Bacillus Thuringiensis Scientific Classification Biology Essay

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Bacillus thuringiensis (Bt) is a gram-positive, spore-forming 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.

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.

OCCURRENCE OF Bt:

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 and some mosquitoes, blackflies' 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.

DIVERSITY OF MOSQUITOCIDAL STRAINS OF BACILLUS THURINGIENSIS:

Table 1: Mosquitocidal B. thuringiensis serotypes and sources of their isolation (modified from J VECT BORNE DIS 42, SEPTEMBER 2005)

H serotype

Subspecies

Source(s) of isolation

H2

finitimus

Soils

H3a,3b.3c

kurstaki

Soils, animal feces, water

H3a,3d,3e

fukuokaensis

soils

H3a,3d

sumiyoshiensis

Soils, animal feces, water

H4a,4c

kenyae

plants

H4a,4b

sotto

soils

H5a,5c

canadensis

soils

H6

entomocidus

Soils, animal feces

H7

aizawai

Soils, animal feces

H8a,8b

morrisoni

soils

H10

darmstadiensis

soils

H10a,10c

londrina

Soils

H11a,11c

kyushuensis

Soils

H12

thompsoni

soils

H14

israelensis

Soils,plants,water insects

H15

dakota

Animal feces

H16

indiana

Soils,waters,animal feces

H17

tohokuensis

plants

H18a,18c

yosoo

plants

H19

tochigiensis

Soils,plants,animal feces

H20a,20c

pondicheriensis

soils

H21

colmeri

Animal feces

H24a,24b

neoleonensis

soils

H27

mexicanensis

Water, animal feces

H28a,28c

jegathesan

soils

H29

amagiensis

Animal feces

H35

seoulensis

Water, animal feces

H42

jinghongiensis

Plant, animal feces

H44

higo

Soil, animal feces

H47

wratislaviensis

soils

H71

jordanica

soils

H5a,5c/H21

canadensis/colmeri

soils

H14/36

israelensis/malaysiensis

Water, animal feces

H14/19

israelensis/insects tochigiensis

soils

H17/19

tohokuensis/tochigiensis

plants

H17/27

Tohokuensis/mexicanensis

Animal feces

H31/49

toguchini/muju

Animal feces

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:

GENETIES 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 (butterfly and moths)

Diptera (mosquitoes and biting flies)

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 black fly 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 bio safety 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 identified 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) identification

MATERIALS AND METHODs:

SAMPLE COLLECTION:

Five different soil samples were collected from:

Rice field - Chengalpet (Rc)

Flower field - Chengalpet(Fc)

Sugarcane field - Chengalpet(Sc)

Rice field - Tambaram(santhoshpuram)(Rt)

Vegetable field - Tambaram(santhoshpuram)(Rt)

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*C for 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.

This procedure was repeated for 10-3,10-4,10-5,10-6,10-7,10-8,10-9 dilutions.

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

MEDIUM PREPARATION:

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

Nutrient agar - 8g/L

CaClâ‚‚-0.103g/L

MnClâ‚‚-0.01g/L

MgClâ‚‚-0.203g /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:

Fifteen glass slides were taken and wiped with ethanol to make it sterile.

The smears of all the fifteen grown samples in the agar plates were prepared in individual glass slides, named accordingly and heat-fixed.

All 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.  (Do not blot.)

      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 :

MATERIALS REQUIRED:

Inoculum loop

15 erlenmeyer flasks (250ml)

Cotton plugs

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

Nutrient agar - 8g/L

CaClâ‚‚-0.103g/L

MnClâ‚‚-0.01g/L

MgClâ‚‚-0.203g /L

INOCULATING SAMPLES FROM NYSM AGAR PLATES TO NYSM BROTH:

     

The inoculums loop was held in the right hand.

the loop was flamed and allowed to cool Do not put down loop.

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

Touch a single colony with the wire loop.

Withdraw loop carefully without touching plate. Do not put down loop or wave it around.

Replace lid of Petri dish.

Lift a universal of sterile nutrient broth in the left hand.

Remove the lid of the universal with the little finger of the right hand which still holds the charged loop.Do not put down the lid.

Flame the neck of the universal.

Insert the loop charged with inoculum into the sterile broth. Touch on the inside of the universal and withdraw.

Flame the neck of the universal, replace lid and place the universal on the bench.

Flame the loop and place on heat resistant mat.

Tighten lid of universal to make secure but do not overtighten.

Incubate under appropriate conditions.

Open the tube and remove a small amount of the colony from the frozen agar surface

with a long handle inoculating needle or pipette. Return the slant to the freezer unless

it has begun to thaw (more than 5 minutes). If the slant thaws, make a fresh subculture

to replace it in the freezer.

Place the inoculum on a PDA plate labelled with the accession number Streak out the

inoculum with a loop or needle.

. Incubate the plate at 30oC until growth appears.

Prepare an LP mount and confirm the identity of the isolate.

Prepare subcultures as needed. If subcultures are sent to other institutions or persons,

record the information on the culture collection data sheet.

Separation & purification of crystal (Delta endotoxin)

Method of Analysis: Gelatin Method.

CULTURE PREPERATION:

Preparation of spore crystal complex

Inoculate - 10ml NYSM O/N

Transfer 100ml to NYSM

Inoculate 7hrs

Inoculate 2ml of 7hr culture into 200ml NVSM (2%) O/N

Check for sporulation (study)

(If no sporulation incubates further)

After sporulation centrifuge 800rmp for 15 minutes.

Discard Supernatant; re suspend the pellet in 1M Nacl.

PURIFICATION OF CRYSTAL BY SUCROSE GELATIN GRADIENT METHOD

Wash twice with NaCl (Centrifuge)

Discard supernatant; resuspend the pellet in 200 of 0.5% gelatin.

Add equal volume of sterile distilled water & centrifuge.

Discard supernatant resuspend the pellet in 20ml of 1.5M sucrose, then add 80ml of the same ond centrifuge at 5000rpm or 3000ghr.Take supernatant, add double the amount of distilled water.

Add centrifuge at 8000rmp for 15 minutes.

Discard the supernatant; add 900ml of 0.05m NaOH in 10mm EDTA & 50ml of DTT.

Centrifuge supernatant contains protein.

Estimate the protein Lowry's method.

ELISA

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