Mr. Vishal Gupta

SupervisorProf. Jitender Sharma, Department of Biotechnology,Kurukshetra University, Kurukshetra -136119Co-Supervisor Dr. Radha Prasanna, Senior Scientist, Division of Microbiology, Indian Agricultural Research Institute, New Delhi-110012

Thesis submission Seminar

Ph.D. (Biotechnology)

Cyanobacteria are an unique group of gram negative photosynthetic prokaryotes with short generation time and tremendous metabolic flexibility. This makes them a favorite model organism for a deeper understanding of several metabolic processes.

They produce compounds with varying bioactivities - cyanotoxins (microcystins, nodularins and hepatotoxins), antibiotics, enzymes with protease activity and several biocidal (algicidal/fungicidal) compounds having agricultural and industrial significance.

Most of the work involving this aspect of toxin production has been done using Microcystis (Tillett et al., 2000, 2001; Ouellette et al., 2006) and the expression of these genes is known to be regulated by complex mechanisms and is influenced by environmental factors.

Anabaena is an important genus, widely distributed in diverse habitats and exploited as a rich source of bioactive compounds, such as microcystins, laxaphycins. Many such compounds exhibiting fungicidal and herbicidal/weedicidal properties are influenced by environmental factors like light intensity, temperature, pH, P levels (Carmichael, 1994) etc, however, their genomics and proteomics is far from understood and there is no published information on genes involved in fungicidal activity.

At present, two molecular systems (NRPS and PKS ) are known to be involved in cyanobacterial toxicity. (Tillett et al., 2000; Christiansen et al., 2003; Rouhiainen et al., 2004).

The fungal diseases are one of the most important causes that drastically reduce the agricultural crop production worldwide (Bhadauria et al., 2009). At present, the most common control measure is to use synthetic fungicides. However, the limitation associated with the excessive use of fungicides is that it may lead to the environmental pollution and toxicity to human being and domestic animals.

Apart from this, there is also a chance for the development of fungicide-resistance in these phytopathogens (Liu et al., 2001). The other control measures such as crop rotation and breeding for resistant plant varieties were also employed; however, its drawback is that it requires complete understanding of the mechanism of fungicide tolerance (Compant et al. 2005). Towards this endeavor, yet another approach is to use microbes, as a biocontrol agent, which can restrict the growth of phytopathogens. This is a simple, cost-effective and user friendly technique which offers the eco-friendly way to reduce the agricultural losses.

Endoglucanases/chitinases/chitosanases are identified as key enzymes which can be employed as a biocontrol agent and restrict the growth of phytopathogens (Kucuk and Kivanc, 2004; Adams, 2004). There are several microbes which produce this enzyme and hence can be employed as a biocontrol agent against phytopathogenic fungi.

The successful use of Pseudomonas sp., Serratia marcescens, Trichoderma sp. Bacillus sp. and Streptomyces sp. producing chitinases/endoglucanases has already been demonstrated to restrict the growth of soil borne phytopathogens (El-Moughy et al., 2011; Ganiger et al., 2009; Quecine et al., 2008; Weller, 2007; Anitha and Rebeeth, 2009).

However, there is still a considerable interest in finding more efficient agents, which can be used effectively for the biocontrol purpose (Anitha and Rebeeth, 2009). In this context, cyanobacteria are one of the possible candidates because of their promise as a biofertilizer and biocontrol agent (Prasanna et al., 2008a; Manjunath et al., 2010, Dukare et al., 2011).

Although the use of cyanobacteria as a biofertilizer has been demonstrated, its use as a biocontrol agent has not been explored much. Identification and characterization of genes that are involved in synthesis of plant growth promoting compounds/biocidal compound/enzyme(s) against bacterial and fungal pathogens, in cyanobacteria may open a new avenue of research. The present study has been therefore, planned with the following objectives:

To identify genes involved in the production of the fungicidal

compound/enzyme(s) in selected Anabaena strain(s)

To analyse the structural organization and regulation of the genes

involved in the production of fungicidal compound/enzyme(s) in

Anabaena strain(s)

To carry out expression analyses of production of fungicidal

compound/enzyme(s) under different physiological conditions (light

quality and intensity & temperature and P levels)

To develop a system for the large scale production of fungicidal

compound(s)

Screening of Anabaena strains for fungicidal activity

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Aspergillus candida

Af, A. fertilissima; As, A. sphaerica; Al, A. laxa and N, nystatin

Fungicidal activity (in terms of zone of inhibition) of two selected Anabaena strains on the lawn of Aspergillus candida

I. Axenization of cultures of both Anabaena strains was done using standard methods of subculturing and antibiotic treatment.

Media: BG-11 media(Stanier et al., 1971)

Temperature: 28 ± 2oC

Light intensity: 52-55 μmol photon m-2 s -1

L : D cycles : 16:8 hours

Cultures were made axenic by repeated subculturing and their purity was checked by microscopic studies and their identification was done using the keys given by Desikachary (1959).

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Microscopy of Anabaena strains. A, A. fertilissima; B, A. laxa; C, A. variabilis; D, A. sphaerica. 100x oil immersion objective.

PCR amplification of selected Anabaena strains using primers directed towards 16S rDNA

16S rDNA based identification

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BLASTN results of 16S rDNA sequencing

Identity (%)

Anabaena sp. Accession number

RPAN 1 A. fertilissima

99 Anabaena sp. X59559

RPAN 8 A. laxa 97 Anabaena sp. X59559RPAN 12

A. sphaerica

99 Anabaena sp. X59559

RPAN 16

A. variabilis

99 Anabaena sp. X59559

BLASTN results of 16S rDNA sequences

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Phylogram generated using nearly complete 16S-rDNA sequences (1456 bp) using fD1 – rD1 primers from Anabaena strains of our study and available NCBI sequences, constructed by Neighbour Joining method (Saitu and Nei, 1998). Boot strap values are indicated at the corresponding nodes.

Identification of a microcystin synthetase gene (mcyA)

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PCR amplification of Anabaena strains with primers directed towards condensation domain of microcystin synthetase gene (mcyA). 1, A. laxa; 2, A. fertilissima; 3, A. sphaerica and 4, A. variabilis. M is the 100 bp DNA ladder.

Strain number

Anabaena strain

Similarity (%)

Cyanobacterium Accession number

RPAN 8 A. laxa 98-99 uncultured cyanobacterium

ABS83285

94-98 Microcystis sp. ABY5516; BAA83992

81-82 Anabaena sp. CAD56453-55

BLASTX results of all the mcyA+ Anabaena strains

Dendrogram based on mcyA sequences using Neighbour Joining method. Boot strap values are indicated at the corresponding nodes.

The mcyA+ Anabaena species studied under this investigation formed a separate subgroup. This is an interesting finding which has tremendous significance in understanding evolutionary significance of mcyA, especially in relation to the global spread and diversity among cyanobacterial species. Thus, Anabaena strain (A. laxa) with microcystin toxin production and fungicidal behaviour may phylogenetically more heterogenetic than other toxic without fungicidal effect (mcyA+

from the previous studies and NCBI data base) and non-toxic with fungicidal effect (A. fertilissima).Such sequences may be useful for identifying such Anabaena strains.

PCR based amplification as a tool for screening ofpromising strain as source

of fungicidal enzyme/compound

Several primers were designed from the conserved region of antifungal enzymes (chitinases, chitosanases and endoglucanases) and compounds (Thioquinolobactin, phenazine, 2-amino benzoic acid, iturin and phenyl acetic acid) from different bacteria such as Serratia sp., Pseudomonas sp., Bacillus sp.

Primer designing from the conserved regions of phenazine, phenyl acetic acid, 2-amino benzoic acid and iturin in different bacteria

Primer designing from the conserved regions of chitinase gene from different bacteria

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PCR amplification of Anabaena strains with primers directed towards chitosanase (A), thioquinolobactin (B) and phenazine (C). Al, A. laxa; Af, A. fertilissima. M is the 100 bp DNA ladder.

•Among all these primers, the specific designed to detect new strains for the production of antifungal chitosanase similar to chitosanase A of Mitsuaria chitosanotabidus showed a single band of approximate size 1400 bp in both A. fertilissima and A. laxa (exhibiting fungicidal activity).•While, only A. laxa showed amplicons of 550 and 370 bp (Fig. 17 B-C) with specific primers directed towards

For further investigation, A. fertilissima was selected for analyses of chitosanase activity study and A. sphaerica as a negative control (with chitosanase activity but not fungicidal activity)A. laxa was selected for identification of fungicidal compound(s).

Identification and characterization of an antifungal enzyme (chitosanase) in A. fertilissima

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Two Anabaena species- A. fertilissima and A. sphaerica were selected towards differential fungicidal behaviour to optimize the light-dark condition for the maximum

production of chitosanase/antifungal activity

The 14d, 28d and 42d old cultures of two Anabaena strains (A. fertilissima ; A. sphaerica ) were grown independently under variable light: dark conditions viz. continuous light (CL) and dark (CD); L:D-8:16 and L:D-16:8. The time dependent measurement of chitosanase and antifungal activities under different light-dark conditions indicated that in A. fertilissima, both these activities were stimulated in the dark phase and found to be maximum at L:D-8:16.

The relatively lower level of protein in A. fertilissima (as compared to A. sphaerica) in the dark phase of L:D-8:16 (particularly at 28d) suggested that the increase in the length of dark phase leads to retardation of growth which may act as a physiological signal to increase the chitosanase/antifungal activities in A. fertilissima.

Cloning and sequencing of putative chitosanase-encoding (cho) gene

Analysed parameters A. sphaerica A. fertilissima

The length of the sequenced clones (bp) 1410 1425

Largest ORF identified (bp) 1086 1101

Length of Amino acid sequence 362 367

Predicted molecular mass (kDa) 40.0 40.6

BLASTP results of sequenced chitosanase specific gene from A. fertilissima and A. sphaerica

BLASTN analysis revealed 100% (A. sphaerica) and 97% (A. fertilissima) identity with a glycoside hydrolase family 3 like (GH3-like) gene of A. variabilis ATCC 29413 strain

This GH3 family domain protein (EC:3.2.1) comprised enzymes with known functions such as: beta-glucosidase (EC 3.2.1.21)

beta-xylosidase (EC 3.2.1.37)

N-acetyl beta-glucosaminidase (EC 3.2.1.52)

glucan beta-1,3-glucosidase (EC 3.2.1.58)

cellodextrinase (EC 3.2.1.74)

exo-1,3-1,4-glucanase (EC 3.2.1).

Incidentally, this enzyme also belongs to the GH3 family protein (PFAM Accession PF00933) in both organisms

BLASTP results of sequenced chitosanase specific gene from A. fertilissima and A. sphaerica in the PDB data base

Enzyme Organism Similarity PDB accession no.

beta-N-hexosaminidase Bacillus subtilis 33 % 3BMX

beta-N-hexosaminidase Vibrio cholerae 32 % 1TR9

Primer complementation and positions

Cho844F5’atcaatcaatggcagatacagatatattcaaacccatattctgtttggctttcctaatttacgcgatgtgcgacttattcttaaaacccctctccaaacctctcccctgcaaggagagaggcttta

attattatgccccttcccttgtagggaaggggttgggggttaggtctgagagaaagttgcacacggcgttaattttgagttttgaattggtattaattacctctattgagggtgtaggataagggctga

gattatttatgctgttaatccagtctttagctacacaactataggttaagcattATGCCAGCATTGCAGAGACTAGAACGCTTTGGAATCGTCCTAATTC

TGGGTATTTCTGGTACTGAGTTGAGTGATGAAGATAAACGCGCTCTGGGTGAATTGAAACCAATAGGGGTAATATTTTT

TGCTAAAAACTTTGTAGATGGTGTACCTTACGAGGTTTGGCTGGAGACTTTTCAGGAGTTACATAGCCAAATACAATTG

GAATATGCAGAACGCGATTCGATGTTTTTTACCTTAGACCATGAGGGAGGACGCGTTGTGAGGACACCTTTACCGATTA

CCAGATTTCCTCAGGCGTTGTTGTTGCGATCGCACGCCCGTGAAGTAGCAAAAGCCACGGCAATTGAATTAAAATCTG

AGGGCATCAACTTATCTTGGTCACCTGTAGCTGATATTTATTCCCATCCGCAAAATCCGGAGATCGGTTCTCGCGCCTTT

GGAAATACTCCTGAAACTGCGGCTACTGGTGCGCGGGAATTGTATTATTTGGGACTGACAGAAGCCGGAATTGTGGGA

TGCGCCAAACATTTCCCCGGACATGGTGATACTAGCAAAGACTCCCATGTGGAATTACCAATCCTCAACCTGACTCCA

GAGGAATTACGAAGGCGAGAACTTATCCCCTTCCAGAAAGCTTTGATTGAAGAAGGGATTCCCCAGCTCATCATGACC

GCCCATATTTTATTTCCCAAAATCGACCCAGATTTACCAGCTACCCTATCCCGCCAGCCCATCCTCAAAACTATACTG

CGGGAAGAACTTGGTTTTCAGGGTGTCGTTGTGTCTGACGACTTAGATATGAAAGCAGTTTCCGATATGTTTATGGAAC

GTGGTACGGTCGCGCGGGCTTTTAATGCTGGCTGTGATTTATTTATTGTTTCTCGCAATATCCACGCGTCTTCTATCGAG

CGTACCTATAAAATTGCCGAAAATTTTGCTGATGCTTTAACTGATGGTAGTCTGGCTGAGTCAGTAGTAGATTCCGCT

AAGGAGAGAATCGAAAGACTATTGGCGGTAACTCCAGAATATTCTGTACAGATGTTAGATAAAGATACTTTAGTACATC

ATGGCGAATTGGCGATCGCTTGTTGTTTTTAAaagtggtccgaagaa-3’ChoR1 Cho1692R

ChoR3

ChoF3

ChoF1

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Multiple sequence alignment of sequenced genes encoding GH3-like chitosanases in both Anabaena species with bacterial chitosanase (choA) genes (Flavobacterium sp. AY856849,

Herbaspirillum sp. AY856850 and Mitsuaria chitosanitabida AY856851)

The pair-wise amino acid sequence alignment revealed 5 insertions and 5 substitutions in the amino acid sequence of A. fertilissima as compared to A. sphaerica

A. sphaerica

A. fertilissima

Protein prediction: Secretory Signal peptide probability: 0.717Signal anchor probability: 0.127 Max cleavage site probability: 0.607 between position 23 and 24

Protein prediction: Non-secretorySignal peptide probability: 0.063Signal anchor probability: 0.007Max cleavage site probability: 0.045between position 24 and 25

Identification of signal peptides and cleavage sites in A. fertilissima and A. sphaerica by hidden markov model algorithm

Confirmation of the functional chitosanase gene in both the Anabaena strains

Subcloning of chitosanase specific PCR products into pIVEX GST fusion expression vector

SDS-PAGE and HPLC based analyses of purified Cho proteins

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Mutation Oligonucleotide Sequence (5’ 3’) Amino acid Exchanges

E22D F:5' CTGGGTATTTCTGGTACTGACTTGAGTGATGAAGATAAAC 3'

R: 5' GTTTATCTTCATCACTCAAGTCAGTACCAGAAATACCCAG 3'

GAG GAC

L68F F: 5' CATAGCCAAATACAATTCGAATATGCAGAACGCG 3'

R: 5' CGCGTTCTGCATATTCGAATTGTATTTGGCTATG 3'

TTG TTC

E121D F: 5' GAATTAAAATCTGACGGCATCAACTTATC 3'

R: 5' GATAAGTTGATGCCGTCAGATTTTAATTC 3'

GAG GAC

E141D F: 5' CGCAAAATCCGGACATCGGTTCTCGC 3'

R: 5' GCGAGAACCGATGTCCGGATTTTGCG 3'

GAG GAC

L161F F: 5' GGTGCGCGGGAATTCTATTATTTGGGACTG 3'

R: 5' CAGTCCCAAATAATAGAATTCCCGCGCACC 3'

TTG TTC

Q211H F: 5' GAACTTATCCCCTTCCACAAAGCTTTGATTGAAG 3'

R: 5' CTTCAATCAAAGCTTTGTGGAAGGGGATAAGTTC 3'

CAG CAC

Q221E F: 5' GAAGAAGGGATTCCCGAGCTCATCATGACC 3'

R: 5' GGTCATGATGAGCTCGGGAATCCCTTCTTC 3'

CAG GAG

Q244E F: 5' GCTACCCTATCCCGCGAGCCCATCCTCAAAAC 3'

R: 5' GTTTTGAGGATGGGCTCGCGGGATAGGGTAGC 3'

CAG GAG

Identification of catalytic residues of chitosanase responsible for antifungal activity

Eight amino acids (7 in mature protein and 1 in the signal peptide) in the Cho protein of A. fertilissima were changed independently to some other amino acids

Synthetic oligonucleotide primers used for site-directed mutagenesis

E. coli clones harboring mutations in cho of A. fertilissima (A, L68E; B, E22D; C, E121D; D, E141D; E, L161E; F, Q211E; G, Q221E; H, Q244D); I, wild type cho -A. fertilissima (positive control); J, wild type cho-A. sphaerica; K, insert-free vector transformed E. coli (negative control).

Conserved catalytic amino acid residues of Cho in M. chitosanitabidus (GenBank Accession no. AB010493) and Cho of A. fertilissima (ChoAf). Shaded bars indicate the conserved amino acids among them, and the two conserved and catalytic amino acid residues (E121 and E141) are boxed.

RAMACHANDRAN PLOT

A BA. sphaerica A. fertilissima

The Ramachandran plot was constructed from the amino acid sequence of both Anabaena species to understand the differences in the backbone dihedral angles ψ against φ of amino acid residues in protein structure.The plot statistics indicated the difference in the total number of amino acid residues (343 and 350 in A. sphaerica and A. fertilissima, respectively). The number of residues in most favoured regions (A, B, L) and additional allowed regions (a, b, l, p), end-residues, proline and glycine were almost similar in both the plots. While, the numbers of residues in the generously allowed and disallowed regions were quite different; the generously allowed and disallowed regions were found 39 % more (dotted region) and 16.66 % lesser, respectively in A. fertilissima Cho compared to that of A. sphaerica Cho. Thus, the increase in low energy level regions in A. fertilissima Cho require additional interactions to stabilize the protein structure. Such type of protein structures may have functional significance and may be conserved within a protein family, which may be related to possible conformational dynamics of the structure, as suggested by (Pal and Chakrabarti, 2002).

RAMACHANDRAN PLOT STATISTICS

Plot Statistics A. sphaerica A. fertilissimaResidues in most favoured regions (A, B, L) 197 198Residues in additional allowed regions (a, b, l, p) 79 79Residues in generously allowed region (-a, -b, -l, -p) 11 18Residues in disallowed regions 12 10Number of non-glycine and non-proline residues 299 305Number of end-residues (excJ. Gly and Pro) 2 2Number of glycine-residues (shown as triangles) 23 23Number of proline residues 19 20Total number of residues 343 350

A. sphaerica Cho A. fertilissima Cho

A. sphaerica A. fertilissima

Further, Ramachandran plot for all residues were plotted which indicates the increase in Ala (1), Gln (3) and Glu (3) residues in A. fertilissima (Fig. 29). Such changes in the amino acid residues confirmed our site-directed mutagenesis results. These changes in amino acid sequence may take place at the time of evolution which leads to the functional Cho in A. fertilissima

A. sphaerica Cho A. fertilissima Cho

A. sphaerica Cho A. fertilissima Cho

A. sphaerica A. fertilissima

A. sphaerica Cho A. fertilissima Cho

A B

C D

Predicted three dimensional models for chitosanase from both Anabaena species. A and C, ribbon shaped model and B and D, ball and stick model, in A. sphaerica and A. fertilissima Cho, respectively.

Difference in the structural properties of chitosanase three dimensional structures of both Anabaena species

Protein 3D structural properties A. sphaerica A. fertilissima

Number of H-bonds 215 220

Number of helices 14 19

Number of strands 18 12

Number of turns 42 48

The predicted three dimensional structures of both chitosanases were compared - which revealed significant changes in the number of H-bonds, helices, strands and turns in both the proteins. Such changes in the protein structure further strengths the functionality of the chitosanase in A. fertilissima Cho. Thus, structural deformities in the protein structure of A. sphaerica may lead to the loss of functioning.

Expression profiling of cho by Quantitative real-time RT-PCR (qRT-PCR) under increasing dark periods

The expression profiling of cho was found to be dependent upon the increase in the length of dark periods and the sequential 0.15, 0.25 and 0.35 fold increase in the expression was observed in L:D-14:10; L:D-12:12 and L:D-10:14 as compared to that of the control (C-MP).

[Gupta, V., Prasanna, R., Natarajan, C., Srivastava, A.K. and Sharma, J.K. (2010). Identification, characterization and regulation of a novel antifungal chitosanase gene (cho) in Anabaena spp. Applied and Environmental Microbiology 76(9): 2769-2777] Impact Factor 4.0

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Optimization of other environmental/nutritional factors on functioning of chitosanase and its specific

properties