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Biochemical and genetic Characterization of a Streptomyces sps from Everest region and chemical characterization of antibiotics produced therefrom.

A Thesis Proposal on

Biochemical and genetic Characterization of a Streptomyces sps from Everest region and chemical characterization of antibiotics produced therefrom.

Principal investigator
Jyotish Yadav
Student from eighth semester
Universal Science College
Pokhara University
Maitidevi, Kathmandu
2007

Supervisor
Upendra Thapa Shrestha
Universal Science College
Pokhara University
RLABB
Maitidevi, Kathmandu

Research Lab
Research Laboratory for AgriculturalBiotechnology and Biochemistry (RLABB)

Maitidevi, Kathmandu


Introduction

Actinomycetes comprise an extensive and diverse group of Gram-positive, aerobic, mycelial bacteria with high G+C nucleotide content (>55%), and play an important ecological role in soil cycle. The name of the group actinomycetes is derived from the first described anaerobic species Actinomyces bovis that causes actinomycosis, the ‘ray-fungus disease’ of cattle. They were originally considered to be intermediate group between bacteria and fungi but are now recognized as prokaryotic microorganisms (Kuster 1968).

The majority of Actinomycetes are free living, saprophytic bacteria found widely distributed in soil, water and colonizing plants. Actinomycetes population has been identified as one of the major group of soil population (Kuster 1968), which may vary with the soil type. They belong to the order Actinomycetales (Superkingdom: Bacteria, Phylum: Firmicutes, Class: Actinobacteria, Subclass: Actinobacteridae). According to Bergey's Manual Actinomycetes are divided into eight diverse families: Actinomycetaceae, Mycobacteriaceae, Actinoplanaceae, Frankiaceae, Dermatophilaceae, Nocardiaceae, Streptomycetaceae, Micromonosporaceae (Holt, 1989) and they comprise 63 genera (Nisbet and Fox, 1991). Based on 16s rRNA classification system they have recently been grouped in ten suborders: Actinomycineae, Corynebacterineae, Frankineae, Glycomycineae, Micrococineae, Micromonosporineae, Propionibacterineae, Pseudonocardineae, Streptomycineae and a large member of Streptomyces are still remained to be grouped (www.ncbi.nlm.nih.gov). Actinomycetes have characteristic biological aspects such as mycelial forms of growth that accumulates in sporulation and the ability to form a wide variety of secondary metabolites including most of the antibiotics.

One of the major groups in actinomycetes is Streptomyces. Streptomyces contains 69-78 mol% of G+C. Substrate and aerial mycelium is highly branched. Substrate hyphae are 0.5-1.0 µm in diameter. In the colony ages aerial mycelia develop into chain of spores (conidia) by the formation of crosswalls in the multinucleated aerial filaments. Conidial wall are convoluted projection which together with the shape and the arrangement of the spore-bearing structure are characteristic of each species of Streptomyces (Anderson et al., 2001). It produces several antibiotics including of aminoglycosides, anthracyclins, glycopeptides, b-lactams, macrolides, nucleosides, peptides, polyenes, polyethers and tetracyclines (Sahin and Ugur, 2003).

Thus investigators turn towards Streptomyces and also other genera of actinomycetes such as Nocardia, Micromonospora, Thermoactinomycetes etc. for isolation of novel antibiotics. No doubt soil is the natural habitat of most of the microorganisms where vast array of bacteria, actinomycetes, fungi and other organisms exist and provided with suitable growth condition and ability to proliferate. Thus most actinomycetes contributing to antibiotic production are screened from soil (Williams and Khan, 1974).

Our prime focus is to find out the novel antibiotic with broad-spectrum antimicrobial activity from Streptomyces isolates of high altitude. And I will do further study in Streptomyces isolates of Khumbu region.

Background

In, RLABB, The first work on the diversity of actinomycestes was started by Singh, D. and Agrawal, V.P. (2002). The research on actinomycetes form Mount Everest was then continued by Pandey, B., Ghimire, P. and Agrawal, V.P. (2004). Still the work is conducting by Baniya R, Guragain M, Sherpa C and Gurung T. Baniya found many actinomycetes with broad-spectrum antimicrobial activity. Among them most of actinomycetes are Streptomyces. Although more research have been done on antibiosis, classification of antibiotic groups and genetic characterization on the basis of 16s rRNA gene have to be done for significant use in medical field. 16s rRNA gene are highly conserved in bacteria. But contain three sequence variable regions among (α, β and γ shown in fig. 1). Among that variation in the sequence of α-(from nucleotide 982 to 998), β-(1102-1122) regions are used of differentiate genus and γ-(158-203) region for the detection of species of bacteria (Anderson et al., 2001). Hence the study will explore more about the genetic property of Streptomyces and chemical property of antibiotics produced by them.

Fig: Secondary structure of 16S rRNA from Streptomyces coelicolor.

Objectives

1. Biochemical and Genetic Characterization of Streptomyces
2. Chemical Characterization of antibiotics from the Streptomyces isolates

Methodology

1. Isolation Purification of Streptomyces

Soil samples will be obtained from Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Isolation of actinomycetes will be performed by soil dilution plate technique using Starch-Casein Agar (Singh and Agrawal, 2002 & 2003). Actinomycetes on the plates will be identified as colored, dried, rough, with irregular/regular margin; generally convex colony as described by Williams and Cross (1971). Streak plate method will be used to purify cultures of actinomycetes (Williams and Cross, 1971, Singh and Agrawal 2002; Agrawal 2003). After isolation of the pure colonies based on their colonial morphology, colour of hyphae, color of aerial mycelium, they will be individually plated on another but the same agar medium.

2. Morphological and Biochemical characterization:

Morphological examination of the actinomycetes will be done by using cellophane tape and cover slip-buried methods (Williams and Cross, 1971; Singh and Agrawal 2002; Singh and Agrawal 2003). The mycelium structure, color and arrangement of conidiophores and arthrospore on the mycelium will be examined under oil immersion (1000X). The observed structure will be compared with Bergay’s manual of Determinative Bacteriology, Ninth edition (2000) for identification Streptomyces spp. Different biochemical tests will be performed to characterize the Streptomyces spp. The tests generally used are gelatin hydrolysis, starch hydrolysis, urea- hydrolysis, acid production from different sugars utilization tests, resistance to NaCl, temperature tolerance test, hydrogen sulphide production test, motility test, triple sugar iron (TSI) agar test, citrate utilization test, indole test, methyl red test, voges-proskauer (Acetoin Production) test, catalase test, oxidase test (Holt 1989; Singh and Agrawal 2002; Singh and Agrawal 2003).

3. Screening of Streptomyces for antimicrobial activity:

3.1 Primary screening
Primary screening of pure isolates will be determined by perpendicular streak method on Muller Hinton agar (MHA). In vitro screening of isolates for antagonism: MHA plates will be prepared and inoculated with Streptomyces isolate by a single streak of inoculum in the center of the petridish. After 4 days of incubation at 28 °C the plates were seeded with test organisms (Bacillus subtilis, Staphylococcus aureus, Enterobacter aerogens, Escherichia coli, Klebsiella species, Proteus species, Pseudomonas species, Salmonella typhi and Shigella species) by a single streak at a 90° angle to Streptomyces strains. The microbial interactions were analyzed by the determination of the size of the inhibition zone.

3.2 Secondary screening
Secondary screening is performed by agar well method against the standard test organism. Fresh and pure culture of each strain from the primary screening will be inoculated in starch casein broth and incubated at accordingly for 7 days in water bath shaker. Growth of the organism in the flask will be confirmed by the visible pellets, clumps or aggregates and turbidity in the broth. Contents of flasks will be filtered through Whatman no.1 filter paper. The filtrate will be used for the determination of antimicrobial activity against the standard test organisms.

4. Genetic Characterization

4.1 DNA extraction: Individual strains will be mass cultured in SC-broth by incubation the broth in shaker water bath for 5-6 days at 28C. Total DNA from corresponding strains will be extracted as described by a modified version of procedure of Kutchma et al. (1998) (Appendix-II).4.2 DNA polymorphisms: DNA polymorphisms among the strains will be studied by Specific-PCR using Universal primers 8F and 1491R that will amplify specifically 16S rDNA. The amplified band will be sequenced for sub species identification (Rivas et al., 2001) (Appendix-III)


5. Fermentation process

Isolates showing the broad-spectrum antimicrobial activity are grown in submerged culture in 250 ml flasks containing 50 ml of broth describe in Sahin & Ugur,2003. The flasks are inoculated with 1ml of active Streptomyces culture and incubated at 28ºc for 7 days with shaking at 500 rpm. After fermentation, fermented broth will filtered through Whatman no.1 filter paper.

6. Extraction of antimicrobial metabolites

Antibacterial compound will be recovered from the filtrate by treating twice with one volume of ethyl acetate (Busti et al., 2006). And after evaporation residue will be used for determination of antimicrobial activity, minimum inhibitory concentration and to perform bioassay of antibiotic (Pandey et al., 2004).

7. Thin Layer Chromatography and Bioautography

Silica gel plates, 10 X 20 cm, 1mm thick, are prepared. They are activated at 150°C for half an hour. Ten micro-liters of the ethyl acetate fractions and reference antibiotics are applied on the plates and the chromatogram is developed using chloroform: methanol (4:1) as solvent system. The plates are run in duplicate; one set is used as the reference chromatogram and the other is used for Bioassay of antibiotic. The spots in the chromatogram are visualized in the iodine vapour chamber and UV chamber (Thangadural et al., 2002 and Pandey et al., 2004).

Expected Outcomes

Being majority of antibiotics producing Actinomycetes are Streptomyces in this research work we have selected two Streptomyces sps with broad spectrum antimicrobial activity. Since these species are from very cold Everest region the organisms as well as antibiotics produced by them may be novel ones.

References

Anderson AS and Wellington EMH (2001) The taxonomy of Streptomyces and related genera. Int J Syst Evol Microbiol 51:797–814

Bergey's manual of determinative bacteriology 2000 Actinomycetales, 9th edition.

Busti E, Monciardini P, Cavaletti L, Bamonte R, Lazzarini A and Sosio et al. (2006) Antibiotic-producing ability by representatives of a newly discovered lineage of actinomycetes. Microbiology 152: 675-683

Holt, J.G. 1989 Bergey's manual of systematic bacteriology, vol 4, ed. S.T. Williams and M.E. Sharpe, Baltimore, Md: Williams and Williams.

Kuster, H.J. 1968 Uber die Bildung Von Huminstoffen durch Streptomyceten. Landwirtsch. Forsch, 21:48- 61

Kutchma, A.J., Roberts, M.A., Knaebel, D.B. and Crawford, D.L. (1998) Small scale isolation of genomic DNA from Streptomyces mycelia or spores. Biotechniques, 24:452-457.

Nisbet, L.J. and F.M. Fox 1991 The importance of microbial biodiversity to biotechnology, In, The biodiversity ofmicroorganisms and invertebrates : its role in sustainable Agriculture, ed.D.L. Hawksworth, 229-224, CAB International.

Pandey, B., Ghimire, P. and Agrawal, V.P. (2004) Studies on Antibacterial Activity of Soil from Khumbu Region of Mount Everest, a paper presented in International Conference on The Great Himalayas : Climate, Health, Ecology, Management and Conservation, Kathmandu, January 12 -15, 2004

Rivas R., Velázquez E., Valverde A., Mateos P.F. and Molina E. M. 2001 A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species Electrophoresis 22, 1086–1089

Singh, D. and Agrawal, V.P. (2002) Microbial Biodiversity of Mount Everest Region, a paper presented in International Seminar on Mountains - Kathmandu, March 6 – 8, 2002 (organized by Royal Nepal Academy of Science and Technology )

Singh, D. and Agrawal, V.P. (2003) Diversity of Actinomycetes of Lobuche in Mount Everest Proceedings of International Seminar on Mountains – Kathmandu, March 6 – 8, 2002 pp. 357 – 360.

Thangadural S, Shukla SK and Anjaneyulu Y (2002) Seperation and detecrion of certain β-lactan and fluoroquinolone antibiotic drugs by thin layer chromatography. Analytical Science 18: 97-100

Williams, S.T. and T. Cross 1971 Actinomycetes. In: J.R. Norris, D. W. Robbins, (eds), Methods in microbiology, vol.4. London, 295-334, Academic Perss, NewYork.

www.ncbi.nlm.nih.gov.

A DEMONSTRATIVE MANUAL FOR BASIC MOLECULAR BIOLOGY PRACTICAL

A DEMONSTRATIVE MANUAL FOR
BASIC MOLECULAR BIOLOGY PRACTICAL


FOR THE STUDENTS OF
DEPARTMENT OF MEDICAL MICROBIOLOGY
NOBEL COLLEGE
POKHARA UNIVERSITY
Jul 1-6, 2007


Prepared by

Kiran Babu Tiwari
Asst. Prof. of Microbiology, USC
Research Scientist, RLABB

In association with
Upendra Thapa Shrestha
Nirajan Bhattarai
Vijayendra Agrawal
(Research Scientists, RLABB)


Reference:
Professor Dr. Vishwanath Prasad Agrawal
Executive Director
Research Laboratory for Biotechnology and Biochemistry (RLABB)
www.uscollege.edu.np/rlabb
http://www.rlabb.com.np/
http://www.vpagrawal.com/


Acknowledgement


We are indebted to
Prof. Dr. Vishwanath P. Agrawal,
Executive Director, Research Laboratory for Biotechnology and Biochemistry (RLABB);
Director, Universal Science College; and
Academician, National Academy of Science and Technology (NAST)
for providing the facilities to conduct the experiments enlisted in this manual.


CONTENTS

TITLE PAGE

ACKNOWLEDGEMENT

CONTENTS

JULY 1:

ORIENTATION ON BASIC MOLECULAR BIOLOGY PROCEDURES

JULY 2:

EXPT 1. ISOLATION AND PURIFICATION OF BACTERIAL GENOMIC DNA

EXPT 2. ISOLATION AND PURIFICATION OF RNA

JULY 3:

EXPT 3. ISOLATION AND PURIFICATION OF BACTERIAL PLASMIDS BY ALKALINE LYSIS METHOD

JULY 4:

EXPT 4. SPECTROPHOTOMETRIC ANALYSIS OF DNA

EXPT 5. RESTRICTION DIGESTION OF DNA

JULY 5:

EXPT 6. AGAROSE GEL ELECTROPHORESIS OF DNA

JULY 6:

EXPT 7. POLYMERASE CHAIN REACTION

Day 1

ORIENTATION ON BASIC MOLECULAR BIOLOGY PROCEDURES

1. Principle of nucleic acids isolation from bacteria

Nucleic acids are present as nucleoprotein complexes in cells. The major problems encountered in isolation of pure and intact DNA or RNA molecules are: degradation of high molecular weight nucleic acids by mechanical damage or by hydrolytic action of nucleases, contamination of DNA preparations with RNA and vice versa and contamination of nucleic acids with proteins, polysaccharides and other high molecular weight compounds. Methods have been devised for isolation of nucleic acids from different sources taking adequate precautions to eliminate or minimize the above problems.

The main steps involved in isolation of nucleic acids are:

(a) Disruption of cells: Disintegration of bacterial cells can be achieved by treating them with cell coat hydrolyzing enzyme, i.e. lysozyme in presence of a detergent. at low temperature in buffers containing EDTA (chelates Mg2+ ions which are required for DNase activity). For isolation of RNA, and inhibitor of RNase such as bentonite, diethylpyrocarbonate, placental RNase inhibitor etc. is included in the extraction buffer.

(b) Dissociation of nucleo-protein complexes: The approach employed is such that the proteins either get dissociated or degraded while nucleic acids remain unaffected and intact. This is generally achieved by using detergents like SDS, phenol or broad-spectrum proteolytic enzymes such as pronase or proteinase K. Alkaline pH and high concentration of salts improve efficiency of the process.

(c) Removal of contaminating materials and precipitation of nucleic acids: Proteins are removed by treatment with phenol or mixture of chloroform-isoamyl alcohol or phenol-chloroform. Upon centrifugation, the denatured proteins form a layer at the interface between upper aqueous and lower organic phases, lipids and other contaminants remain in the same organic phase while nucleic acids are recovered in the aqueous phase from which they are precipitated with ethanol. For isolation of DNA, the contaminating RNA is removed by selective salt precipitation or treatment with DNase-free RNase. Conversely while isolating RNA, the preparation is incubated with DNase for eliminating DNA as an impurity.

(d) General precautions while handling nucleic acids:
1. All glasswares and solutions (except organic solvents) should be sterilized.
2. Gloves should be worn to avoid contamination of the experimental material and apparatus with nucleases with occur in fair abundance in skin exudates.
3. Phenol causes severe burns and phenol-containing solutions should, therefore, be handled with care. Thoroughly rinse the burns with large volume of water. Do not use ethanol.
4. For work with RNA, rinse all glasswares with 1% diethylpyrocarbonate solution to inactivate RNase and then autoclave them.

2. Basic components for Molecular Biology experiments

LB (Lauria-Bertani) broth: 0.5% NaCl, 1% Tryptone, 0.1% Yeast extract
Overnight Log- phase culture
Solution I (Lysis buffer): 25mM Tris, 50mM Glucose, Lysozyme [10mg/ml stock; GNB (Gram Negative Bacteria) 0.5mg/ml, GPB (Gram Positive Bacteria) 3-5mg/ml], pH 8.0
Solution II (Lysis): (a) for plasmid isolation: 0.2M NaOH, 10% SDS (b) for genomic DNA isolation: 10% SDS only.
Proteinase K: 10mg/ml stock, 10-20mg/65°C/3hrs
Solution III: 3M Sodium acetate, pH 4.8 by acetic acid
Phenol: distilled, protein precipitant
Chloroform: protein precipitant, phenol solvent
RNase: 5mg/ml stock, 1-2ml
Absolute ethanol/Isopropanol: DNA precipitant, 2.5vol of ethanol or 1vol of isopropanol
70% ethanol: washing DNA precipitant
TE buffer: DNA resuspending buffer, 10mM Tris, 1mM EDTA, pH 8.0; Autoclave before use
TAE buffer: Electrophoresis, 50X, Tris, 24.2gm; acetic acid, 5.71ml; EDTA (0.5M), 11.1ml; DW, 100ml; pH 8.0; Autoclave before use
Loading dye: 6X; 50% Glycerol, 6.0ml; 2%BPB (Bromophenol Blue), 1.0ml; DW, 3ml; Always use sterile DW
Ethidiun Bromide (EtBr)*: 10mg/ml stock (Final concentration: 0.5mg/ml)
λ/HindIII Marker: 23.13Kb, 9.42Kb, 6.56Kb, 2.32Kb, 2.07Kb, 0.56Kb and 0.13Kb
Restriction enzymes: EcoRI and HindIII (10U/ml each)
Restriction enzyme buffers for EcoRI and HindIII (10X each)
Sterile Double distilled water (DDW)
Water bath
Cold centrifuge (upto 20000rpm)
Micropipettes and sterile tips

*Note: EtBr is carcinogen, so, handle with gloves

Day 2

Expt. 1. Genomic DNA extraction from bacteria

-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT (Microfuge tube) and spin at 5000rpm for 10min.

-Remove the supernatant and spin once with same volume as above to collect more cell mass.

-Remove the supernatant.

-Add 100µl Sol. I and keep for 30min at RT (Room temperature).

-Add 1/10 vol. of 10% SDS and swirl to mix.

-Add Proteinase K (1-2 µl) and incubate for 30min at 37ºC with gentle shaking.

-Add 1-2µl of RNase and incubate for 10min at RT.

-Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10min at RT.

-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.

-Add equal vol. of 3M sodium acetate, mix and stand it for an hour in cold.

-Spin at 10000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.

-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.

Expt. 2. RNA extraction from bacteria

-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.

-Remove the supernatant and spin once with same volume as above to collect more cell mass.

-Remove the supernatant.

-Add 100µl Sol. I and keep for 30min at RT.

-Add 1/10 vol. of 10% SDS and swirl to mix.

-Add Proteinase K (2 µl) and incubate for 30min at 37ºC with gentle shaking.

-Add equal vol. of Phenol:Chloroform (3:1) solution and mix gently.

-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.

-Add equal vol. of 3M sodium acetate and/or two vol. of absolute ethanol. Mix and stand it for 1-2 hour in cold.

-Spin at 15000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.

-Dissolve the pellet collected during spin in 50µl TE (pH 7.0) and store in deep freeze.

Day 3

Expt 3. Plasmid DNA extraction from bacteria

-Take 1.5ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000rpm for 10min.

-Remove the supernatant and spin once with same volume as above to collect more cell mass.

-Remove the supernatant.

-Add 100µl Sol. I and keep for 30min at RT.

-Vortex for a while and add Proteinase K (2µl) and incubate for 30min at 37ºC with gentle shaking.

-Add freshly prepared Sol. IIa (200µl) and mix gently (Do not vortex).

-Add ice cold Sol. III (150µl) and mix gently (Do not vortex).

-Add 1-2µl of RNase and incubate for 10min at RT.

-Add equal vol. of Phenol: Chloroform (1:1), mix gently and keep for 10min at RT.

-Centrifuge at 8000rpm for 10min at 4ºC and collect the supernatant in a new sterile MFT.

-Add equal vol. of isopropanol and stand it for an hour in cold.

-Spin at 13000rpm at 4ºC for 15min and wash the pellet with 70% ethanol.

-Dissolve the pellet collected during spin in 50µl TE and store in deep freeze.

Day 4

Expt. 4. Spectrophotometric analysis of DNA

1. Remove the DNA preparation from the freeze and thaw it.

2. Transfer 495μl TE buffer to a quartz cuvette and add 5μl of the DNA preparation. Mix well.

3. Set the spectrophotometer to 260nm and blank the instrument with TE buffer.

4. Measure the absorbance (A260) of the DNA dilution.

5. Repeat steps 3 & 4 with the spectrophotometer set at 280 nm.

6. Calculate the DNA concentration from A260. [µg/ml = A260 X dilution X 50].

7. Calculate A260/A280 in order to estimate the purity of the DNA preparation (Pure DNA has a A260/A280 ratio of 1.8 – 2.0).

Expt. 5. Restriction digestion of DNA

1. Dilute the DNA extracts, Marker/s and enzymes in suitable solvents accordingly.

2. Calculate the volume for digestion reaction as shown in the table given below.

Components............ Rxn. .........1 Rxn. 2
λ DNA (1µg/µL) ............10 µL ..............-
DNA extract (1µg/µL) ...- ....................10 µL
HindIII (10U/µL) .........1 µL ................1 µL
HindIII buffer (10X) ....1 µL ................1 µL
DW ..................................8 µL ................8 µL
Total ...............................20 µL ..............20 µL

3. Perform digestion reaction by mixing the components in sterile MFT.

4. Briefly centrifuge the contents and keep at 37ºC for 6hrs.

5. Perform agarose gel electrophoresis to interpret the results.


Day 5

Expt. 6. Agarose electrophoresis of DNA

1. Prepare 0.8% agarose gel in 1X TAE buffer (28ml).

2. Dissolve agarose completely in micro-oven and cool to 600C.

3. CAREFULLY, add EtBr into the gel solution to final concentration of 0.5µg/ml.

4. Position a comb in the mold. Pour into gel mold and let it cool for 30 minutes.

5. Pour the TAE buffer into the gel buffer reservoir.

6. Prepare sample taking 20µl of DNA sample and mix with 4µl blue juice.

7. Carefully remove the comb.

8. Load the DNA (15 µl) in the wells, flanking wells with similarly processed DNA size standard.

Note: the amount of the sample that can be loaded in a well depends on the thickness of the gel as well as dimensions and placing of the comb.

9. Put the lid on the gel apparatus; attach the electrodes and adjust voltage to 100 volts.

10. Allow the gel to run until line of blue juice is visible near the end of the gel.

11. Turn off the current and visualize the gel in UV transilluminator.

12. Interpret the results.


Day 6

Expt 7. RAPD-PC(Randomly Amplified Polymorphic DNA – Polymerase Chain Reaction)

1. Thaw the DNA extract and dilute in sterile TE to final concentration with10ng/µl.

2. Prepare reaction mixture as given below:

Components---------- Volume (µl)
PCR buffer (pH 8.3) ----------5
dNTP (2.5mM each) ---------4
Taq polymerase (1U/µl) -----1
Template DNA (10ng/µl) ----1
RAPD-Primer (10µM) -------8
10% DMSO ------------------5
DDW ------------------------26
Final Volume-----------------50

3. Operate the thermal cycler program as given below:
Initial denaturation------- 94ºC-------5mi -----Single step
Denaturation -------------94ºC -------1min
Annealing ----------------36ºC -------1min ----30 Cycles
Extension ----------------72ºC -------2min
Final extension -----------72ºC -------5min ----Single step

4. Perform agarose gel electrophoresis as described in Expt. 6.

OPTIMIZATION OF RAPD-PCR FOR GENETIC FINGERPRINTING OF BACILLUS THURINGIENSIS

Gyan Sundar Sahukhal1, Upendra Thapa Shrestha1, Kiran Babu Tiwari1, Binod Lekhak2, Anjana Singh2, Viswanath Prasad Agrawal*
(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;
(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

* Corresponding author: gyan633413@gmail.com

ABSTRACT

A random amplified polymorphic DNA fingerprinting assay has been optimized that discriminate different B. thuringiensis isolates. Random amplification of polymorphic DNA (RAPD) is proving to be a useful technique used for screening diversity, particularly at intraspecific levels, including many population studies. Relationships between species' may be determined by comparing their unique fingerprint information, which are expected to be identical among related species. The technique can be troublesome and time consuming to establish due the essentially empirical approach to optimization. By standardization of certain parameters and use of a commercially available PCR buffer, a particularly promising primer was identified and a RAPD condition for a highly discriminatory and reproducible characterization of B. thuringiensis isolates was achieved. In addition, a technique to obtain reproducible RAPD fingerprints of B. thuringiensis without the need to purify genomic DNA described.

Delta-endotoxin Immuno Cross-reactivity of Bacillus thuringiensis Isolates Collected from Khumbu Base camp of Mount Everest Region

(Journal of Food Science and Technology Nepal. 2: 128-131)

Upendra Thapa Shreshtha(1), Gyan Sundar Sahukhal(1), Subarna Pokhrel(1), Kiran Babu Tiwari(1), Anjana Singh(2), Vishwanath Prasad Agrawal(1*)

(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;

(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np

ABSTRACT

Bacillus thuringiensis strains were isolated from soil samples collected from Khumbu Base Camp of the Everest region and characterized by standard methods. Crystal protein (d-endotoxin) was extracted from the crystal protein producing strains (46 from Phereche and 40 from Sagarmatha National Park) from stationary phase culture broth and tested for insect bioassay. Crystal proteins were further purified by Native-PAGE. Among ten randomly selected isolates, one isolate showed the highest insecticidal activity against Dipteron insects. Its crystal protein had molecular weight about 120-130KD (revealed by SDS-PAGE) and was used to produce polyclonal antibody in New Zealand’s white rabbits. The presence of polyclonal antibody was confirmed by Ouchterlony double diffusion method. Indirect ELISA was optimized coating 6-8mg crystal protein per well in microtitre plate. The optimal dilution of the polyclonal antibody was 1000 folds corresponding to OD450 = 0.045 for color observation. Of the total 86 crystal protein producing isolates, crystal proteins from 31 isolates (36.05%) were 25-30% crossreactive, two groups of 6 isolates (6.97%) were 75-80% and 85-90% crossreactive respectively, and 4 isolates (4.65%) were 80-85% crossreactive with the polyclonal antisera. Only 3 isolates (3.49%) were more than 90% crossreactive. The Discriminatory Index (D) of the Indirect ELISA was 0.92.

Keywords: Khumbu region, d-endotoxin, Crystal protein, Insect-bioassay, Immunodiffusion, Immunocrossreactivity.

INTRODUCTION

Microbial insecticides are especially valuable as their toxicity to non target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safer for both the pesticide user and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium Bacillus thuringiensis fulfills the requisites of a microbiological control agent against agricultural pest and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides include various Lepidoptera (butterfly), Diptera (fliesand mosquitoes), and individual Coleopteran (Beatle) species and some strains kill off nematodes (Schnepf et al , 1998) where as B. thuringiensis var. kurstaki HD1 is highly potent strain due to its wide spread insecticidal properties (Dulmage, 1970).

Insect bioassay and rocket immunoelectrophoresis are currently used to detect and measure the levels of crystal proteins. Though the rocket immunoelectrophoresis is more sensitive than insect bioassay, it requires a considerable amount of antigen, at least 10 mg/ml (Wie et al, 1982). Immunodiffusion and ELISA are more practical and reliable methods. ELISA measures changes in enzyme activities proportional to the antigen or antibody concentrations. It is a highly versatile and sensitive analytical procedure for qualitative and quantitative determination of antibodies and almost any kind of antigens. The method discriminates different epitopes very efficiently provided that antibodies of high specificity and affinity are available. The detection limits of the assay may be well below 1 ng/ml (Perlmann and Perlmann, 2001). Hence ELISA can be effectively used to study cross reactivity of a given type of antigen. Identical antigens possess 100% crossreactivity with the given antisera and non identical ones don’t show any degree of crossreactivity. Thus the diversity of the given antigen and hence organisms in a given complex population can be studied by their cross reactivities. In order to study crystal protein diversity of B. thuringiensis strains, Indirect ELISA procedure was optimized in this study.

METHODOLOGY

Soil sampling, isolation and biochemical characterization: Soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu Base Camp of Everest region and were transported to RLABB, where the study was carried out from March 2005 to December 2005 in joint collaboration with Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal. Bacillus thuringiensis were isolated by acetate selection method (Travers et al, 1987). The isolated organisms were identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).

Collection of Mosquito larvae: Mosquito (Lepidoptera) Larvae were collected from the ditches in local area of Bode, Bhaktapur, Nepal for insect bioassay. The larvae were identified as Culex spp. by zoologists at the Central Department of Zoology, Tribhuvan University, Kirtipur and bioassay was performed as described by Pang (1994).

Extraction and purification of crystal proteins: B. thuringiensis strains were incubated in Brain Heart Infusion broth (Calf brain infusion 200 g/l, beef heart infusion 250 g/l, protease peptone 10 g/l, dextrose 2 g/l, sodium chloride 5 g/l, disodium phosphate 2.5 g/l, final pH 7.4 ±0.2 at 25°C) and sterilized by autoclaving (15 lbs pressure 121oC, 15 min) at 30°C for 3-7 days till autolysis. Spores and crystals were separated by centrifugation (10,000 rpm, 20 min, 4 oC), and then washed four times with phosphate buffer (pH 7, 0.05M). The pellet was finally suspended overnight in carbonate buffer of pH 10.5 (0.05 M sodium carbonate, 0.01M b-mercaptoethanol, 1mM EDTA, 1mM PMSF) with constant shaking at 23-26oC, and centrifuged (10000 rpm, 20 min, 4 oC). Protein content in the supernatant was determined by Bradford assay (1976). In order to determine which proteins are responsible for the biological activities, they were electrophoresed under non denaturing conditions by Native PAGE (Blackshear, 1984) and the major bands were sliced , grinded in a minimum volume of phosphate-buffered saline (10 mM sodium phosphate, 150 mM NaCl, pH 7.2) , centrifuged (10000 rpm, 20 min, 4 oC) and concentrated using 20% TCA.

Insect bioassay and Molecular weight determination: For insect bioassay 10 larvae were taken in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast. Five ml of B. thuringiensis stationary phase culture was added and allowed to stand for 3 days. The number of deaths was recorded for one, two and three days. For the purified crystal protein bioassay, proteins (30µg/ml per assay) from each band were tested for insecticidal property as done by Pang (1994). Proteins from insect bioassay positive band was electrophoresed to determine molecular weight using molecular weight markers (lysozyme 14 KD, casein 22 KD, BSA 66KD) according to themethod of Laemmli (1970).

Polyclonal antiserum production: Proteins from insect bioassay positive band was resuspended in saline to a concentration of 1.0 mg/ml. The suspension was emulsified in an equal volume of Freund’s complete adjuvant (Difco, USA). A pair of New Zealand’s white rabbit was injected with 500 µg of the emulsified proteins (1.25 ml) by the intramuscular route in hind limbs. The booster injections with incomplete adjuvant were given three times in 14 days interval. The animals were bled 7 days after third booster dose. Polyclonal antiserum was pooled and decomplemented by incubation at 56°C for 30 min. Aliquots of antiserum (0.1 to 0.5 ml) were stored at -20°C until assayed.

Immunodiffusion and ELISA: The presence of polyclonal antibody was confirmed in a 1% agarose gel (0.05 M phosphate buffer, pH 7.2) by the Ouchterlony method (Talwar and Gupta, 1997) where 50 µl undiluted and diluted (1:10,1:100, 1:1000,1:10000 dilutions) antisera was poured in a centre well surrounded by 5 wells in petri-plate. 50 µl crystal protein antigen preparations (1 mg/ml) from SNP and Phereche isolates were applied in the surrounding wells against antiserum. The petri-plate was incubated at 4oC for 72 hours in a moist chamber. Immuno crossreactivity of B. thuriengensis crystal proteins was studied by indirect ELISA, where optimal dilutions of polyclonal antiserum and its second antibody (anti rabbit IgG conjugated with Horse Radish Peroxidase, Sigma, USA) was determined by chequerboard titration method (Trottier et al, 1972; Voller et al, 1976). To coat 96-well polystyrene microtitre plate, 100 µl crystal protein antigen (6µg) prepared in PBS (137 mM NaCl, 1.76 mM KH2PO4, 8.1 mM Na2HPO4, 2.7 mM KCl; pH 7.2 ) was applied in each well, and incubated overnight at 4oC (Trottier et al, 1972).Uncoated protein was washed 3 times using washing buffer (0.05%Tween 20 in PBS). For blocking, 200µl of 2% BSA in PBS was applied in each well and allowed to stand for 1 hour at room temperature. Blocking was done twice. After 3 times washing, each well was incubated with 100 µl polyclonal antibody of different dilutions for 2 hours at room temperature, and unbound polyclonal antibody was washed 4 times. The microtitre plate was then incubated with 100 µl of second antibody of different dilutions per well at room temperature for 1 hour and washed 4 times. Finally it was incubated with 100 µl tetramethyl benzidine substrate solution (0.01% tetramethyl benzidine in citrate phosphate buffer of pH 4.9, 2µl of 30% H2O2), reaction was allowed proceed for half an hour, stopped adding 1N HCl and the intensity of color was read at 450 nm within 10 min in ELISA reader (Dynatech MR-250).

Calculation of discriminatory index value (D): D value was calculated according to the formula of Hunter and Gaston (1988).Where, N=Numbers of isolates;S=Number of different polymorphic types.

RESULTS AND DISCUSSION

Total 109 B. thuringiensis isolates were obtained from soil samples of Phereche and SNP. Sixty three and 46 isolates were obtained from 52 Phereche and 39 SNP soil samples respectively. All the isolates were Gram positive rods. Of the 109 total isolates, only 86 isolates (79.63%) were positive on crystal protein staining, which were further proceeded for immunological characterization (Bergey’s Manual, 1986).Molecular weight determinationFive bands were observed for S6 preparation having molecular weight of 40, 58, 71, 83 and 107 KD in SDS-PAGE and a single band (Mol. Wt. -120-130KD) was observed in Native PAGE. This indicates that S6 crystal protein is composed of 5 different protein subunits. Similar result was observed by Drobniewski and Ellar (1989) and Pfannenstiel et al (1986) in their work on B. thuringiensis.Insect bioassayThe result of insect bioassay of crystal protein from SNP and Phereche B. thuriengensis isolates is displayed in Figure 1. For insect bioassay, cultures were grown to stationary phase which is suitable for the sporulation and production of crystal protein (Hunter and Gaston, 1988), and were used for the bioassay on mosquito larvae. Insecticidal activity of S6 endotoxin (30µg/ml) partially purified from autolysed B. thuringiensis broth by alkaline solution method following purification in Native PAGE is found to be 100 % (10/10) efficient, and hence it was used for polyclonal antibody production. Purification by Native PAGE has also been reported by Pang (1994). These crystal proteins are solubilized in the alkaline environment of Lepidoptera larval midgut, and then processing by midgut proteases results in a relatively stable, mature toxin (Van Rie et al., 1990). Activated cry toxins have two known functions, receptor binding and ion channel activity. The activated toxin binds readily to specific receptors on the apical brush border of the midgut microvilli of susceptible insects. Binding is a two-stage process involvingreversible and irreversible steps. The latter steps may involve a tight binding between the toxin and receptor, insertion of the toxin into the apical membrane, or both. It has been generally assumed that irreversible binding is exclusively associated with membrane insertion. Soon after insertion, toxins made pores on membrane and enter to body system. As soon as they enter to body they stop feeding and alternately death of insects occurs due to blood poisoning (Schnepf et al, 1998).Immunodiffusion and ELISADouble immunodiffusion performed in agarose gel gives clearly visible precipitin bands against crystal proteins of B. thuringiensis isolates from both SNP and Phereche upto 1:100 dilution of polyclonal antisera tested.(Fig. 2). The method gives reproducible results to detect crystal protein antigen with 100% specificity. For performing ELISA (Fig. 3), first and second antibody of dilutions 1:1000 and 1:2000 were optimal respectively. Below OD450 = 0.045, there was not clearly visible change in color to naked eyes. Trottier et al (1992) chose the optimal dilution of first and second antibodies difference between ELISA value with and without antigens with all other conditions remaining same. Between each ELISA step, plates were washed five times with a microtitre plate washer in order to prevent the false positive result (Trottier et al, 1992). The discriminatory index value, D (Table 1), was 0.92 (N=86, S=22). Hence the Indirect ELISA method divided all isolates into 13 groups with 92% confidence (Table-1). In which only 3 isolates (3.49%) were above 90% cross-reactive. The result clearly shows that B. thuringiensis isolates from Khumbu Base Camp are highly diverse for their crystal protein antigenicity. The S6 isolate showing potent insecticidal property tested against lepidopteron insects need to be studied further in larger trials so that it can have applicability to reduce the massive crop yield loss in this region.

CONCLUSION:

B. thuringiensis is diversed into 13 different groups in Khumbu Base Camp of Mount Everest region. Crystal protein from one of the most potent strain (S6) is 100% effective against lepidopteron insects.

ACKNOWLEDGEMENT

We express our full gratitude to CNR (Italy’s National Research Council) for supporting this work and especially thank to Mr. Yogan Khatri, Mr. Deepak Singh and Rajendra Aryal for collecting soil samples from Mount Everest region. Finally my (Upendra Thapa Shrestha) special regards goes to my dear parents for their everlasting support.

REFERENCES

Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol 1999; 65: 3714-6.

Bergey’s Manual of Systematic Bacteriology, Volume 2, 1986).Pang AS. Production of antibodies against Bacillus thuringiensis delta-endotoxin by injecting its plasmids. Biochem Biophys Res Commun 1994; 202: 1227-34.

Blackshear PJ (1984). Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54.

Drobniewski FA and Ellar DJ. Purification and properties of a 28-kilodalton hemolytic and mosquitocidal protein toxin of Bacillus thuringiensis subsp. darmstadiensis 73-E10-2. J Bacteriol 1989;171: 3060-7.

Dulmage HT Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol 1970; 16: 385-9.

Hunter PR and Gaston MA. Numerical Index of the Discriminatory Ability of Typing Systems: an Application of Simpson's Index of Diversity. Journal of Clinical microbiology 1988; 26: 2465-6.

Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970; 227:680-5.Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 1970; 227:680-5.

Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.

Perlmann P and Perlmann H (2001) Enzyme-Linked Immunosorbent Assay ENCYCLOPEDIA OF LIFE SCIENCES 2001 Nature Publishing Group.Travers RS, Martin PA and Reichelderfer CF Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol 1987; 53: 1263-6.

Pfannenstiel MA, Couche GA, Ross EJ and Nickerson KW. Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin. Appl Environ Microbiol 1986;52: 644-9.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

Talwar GP and Gupta SK (1997). A hand book of practical and clinical immunology, second edition, volume 1, CBS publishers and distributors, Daryaganj, New Delhi.

Trottier YL, Wright PF and Lariviere S Optimization and standardization of an Enzyme-Linked Immunosorbent Assay protocol for serodiagnosis of Actinobacillus pleuropneumoniae serotype 5. J Clin Microboil 1992;30(1):46-53.

Van Rie, J., S. Jansens, H. Ho¨fte, D. Degheele, and H. Van Mellaert. Receptors on the brush border membrane of the insect midgut as determinants of the specificity of Bacillus thuringiensis delta-endotoxins. Appl. Environ. Microbiol 1990;56:1378-85.

Voller A., Bidwell D and Bartlett A. (1976) Microplate enzyme immunoassays for the immunodiagnosis of virus infections Manual of clinical immunology. American Society for Microbiology p. 506-512.

Wie SI, Andrews R JR, Hammock B, Faust R.M and Bulla LA () Enzyme-Linked Immunosorbent Assays for Detection and Quantitation of the Entomocidal Parasporal Crystalline Protein of Bacillus thuringiensis subspp. kurstaki and israelensist Appl Environ Microbiol: 1982, 891-4.

Strong mosquitocidal Bacillus thuringiensis from Mt. Everest

Upendra Thapa Shreshtha1, Kiran Babu Tiwari1, Anjana Singh2, Vishwanath Prasad Agrawal1*
(1) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Universal Science College, Maitidevi, Kathmandu, Nepal;
(2) Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

*Address for Correspondence: Prof. Dr. Vishwanath Prasad Agrawal, RLABB, Tel: +977-1-4442775, E-mail: vpa@wlink.com.np

Microbial insecticides are especially valuable as their toxicity to non-target animals and humans is extremely low compared to other commonly used chemical insecticides. They are safer for both the pesticide users and consumers of pesticide treated crops (Neppl, 2000). The soil bacterium Bacillus thuringiensis fulfills the requisites of a microbiological control agent of agricultural pests and vectors that cause massive crop destruction (Ben-Dov et al, 1999). The main target pest of B. thuringiensis insecticides includes various Lepidoptera (butterfly), Diptera (flies and mosquitoes), Coleopteran (Beatle) and some strains of nematodes (Schnepf et al, 1998). The kurstaki HD1 strain of B. thuringiensis is shown to possess wide spread insecticidal properties (Dulmage, 1970).

To study B. thuringiensis population from high altitude, soil samples were collected from Sagarmatha National Park (SNP) and Phereche of Khumbu region situated in the base camp of Mt. Everest, and processed in Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). B. thuringiensis strains were isolated from soil samples by acetate selection method (Travers et al, 1987, Shrestha et al. 2006) and identified by standard microbiological techniques including colonial and morphological characteristics, and biochemical tests (Bergey’s Manual, 1986).

Out of 86 δ-endotoxin positive isolates (total 146), 10 randomly selected ones were used for insect bioassay. Larvae, collected from the ditches in local area of Bode, Bhaktapur, Nepal, were reared in a jar containing 100 ml of sterilized water containing 0.3 ml of 5% Brewer's Yeast and 5 ml of B. thuringiensis stationary phase culture and allowed to stand for 3 days. Although all isolates tested were effective against the larvae, isolate S6 was the most effective of all (Fig. 1).

The isolates, obtained from the soil samples above 4000m altitude where mosquitoes are not expected, may be novel. The S6 isolate showing potent insecticidal property tested against dipterans need to be studied further in larger trials so that it can have applicability to reduce the mosquitoes and different diseases caused by these vectors (Malaria, Filaria, Kalazar etc).

Fig 1: % Mortality of larvae

REFERENCES:
Ben-Dov E, Wang Q, Zaritsky A, Manasherob R, Barak Z, Schneider B, Khamraev A, Baizhanov M, Glupov V, Margalith Y. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains. Appl Environ Microbiol 1999; 65: 3714-6.

Bergey’s Manual of Systematic Bacteriology, Volume 2, 1986.

Dulmage HT. Production of spore-delta-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J Invertebr Pathol 1970; 16: 385-9.

Neppl CC (2000). Managing Resistance to Bacillus thuringiensis Toxins. Environmental Studies University of Chicago.

Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR and Dean DH. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

Shrestha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP. Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal 2006; 2: 128-131.

Travers RS, Martin PA and Reichelderfer CF. Selective Process for Efficient Isolation of Soil Bacillus spp. Appl Environ Microbiol 1987; 53: 1263-6.

Cloning of cry3 fragment in Escherichia coli

Shyam K. Shah1, Kiran Babu Tiwari1,2, Upendra Thapa Shrestha2, Subarna Pokhrel3, Eitan Ben-Dov4 and Vishwanath P. Agrawal1,2*
(1) Department of Biochemistry, Universal Science College, Pokhara University, Kthmandu, Nepal;
(2) Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal;
(3) Department of Enzyme Engineering, Seoul National University, Korea;4Department of Life Sciences, Ben-Gurion University of the Negev, Be’er-Sheva 84105, Israel*

Correspondence Address: Dr. Vishwanath P. Agrawal, Professor of Biochemistry, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal.Email: vpa@wlink.com.np. Contact: +977-1-2110043

ABSTRACT

Bacillus thuringiensis was isolated and purified from the soil sample collected from Khumbu, Mt. Everest base camp. Total DNA was extracted and PCR was done using nine universal primers for cry1 to cry9. A specific band of about 300bp was amplified with universal primer 3. DNA library was created by digesting the DNA with HindIII, ligation into pUC18 followed by transformation of Escherichia coli HB101. Using universal primer 3 for PCR, 100 out of 1000 clones prepared were screened and three were found to posses cry3 specific fragment of the same size as before. The cry3 specific fragment was cloned, extracted, purified and sent for sequencing. As the bacterium was isolated from high altitude, the gene may be novel with promising for biological control and management of insects.

INTRODUCTION

Bacillus thuringiensis is an aerobic, ubiquitous, gram positive, spore forming bacterium that forms an insecticidal parasporal crystal protein (δ-endotoxin). The crystal protein can be used to control certain insect species among the orders lepidoptera, diptera, coleopteran (Beegle and Yamamoto, 1992) and, hence, is a useful alternative to synthetic chemical pesticide applied in commercial agriculture, forest management and mosquito control. The genes encoding δ-endotoxin production have been cloned in other bacteria and transferred into crop plants. This enables genetic improvement in the potency and host spectrum of B. thuringiensis strains and development of crop varieties that produce δ-endotoxin within their own tissues (Schnepf et al. 1998).The toxins are specific and have no detrimental effects on mammals or birds and are easily degraded in environment. In susceptible insects, the toxin is dissolved in the mid gut, releasing pro-toxin that are proteolytically converted into smaller toxin polypeptides (McGaughey and Whalen, 1992). Following activation, these toxins bind with high affinity to receptors on the epithelium. After binding, the toxins generate pores in the cell membrane, disturbing cellular osmotic balance and causing the cell to swell and lyses. Recently, the crystal proteins and their genes have been classified based on their structure, antigenic properties and activity spectrum.In Nepalese context, though isolation and characterization of B. thuringiensis from different soil samples and their insect toxicity have been studied and tested, molecular characterization of the bacteria has to be explored yet, especially from extreme environments in order to find novel strains. To study B. thuringiensis population from high altitude, soil samples were collected from Khumbu, Mt. Everest base camp and the bacteria were isolated in Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB). Further, biochemical and insecticidal properties had already done in RLABB.

MATERIALS AND METHODOLOGY

Bacterial strains, plasmids and media: Bacillus thuringiensis of unknown strain was obtained from RLABB, Nepal. Escherichia coli HB101 is used as host strain for expression of Bacillus thuringiensis gene in Luria-Bertani medium (Bacto tryptone, 10g; yeast extract, 5g; NaCl, 10g for a liter). Plasmid pUC18 was used as cloning vector.

Isolation of total DNA: DNA from Bacillus thuringiensis were isolated by incubating a 50 ml Bacillus thuringiensis culture overnight at 37°C in LB medium with vigorous shaking. Cells were pelleted by centrifugation for 5 min at 8000 rpm and resuspended in sterile SSC buffer. Then cells were lysed using lysozyme (10mg/ml) and Sodium deducible sulphate (10%). DNA was extracted by phenol: chloroform extraction method (Sambrook et al 1989). Then all DNAs preparations were purified by gel extraction method using Kit (Genei, Banglore ,India).

PCR using universal primers (Ben-Dov et al 1997): Universal primers for cry1 (Un1), cry2 (Un2), cry3 (Un3), cry4 (Un4), cry5 (Un5), cry6 (Un6), cry7 (Un7), cry8 (Un8) and cry9 (Un9) were used for PCR. Amplification was carried out in a DNA programmable Thermal controller (MJ Research, Inc.) for 35 reaction cycles. Each reaction mixture was carried out in 50µl system containing 2µl of template DNA mixed with 5µl of 10X reaction buffer, (2.5mM) dNTPs, 10 pmole each primer and 0.5U/µl of Taq polymerase (Genei, Banglore, India).Template DNA was denatured for 1 min at 94°C and annealed to primers at 50°C for 1 min 30s. Extension of PCR products were achieved at 72°C for 2 min. The PCR products were analyzed by 2% agarose gel electrophoresis.

Restriction digestion and ligation of Bt DNA and vector (pUC18): The vector was restricted by 2.5 µl of HindIII (5U/µl) with 10µl of 10X buffer in 100 µl digestion mixture and dephosphorylated with 5U of Calf-intestinal alkaline phosphatase. Similarly, 40 µl of Bt DNA was digested with 5 µl of HindIII (5U/µl) with 10 µl of 10X buffer C in 100 µl digestion mixture(Sambook et al 1989). The vector and inserts were purified using DNA purification kit and then ligated (50ng of vector and 257.078 ng of inserts using 0.5U/µl of T4 DNA ligase in a volume of 10µl) (Johnson et al 1996).

Transformation: Transformation was carried out as described by (Schnepf and Whiteley 1981). E. coli HB101 transformants were selected on media containing ampicillin at 100µg/ml.

Screening of bacterial colonies: Individual transformant was mass cultured in 5ml LB broth and plasmid was extracted by alkaline extraction procedure (Birnboim and Doly, 1979). The recombinant plasmids were screened through PCR method using universal primer 3 (Un3) as described above.

Isolation and purification cry3 fragment: The cry3 fragment (~300bp) was amplified by PCR and electrophoresed (2% agarose gel). The product was extracted, purified and stored in deep freeze till sending for sequencing.

RESULT:

Among the nine universal primers used for PCR of the B. thuringiensis DNA, the cry3 gene fragment (~300) was found to be amplified using Un3 primers (Fig. 1). A total of 1000 individual E. coli chimeras were isolated and stored in deep freeze. So far 100 clones were screened and three were found to have cry3 specific fragment.

Fig. 1. Agrose gel (2%) electrophoresis of PCR products amplified from total DNA from B. thuringiensis strain with nine universal primers (Un). Lanes: C, negative control; M, Marker (λ DNA/HindIII); 1, Un1; 2, Un2; 3, Un3; 4, Un4; 5, Un5; 6, Un6; 7, Un7; 8, Un8 and 9, Un9.

Fig 2. Agarose (2%) gel electrophoresis of PCR products with Un3 primers. Template plasmids were from recombinant E. coli. Lanes: M, marker (λ DNA/HindIII); C, negative control; 1, plasmid containing cry3 specific fragment; 2, plasmid without cry3 specific fragment.

DISCUSSION:

The crystal proteins of B. thuringiensis have been extensively studied because of their pesticidal properties and their high natural level of production. The increasingly rapid characterization of new crystal protein genes has resulted in a variety of sequences and activities of the crystal proteins. Isolation and characterization of B. thuringiensis from different soil sample and their insect toxicity have been studied and tested; molecular characterization of cry protein gene has not done yet in Nepal. Hence, this study was planned with a hope to find novel B. thuringiensis that are cold tolerant, as it was isolated from a high altitude soil samples (Khumbu, Mt. Everest base camp).The bacteria were found to possess cry3 gene as universal primer gave the specific amplification product on PCR among the nine universal primers tested. The size of the cry3 fragment product (~300bp) is markedly lesser than reported elsewhere (589 to 604bp; Ben-Dov et al, 1997). The smaller size of the product may be due to loss of a part of the fragment during recombination events or may be novel cry3 gene fragment in the B. thuringiensis population in the Mt. Everest base camp. In a study, the crystal proteins extracted from some B. thuringiensis from the soil samples, where no mosquitoes are found, were found to be more mosquitocidal compared to that obtained from B. thuringiensis isolated from Kathmandu valley (Shrestha et al, 2006). More universal primers are to be tested against the B. thuringiensis DNA which may explore other cry genes. The absence of the PCR products doesn’t necessarily imply that the strain is devoid of respective gene. A strain may contain a novel gene not detectable with the universal primers for those bacteria, which are isolated from extreme ecological niches. The amplified product was concentrated and purified. The product is being sent for sequencing to know whether it is novel or not. When found to be novel, a piece of the fragment can be used to synthesize specific primers and/or probe to detect the strain in the given population.

REFERENCES:

Beegle CC and Yamamoto. History of Bacillus thuringeinsis, Berliner research and development. Can Entomol 1992; 124: 587-616.

Ben-Dov E, Zaritsky A, Dahan E, Barak Z, Sinai R, Manasherob R, Khamraev A, Troitskaya E, Dubitsky A, Berezina N and Margalith Y. Extended screening by PCR for seven cry-group genes from field-collected strains of B. thuringiensis. Appl Environ Microbiol 1997; 63: 4883-4890.

Birnboim HC and Dolly JA. Rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acid Res 1979; 7: 1513-22.

Johnson TM, Rishi AS, Nayak P and Sen S. Cloning of a cryIIIA endotoxin gene of B. thuringiensis var. tenebrionis and its transient expression in indica rice. J Biosci 1996; 21: 673-685.

McGaughey WH and Whalen ME. Managing insect resistance to Bacillus thuringeinsis Toxins. Science 1992; 258: 1451-5.

Sambrook J, Fritsch EF and Maniatis T. Molecular cloning: A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York. 1989.

Schnepf HE and Whiteley HR. Cloning and expression of Bacillus thuringeinsis crystal protein gene in E. coli. Biochemistry 1981; 78: 2893-7.

Schnepf HE. et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62: 775-806.

Shreshtha UT, Sahukhal GS, Pokhrel S, Tiwari KB, Singh A and Agrawal VP. Delta- endotoxin immuno cross-reactivity of Bacillus thuringiensis isolates collected from Khumbu base camp of Mount Everest region. J Food Sci Technol Nepal 2006; 2: 128-131.

Thermostable glucose isomerase from psychrotolerant Streptomyces species

Bidur Dhungel1, Manoj Subedi1, Kiran Babu Tiwari1,2, Upendra Thapa Shrestha2, Subarna Pokhrel3 and Vishwanath Prasad Agrawal1, 2

(1) Universal science College, Pokhara University, Kathmandu, Nepal
(2) Research Laboratory for Agricultural Biotechnology and Biochemistry, Kathmandu, Nepal
(3) Department of Enzyme Engineering, Seoul National University, Korea

Corresponding Address: Dr. Vishwanath P. Agrawal, Professor of Biochemistry, Universal Science College, Pokhara University, Kathmandu. Email: vpa@wlink.com.np

ABSTRACT

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.

INTRODUCTION

Glucose isomerase (EC 5.3.1.9), GI, is an intracellular bacterial enzyme1 that catalyzes the reversible isomerization of glucose to fructose or xylose to xylulose2. Fructose is the sweetest of various naturally occurring sugars and there has long been a demand for it as alternative to sucrose. GI is one of the three highest tonnage value enzymes, amylases and proteases being the other two.3 The enzyme is used to produce high-fructose corn syrup from corn starch. This process involves several separate enzymatic steps, including liquefaction of corn starch by a-amylase, saccharification by glucoamylase, and isomerization by glucose isomerase.4

Most commercially available GI has been isolated from mesophilic microorganisms, including Streptomyces, Actinoplanes, Flavobacterium and Bacillus spp.3 GIs are homotetramer with 45kDa or 49kDa, the former being more conservative.5- 9 Most of the GIs are not highly thermostable (limited to 60ºC only) and less active at neutral. Thermostable GIs with neutral or slightly acidic pH optima have a potential for industrial applications. The thermo-acid-stable GI allow for faster reaction rates, higher fructose concentration at equilibrium, higher process stability, decreased viscosity of substrate and product streams, and reduced by-product formation.10

Owing to the industrial significance of the enzyme, GI from various microorganisms has been studied and their catalytic and physicochemical properties have been reviewed.11,12 Thermophilic microorganisms produce industrially thermostable enzymes which have been evolved and adapted to the extreme environment of their natural habitats. As the Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB) has been studying and exploiting the actinomycetes’ population diversity from high altitude ecological niche, this work was done on D-glucose isomerase of the psychrotolerant Streptomyces spp. isolated from the soil sample collected from Khumbu, Mount Everest base camp.

METHODOLOGY

Culture: Streptomyces spp. Lob 15.4, isolated from soil samples collected from Lobuche, Mt. Everest base camp, was revived by inoculating spores into a 250 ml conical flask containing 50 ml of culture medium (1% tryptone, 0.7% yeast extract, 1% xylose and 0.1%MgSO4.7H2O, pH 7.0-7.2) followed by incubating at 28ºC in a shaker waterbath (200rpm) for 4 days.13

Enzyme preparation: Streptomyces cells were collected by centrifugation, washed several times with deionised water and homogenized in vertexer in 0.1M phosphate buffer (pH 7.0) containing 5mM MgSO4, 0.5mM CoCl2 and 1mM PMSF. Cells were disrupted in a bath sonicator for 30 min with ice and centrifug at 10000 rpm for 20 min at 4ºC to obtain enzyme supernant.13

Enzyme assay: A 100ml of the enzyme was incubated in 900ml phosphate buffer (pH 7.0) containing 5mM MgSO4, 0.5mM CoCl2 and 0.8M glucose at 37ºC for 40 minutes, followed by keeping the tubes in an ice bath. The amount of the product, fructose, was determined by Seliwanoff’s method.10

Protein determination: Protein content in the supernatant was determined by Bradford assay.14

Enzyme purification: Ammonium sulphate was added to the crude enzyme extract to 45% saturation, incubated for an hour at 4ºC with gentle mixing. The precipitate was collected by centrifugation at 10,000 rpm for 20 min at 4ºC and dissolved in 0.1M phosphate buffer (pH 7.0) containing 5mM MgSO4, 0.5mM CoCl2. The ammonium sulphate concentration was increased stepwise to 60%, 75% and finally to 90% saturation; and the precipitates were harvested accordingly. The fraction containing glucose isomerase activity was pooled and dialyzed overnight against 0.1M-phosphate buffer (pH 7.0).15 Then, a Sepharose 4B column (3.2 by 38.5cm) was prepared and equilibrated with 0.05M phosphates buffer containing 0.15M NaCl. The dialyzed enzyme was applied to the column and eluted with the phosphate buffer. Fraction containing glucose isomerase activity was collected, concentrated with ammonium sulphate and dialyzed against 0.1M phosphate buffer (pH 7.0).15 The purification steps were monitored by SDS-PAGE16 and Native-PAGE17.

Optimization: Optimization was done in phosphate buffer (pH 7.0) containing 5mM MgSO4, 0.5mM CoCl2 at 37ºC for 40 min. unless mentioned otherwise. Glucose (substrate) from 0.1-1.0M and enzyme from 6-124µg were mixed in the respective optimization reaction. Phosphate buffers from pH 4-10 were used to optimize pH of the respective reaction with 0.6M glucose. Time and temperature were optimized by incubation the respective reaction mixtures for 10 to 60 minutes and 30 to 90ºC in phosphate buffer with pH 6.9. To optimize Mg2+ and Co2+ concentrations, 0.05-10mM ions were mixed in the respective reaction buffer. Amount of product (fructose) produced was determined by Seliwanoff’s method.10

Half-life and Thermal stability were determined by measuring residual activity under optimum assay condition after pre-incubation of the enzyme- at 100ºC for 5-30 minutes for half life and at 40-90ºC for 30 to 150 minutes for thermal stability.15

RESULT

A maximum enzyme activity of broth was obtained after 96h (4days) of cultivation in media. Fraction collected during 90% saturation of ammonium sulphate showed glucose isomerase activity and was further purified by column chromatography to 7.1 fold increase in activity (Fig. 1). The purified fraction had specific activity 0.490U/mg (Fig. 2). Native-PAGE revealed that the enzyme had mol. wt. about 200kD. The Michaelis constant (KM) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg (Fig. 3), respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M (Fig. 43), 62.14μg/100μl (Fig. 5), 6.9 (Fig. 6), 70ºC (Fig. 7), and 30 minutes (Fig. 8), respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM (Fig. 9) and 0.5mM (Fig. 10), respectively. The enzyme had half life 30 minutes at 100ºC (Fig. 11). The enzyme was quite thermostable (Fig. 12).

DISCUSSION:

The production of glucose isomerase from Streptomyces species has been documented by several investigators.18,19 The sample sonicated for 20minutes showed maximum enzyme activity.(13)

Chou and Anderson also found glucose isomerase activity in 90% ammonium sulfate saturation.20 To get more purified form of the enzyme, the chromatography has to be done several times. Chen and Anderson reported that the enzyme was purified using DEAE –Sephadex A-50 and the purification was about 12.6 fold over the crude.20 The enzyme activity was stable at 37ºC; therefore, all steps of purification were performed at that temperature. The purified fraction had specific activity 0.490U/mg and crude enzyme had 0.069 U/mg, suggesting a 7.1 fold increase in specific activity of the purified enzyme over crude was observed.

Homogeneity of the purified enzyme was determined by Sodium dodecyl sulphate -polyacrylamide gel electrophoresis (SDS-PAGE). The purified enzyme was homogeneous by the detection of a single protein band on SDS-PAGE and Native-PAGE. Approximately, mol. wt. of 200kD protein band on Native-PAGE and about 50kD on SDS-PAGE was determined, suggesting that the enzyme to be tetramer.

The enzyme also called as Xylose isomerase (XI) as it converts xylose to xylulose besides converting glucose to fructose. Hence, xylose was used as the inducer of the enzyme in the culture medium. The enzyme was, then, optimized using glucose as a substrate. The optimum glucose concentration was 0.6M. The Michaelis constant (KM) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. In the other studies the KM value upto 0.2M10 to 0.167M21 was also been reported. Lama et al. reported Vmax of 6.3U/mg.21 The enzyme with lower KM and higher Vmax towards substrate is more preferred for exploitation of enzyme behavior, suggesting the enzyme extract in the work may not be as competent to that obtained from mesophilic or thermophilic bacteria which are more suitably adapted to higher temperature.

The optimum pH is the ranges between pH 7.0 to 9.0.4 The optimum pH of the glucose isomerase is slightly acidic, pH 6.9. It was apparently lower than that of enzymes from other Streptomyes species.22 Therefore, a low pH optimum is an attractive property for enzyme application because the use of the enzyme at neutral or low pH prevents the formation of by-product, psicose.

Most of glucose isomerase isolated to date showed an optimum temperature around 80ºC.13 Most of the industrially exploitation of the enzyme is done at 60ºC, as Hodge indicated that degradation of ketoses occurs at high temperatures, characterized by pronounced discoloration of an aqueous sugar solution. Interestingly, in this study, the optimum temperature of the enzyme from the cold tolerant bacteria was 70ºC. This may be due to conservation of the gene in bacterial population.6-9 Lama et al. also documented that the kinetic characteristics for XI or GI were similar to XI from distantly related bacteria.21 The optimum temperature of the GI explored that psychrotolerant organisms may have thermostable proteins.

The optimum reaction time of the enzyme was 30min, similar to most of the enzymes from diversed bacteria.1,4,20 Compared to the half life reported by Chou et al13, 120h at 70ºC, the half life of the GI in this work was 30 minute at 100ºC, suggesting to be a quite thermostable one.

Glucose isomerases typically require the presence of divalent metal cations such Mg2+ or Co2+ as essential cofactors for their catalytic activity.23 Treatment of purified enzyme with EDTA resulted in an almost complete loss of enzyme activity. However, the activity could be restored by the addition of metal ions. In particular, increasing amounts of Mg2+ or Co2+ (each up to 10mM) were able to restore only 60-80% of the original xylose isomerase activity. Lama et al. reported, for glucose isomerase, 10mM Mg2+ was required to restore 80% of the original activity.21 As is common with other isomerases, 10mM Mg2+ plus 1mM Co2+ restored total glucose isomerase activity. However, the lower values of the cations observed in this study, suggested that the enzyme might be adapted in the ecological niche.

ACKNOWLEGMENT:

We express our especial thank to Mr. Yogan Khatri, Mr. Deepak Singh and Rajendra Aryal for collecting soil samples from Mount Everest region and all the staffs of RLABB.

REFERENCES:

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2. Tsumura N and T Sato (1961). Enzymatic conversion of D-glucose to D-Fructose: Identification of active bacterial strain and conformation of D-Fructose formation. Agric Biol Chem 25:616-619.

3. Bhosale SH., MB Rao and VV Deshpande (1996). Molecular and industrial aspects of glucose isomerase. Microbiol Rev 60:280-300.

4. Lee C and JG Zeikus (1991). Purification and characterization of thermostable glucose isomerase from Clostridium thermosulfurogenes and Thermoanaerobacter stain B6A. Biochem J 274: 565-571.

5. Kwon, HJ., Kitada, M., Horikoshi K. (1987).Purification and properties of D-xylose isomerase from alkaliphilic Bacillus no KX-6. Agric Biol Chem 51:1983-1989.

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10. Chen, WP., AW. Anderon, and YW. Han.(1979) Production of glucose isomerase by Streptomyces flavogriseus. Appl. Environ. Microbiol. 37:785-787.

11. Chen WP (1980) Glucose isomerase (a review). Process Biochem June/July: 30-35.

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14. Bradford MM (1976) A rapid and sensitive method for quantization of microgram quantities of protein using the principles of protein-dye binding. Anal Biochem 72: 248-254.

15. Liu SY, J Wiegel and FC Gherardini (1996) Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/ SL-YS 489. J Bacteriol 178: 5938-5945.

16. Laemmli, UK (1970) Cleavage of structural proteins during assembly of The head of bacteriophage T4. Nature 277: 680-685.

17. Blackshear PJ (1984) Systems for polyacrylamide gel electrophoresis. In Methods in enzymology (Jakoby WB eds.) vol 104: 237-255.

18. Takasaki Y (1966) Studies on sugar isomerizing enzyme. Production and utilization of glucose isomerase from Streptomyces spp. Agric Biol Chem 30: 1247-1253.

19. Strandberg GW and KL Smiley (1971). Free and immobilized glucose Isomerase from Streptomyces phaeochromogenes. Appl Microbiol 21: 588-593.

20. Antrim RL, W Colilla and BJ Schnyder (1979). Glucose isomerase production of high fructose syrups. Appl Microbiol Biotechnol 2: 97-155.

21. Lama L, V Nicolaus, V Calandrelli, I Romano, R Basile and A Gambacorta (2001). Purification and characterization of thermostable xylose (glucose) isomerase from Bacillus thermoantarcticus. J Microbiol Biotechnol 27: 234-240.

22. Bucke C (1997). Industrial glucose isomerase In A Wiseman (ed.). Topics in enzyme and fermentation biotechnology. Ellis Horwood Limited. pp. 147-171.

23. Whitlow M, AJ Howard, BC Finzel, TL Poulos, E Winborne and GL Gilliland (1991). A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6A Streotomyces rubiginous structures with xylitol and D-xylose. Proteins 9: 153-173.

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