises 1989 "Rules for the Manufacture, Use, Import, Export and Storage of Hazardous Microorganisms, genetically Modified Organisms and Cells" (1989 Rules).

Department of Biotechnology guidelines, the 1990 "Recombinant DNA Safety Guidelines" (1990 DBT Guidelines)

1994 "Revised Guidelines for Safety in Biotechnology" (1994 DBT Guidelines)

1998 "Revised Guidelines for Research in Transgenic Plants and Guidelines for Toxicity and Allergenicity Evaluation of Transgenic Seeds, Plants and Plant Parts" (1998 DBT Guidelines).

Guidelines for the conduct of confined field trials of regulated, GE crops, 2008

Standard Operating Procedures for confined field trials 2008

Guidelines and protocols for food and feed safety assessment of GE crops, 2008.

Bt strains show genetic diversity with different toxic potential mostly due to plasmid exchange between strains (Thomas et al. 2001). Hence, each habitat may contain a novel Bt strain awaiting to be discovered which has a toxic effect on a some target insect group. The aim of this study is to isolate, identify, characterize Bt strains from different soil samples, and to quantify and qualify their cry protein content to study the best strain among all these isolated strains which can be used as a effective biopesticide against mosquitoes and black flies' midges. 

OBJECTIVES:

bti(bacillus thuringiensis var israelensis)

Isolation of Bacillus thuringeinsis (bti) from different soils.

10 cultures will be identified with specific medium (verify with std.)

Culturing of Bacillus thuringiensis

Visualizing by gram's staining

protein(delta-endotoxin) purification for best strains

protein(delta-endotoxin) quantification by Lowry's method

Quantification of protein(delta endotoxin) by ELISA

SDS-PAGE for protein(delta endotoxin) profiling.

MATERIALS AND METHODS:

SAMPLE COLLECTION:

Five different soil samples

Rice field - Chengalpet (Rc)

Flower field - Chengalpet(Fc)

Sugarcane field - Chengalpet(Sc)

Rice field - Tambaram(santhoshpuram)(Rt)

Vegetable field - Tambaram(santhoshpuram)(Rt)

Were collected by scraping off surface material with a sterile spatula and about 10 g samples were obtained from 2-5 cm depth. All samples were placed in sterile plastic bags aseptically and stored at 4°C until processed.

ISOLATION FROM SOIL SAMPLE:

PREPARATION OF SOIL SAMPLE STOCK:

0.85% Saline preparation: 850ml of saline was prepared by adding 7.225g of NaCl in 850ml of nano pure water.

10g of the Rice field Chengalpet (Rc) soil sample was added to sterile 250 ml conical flasks all containing 100ml of 0.85% saline solution.

Kept in shaker at 27 ͦ Cfor 1hour and 10ml of the supernatant was transferred to a sterile test tubes named as Rc.

The procedure was repeated for remaining 4 soil samples and named as Fc for flower field Chengalpet, Sc for sugarcane field Chengalpet, Rt for Rice field Tambaram, Vc for vegetable field Tambaram .

SERIAL DILUTION:

All the above 5 samples were serially diluted as by the below procedure.

10 sterile test tubes were taken

1ml of Rc stock sample was pipette out to a test tube containing 9ml of 0.85% saline and mixed gently. Marked as 10-1 dilution.

From this 1ml was pipetted into 9ml of 0.85% saline and marked as 10-2 dilution.

Likewise 10-3, 10-4, 10-5, 10-6, 10-7, 10-8, 10-9 dilutions of Rc was also done.

All the above steps was completely repeated for all the other four (Fc, Sc, Rt, Vt) samples.

MEDIUM PREPARATION:

Nutrient yeast extract mineral salt agar medium (NYSM-solid medium) [composition (wt/v %):

Nutrient agar - 8g/L

CaClâ‚‚.2Hâ‚‚O - 0.103g/L

MnClâ‚‚.4Hâ‚‚O - 0.01g/L

MgClâ‚‚.6Hâ‚‚O - 0.203g/L

Tetracycline - 15 mg/L

Materials Required:

Erlenmeyer flask - the sloped sides of the flask are ideal for preparing and autoclaving media.

Scale and weighing papers or trays/"boats" for measuring ingredients.

Empty Petrie plates, which should be sterile and packaged in a plastic bag.

Preparing the media

All dry ingredients except the agar was measured and placed into a 2L Erlenmeyer flask.

1000 ml of nano pure water was added and shook/stirred vigorously to get most of the dry material into solution.

Small aliquot of the solution was taken and the pH was checked in a pH-meter and the pH was adjusted to 7-7.4.

Agar was added and continued mixing - the agar will not go into solution at this stage, but it's important that it not form a large clump on the bottom.

The opening of the flask was covered with tin foil and placed it in an autoclavable metal bin with some water in the bottom (~1cm deep).

The media was autoclaved for a minimum of 20' on the liquid cycle. Pressures typically range from 15-20psi.

After autoclaving, gently the flask was swirled while holding it in water-proof oven or heat-proof gloves. This action is necessary to insure even distribution of the agar in the media; else it often remains denser near the bottom.

The media needs to temper before it is poured into plates. Thus the flask was placed on a heat-proof surface and let it cool. Large volumes (1L or more) should be swirled every 10-20 minutes to redistribute the media within the flask.

The plates were got ready in a laminar air hood. All plates was stacked and poured from bottom to top, lifting the lid of sequential plates (and those above it) to pour the media. Wore the oven-safe mitts while pouring.

The bottom half of the plate should be ~1/2 full, approximately 25mL of media per plate. Plates poured singly generally solidified within 1/2 hour at room temperature.

Solidified plate's were stacked right-side up and slide the original bag in which the empty Petrie plates came.

The plates were turned over so the plates now will be facing agar-side up, exactly as how they should be stored.

Labeled these plates with the type of media and date poured

PLATING PROCEDURE:

The samples of dilutions 10^-1, 10^-4, 10^-6 of each soil samples were taken.

0.1 ml of 10^-1 dilution of Rc sample was poured at the centre of one pre poured agar plate (this center placement makes it easier to spread the sample and to keep the sample away from the edge of the plate)

The sample were spread immediately and spread over the surface by rotating the plate or by a rotating bent glass tube on the surface in clock wise direction.

The procedure was repeated for all 10^-1, 10^-4, 10^-6 dilutions of all samples individually on separate pre pour NYSM agar plates.

Totally 15 such plating was done and all the plates were incubated over night at 28 ͦ C to obtain maximum growth.

STAINING:

PRINCIPLE:

The staining technique is based on the difference between the cell wall compositions of different bacteria. Bacterial cell wall may have higher lipid content or the protein content. Also the stains used in Gram staining have different affinity for these components and they bind with them reversibly or irreversibly. Hence Gram positive bacteria bind the stain irreversibly and cannot be decolorized by alcohol also where as Gram negative bacteria bind the stain reversibly and give it away when washed with water and alcohol. Then they take up the secondary stain become pink stained.

Figure 5: Comparison of the Gram positive and Gram negative bacterial cell walls. (Taken fromhttp://www.micro.cornell.edu/cals/micro/research/labs/angert-lab/low.cfm)

PROCEDURE:

A uniform smear of the samples was prepared on individual glass slides and named accordingly and heat-fixed.

The slides were stained as follows:

      a. Flooded with crystal violet for one minute.

      b. Excess dye was poured off and washed gently in tap water and drained the slides against a paper towel.

      c. The smears were exposed to Gram's iodine for one minute by washing with iodine, then adding more iodine and leaving it on the smear until the minute is over.

      d. Washed with tap water and drained water off carefully. 

      e. Washed with 95% alcohol for 30 seconds.

      f. Washed with tap water at the end of the 30 seconds to stop the decolorization and drained.

      g. Counterstained with 0.25% safranin for 30 seconds.

      h. Washed, drained, blotted, and examined under oil immersion microscope at 100X resolution.

SUBCULTURING: Isolates appearing as slender rods with a sub terminal, ovalendospore in a swollen sporangium were alone sub cultured, which are as follows :

Rice field-Chengalpet (Rc), replica-2, 10ˉ4 dilution.

Sugarcane field-Chengalpet (Sc), replica-1, 10ˉ4 dilution.

Flower field-Chengalpet (Fc), replica-2, 10ˉ1 dilution.

Rice field - Chengalpet (Rc), replica-2, 10ˉ6 dilution.

Flower field-Chengalpet (Fc), replica-1, 10ˉ4 dilution.

MATERIALS REQUIRED:

Inoculum loop

15 erlenmeyer flasks (250ml)

Cotton plugs

Nutrient yeast extract mineral salt medium (NYSM-Liquid medium) [composition (wt/v %)]:

Nutrient broth - 8g/L

CaClâ‚‚.2Hâ‚‚O - 0.103g/L

MnClâ‚‚.4Hâ‚‚O - 0.01g/L

MgClâ‚‚.6Hâ‚‚O - 0.203g/L

Tetracycline - 15 mg/L

INOCULATING SAMPLES FROM NYSM AGAR PLATES TO NYSM BROTH:

     

The inoculum loop was held in the right hand and was flamed in a sprit lamp and allowed to cool.

With the left hand, the lid was lifted a little of the lid of Petri dish containing the inoculum.

Touched a single colony with the wire loop and it was withdrawn carefully without touching the plate.

The lid of Petri dish was replaced carefully.

A universal of sterile LB broth was taken in the left hand.

The lid of the universal was removed with the little finger of the right hand which still holds the charged loop.

The neck of the universal was flammed.

The loop charged with inoculum was inserted into the sterile broth. Touched on the inside of the universal and withdrawn.

The neck of the universal was flamed, replaced lid and placed the universal on the surface of the Laminar Air Flow Hood.

Flamed the loop and placed it on heat resistant mat.

Tightened the lid of universal to make secure.

This procedure was performed for all the five samples.

Incubated at 37 ͦ C overnight.

CRYSTAL PURIFICATION:

Separation and purification of crystal (delta endotoxin)

Method of analysis (gelatine method)

Culture preparation:

The spore crystal complex was prepared.

10ml of NYSM medium was inoculated.

100ml was transferred to NYSM and inoculated for 7hrs.

2ml of the culture was inoculated into 200ml of NYSM (2%) O/N

After sporulation, the culture was centrifuged at 8000rpm for 15 min.

The supernatant was discarded, and the pellet was resuspended in 1M NaCl and centrifuged at 7000 rpm for 20 minutes.

Purification of crystal by sucrose gelatine gradient method

Washed twice with NaCl (centrifuged).

The supernatant was discarded, and the pellet is resuspended in 200ml of 0.5% gelatine

Equal volume of sterile distilled water is added and centrifuged at 7000 rpm for 10 minutes.

The supernatant is discarded, and the pellet is resuspended in 20ml of 1.5M sucrose, then 80ml of same one is added and centrifuged at 7000rpm for 10 minutes.

The supernatant was taken and double the amount of distilled water was added.

Centrifuged at 8000rpm for 15 min.

The supernatant is discarded, 900ml of 0.05M NaOH was added in 10mm EDTA and 50ml 0f DTT.

The supernatant containing protein was centrifuged at 7000 rpm for 1hr.

The protein was estimated by Biophotometer

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