Mr.

Vishal Gupta
Thesis submission Seminar Ph.D. (Biotechnology)
Supervisor Prof. Jitender Sharma, Department of Biotechnology, Kurukshetra University, Kurukshetra -136119 Co-Supervisor Dr. Radha Prasanna, Senior Scientist, Division of Microbiology,

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

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 tohumanbeing 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

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

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

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

Aspergillus candida Af

As

Av As Af

N Al

Al

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

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 mol 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).

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

16S rDNA based identification

A 1 2
3 4 M

B 1 2
1500 1000 500 bp

M 1500 1000 500

bp 3 4

M 1

1200 1000 500 bp

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

Strain Anabaena number strain

BLASTN results of 16S rDNA sequences

RPAN 1 A. fertilissima RPAN 8 A. laxa RPAN 12 A. sphaerica RPAN 16 A. variabilis

BLASTN results of 16S rDNA sequencing Identity Anabaena sp. Accession (%) number 99 Anabaena sp. X59559 97 99 99 Anabaena sp. Anabaena sp. Anabaena sp. X59559 X59559 X59559

C 1

C 2

C 3

C 4

C 5

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

Identification of a microcystin synthetase gene (mcyA)

M 1 2 3 4

10 20 10 00 50 0 30 0 b p

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.

BLASTX results of all the mcyA+ Anabaena strains

Strain Anabaena strain Similarity (%) Cyanobacterium number RPAN 8 A. laxa 98-99 uncultured cyanobacterium 94-98 Microcystis sp. 81-82 Anabaena sp. Accession number ABS83285 ABY5516; BAA83992 CAD56453-55

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

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
M Af Al M Al 1000 1500 1000 500 bp A 1000 500 500 M Al

bp

bp

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.

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

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
A 1.2 1 0.8 0.6 0.4 10 14d 28d 42d 20 15 B 25 14d 28d 42d

) m ( t b h i f e n o Z
C-M P C-L P C-D P L -8:16 :D C L

) l m / U I ( y v c e n a s o t i h C

0.2 0

5 0 C-M P C-LP C-DP L:D -8:16 CL N

0.9 0.8 0.7 0.6 0.5 0.4 A. fertilissima A. sphaerica

) l / g m ( c n i e t o r P

0.3 0.2 0.1 0

CP -M

CP -M

CP -M

CP -D

CP -D

CP -D

L C

L C

CP -L

CP -L

D 1 : : L -8 6

D 1 : : L -8 6

CP -L

14d

28d

42d

D 1 : : L -8 6

L C

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

Cloning and sequencing of putative chitosanaseencoding (cho) gene

Analysed parameters The length of the sequenced clones (bp) Largest ORF identified (bp)

A. sphaerica 1410 1086

A. fertilissima 1425 1101 367 40.6

Length of Amino acid sequence 362 Predicted molecular mass (kDa) 40.0

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).

BLASTP results of sequenced chitosanase specific gene from A. fertilissima and A. sphaerica in the PDB data base
Enzyme Organism Similarity PDB accession no. 33 % 32 % 3BMX 1TR9

beta-Nhexosaminidase beta-Nhexosaminidase

Bacillus subtilis Vibrio cholerae

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

Primer complementation positions

Cho844F ChoF1

and

5atcaatcaatggcagatacagatatattcaaacccatattctgtttggctttcctaatttacgcgatgtgcgacttattcttaaaacccctctccaaacctctcccctgcaaggagagaggcttta attattatgccccttcccttgtagggaaggggttgggggttaggtctgagagaaagttgcacacggcgttaattttgagttttgaattggtattaattacctctattgagggtgtaggataagggctga gattatttatgctgttaatccagtctttagctacacaactataggttaagcattATGCCAGCATTGCAGAGACTAGAACGCTTTGGAATCGTCCTAATTC TGGGTATTTCTGGTACTGAGTTGAGTGATGAAGATAAACGCGCTCTGGGTGAATTGAAACCAATAGGGGTAATATTTTT TGCTAAAAACTTTGTAGATGGTGTACCTTACGAGGTTTGGCTGGAGACTTTTCAGGAGTTACATAGCCAAATACAATTG GAATATGCAGAACGCGATTCGATGTTTTTTACCTTAGACCATGAGGGAGGACGCGTTGTGAGGACACCTTTACCGATTA CCAGATTTCCTCAGGCGTTGTTGTTGCGATCGCACGCCCGTGAAGTAGCAAAAGCCACGGCAATTGAATTAAAATCTG AGGGCATCAACTTATCTTGGTCACCTGTAGCTGATATTTATTCCCATCCGCAAAATCCGGAGATCGGTTCTCGCGCCTTT GGAAATACTCCTGAAACTGCGGCTACTGGTGCGCGGGAATTGTATTATTTGGGACTGACAGAAGCCGGAATTGTGGGA TGCGCCAAACATTTCCCCGGACATGGTGATACTAGCAAAGACTCCCATGTGGAATTACCAATCCTCAACCTGACTCCA GAGGAATTACGAAGGCGAGAACTTATCCCCTTCCAGAAAGCTTTGATTGAAGAAGGGATTCCCCAGCTCATCATGACC GCCCATATTTTATTTCCCAAAATCGACCCAGATTTACCAGCTACCCTATCCCGCCAGCCCATCCTCAAAACTATACTG CGGGAAGAACTTGGTTTTCAGGGTGTCGTTGTGTCTGACGACTTAGATATGAAAGCAGTTTCCGATATGTTTATGGAAC GTGGTACGGTCGCGCGGGCTTTTAATGCTGGCTGTGATTTATTTATTGTTTCTCGCAATATCCACGCGTCTTCTATCGAG CGTACCTATAAAATTGCCGAAAATTTTGCTGATGCTTTAACTGATGGTAGTCTGGCTGAGTCAGTAGTAGATTCCGCT AAGGAGAGAATCGAAAGACTATTGGCGGTAACTCCAGAATATTCTGTACAGATGTTAGATAAAGATACTTTAGTACATC ATGGCGAATTGGCGATCGCTTGTTGTTTTTAAaagtggtccgaagaa-3
ChoR1 Cho1692R ChoR3 ChoF3

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

Identification of signal peptides and cleavage sites in A. fertilissima and A. sphaerica by hidden markov model algorithm
A. sphaerica Protein prediction : Non -secretory Signal peptide probability : 0.063 Signal anchor probability : 0.007 Max cleavage site probability : 0.045 between position 24 and 25

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

Confirmation of the functional chitosanase gene in both the Anabaena strains

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

) N c l G 1 ( ) N c ) N l2 c G l G (2 1 ( ) N c l G ( ) N 3 c l G 3 ( ) N c l G ( ) N cN lc G 4 (l )4 (G

Standard M 66 1 2

43 A. sphaerica 14.3 (kDa)

A. fertilissima

Retention time (min)

SDS-PAGE and HPLC based analyses of purified Cho proteins

) N c l G ( ) N c l G (

) N c l G ( ) N c l G ( 6 6

5 5

Identification of catalytic residues of chitosanase responsible for antifungal activity Synthetic oligonucleotide primers used for site-directed mutagenesis Mutation Oligonucleotide Sequence (5 3) Amino acid Exchanges
E22D F:5' CTGGGTATTTCTGGTACTGACTTGAGTGATGAAGATAAAC 3' R: 5' GTTTATCTTCATCACTCAAGTCAGTACCAGAAATACCCAG 3' L68F E121D E141D F: 5' CATAGCCAAATACAATTCGAATATGCAGAACGCG 3' R: 5' CGCGTTCTGCATATTCGAATTGTATTTGGCTATG 3' F: 5' GAATTAAAATCTGACGGCATCAACTTATC 3' R: 5' GATAAGTTGATGCCGTCAGATTTTAATTC 3' F: 5' CGCAAAATCCGGACATCGGTTCTCGC 3' R: 5' GCGAGAACCGATGTCCGGATTTTGCG 3' L161F Q211H F: 5' GGTGCGCGGGAATTCTATTATTTGGGACTG 3' R: 5' CAGTCCCAAATAATAGAATTCCCGCGCACC 3' F: 5' GAACTTATCCCCTTCCACAAAGCTTTGATTGAAG 3' R: 5' CTTCAATCAAAGCTTTGTGGAAGGGGATAAGTTC 3' Q221E Q244E F: 5' GAAGAAGGGATTCCCGAGCTCATCATGACC 3' R: 5' GGTCATGATGAGCTCGGGAATCCCTTCTTC 3' F: 5' GCTACCCTATCCCGCGAGCCCATCCTCAAAAC 3' R: 5' GTTTTGAGGATGGGCTCGCGGGATAGGGTAGC 3' CAG GAG CAG GAG CAG CAC TTG TTC GAG GAC GAG GAC TTG TTC GAG GAC

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

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

A. 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 against ofamino acid residues inprotein 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

RAMACHANDRAN PLOT STATISTICS

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

Plot Statistics

A. sphaerica

A. fertilissima

A. sphaerica Cho Further, Ramachandran plot for all residues were plotted which indicates the increase in Ala (1), A. fertilissima Cho 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

A. fertilissima

A. sphaerica Cho

A. fertilissima Cho

A. sphaerica Cho

A. fertilissima Cho

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 A. sphaerica A. properties fertilissima

Number of H-bonds Number of helices Number of strands Number of turns

215 14 18 42

220 19 12 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 realtime RT-PCR (qRT-PCR) under increasing dark periods
0.6 0.5 0.4 0.3 0.2 0.1 c d l f n o i s e r p x E 0 L:D (14:10) L:D (12:12) L:D (10:14) L:D (8:16) A. fertilissima A. sphaerica

[Gupta,

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:D12:12 and L:D-10:14 as compared to that of the control (C-MP).

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

Optimization of other environmental/nutritional factors on functioning of chitosanase and its specific properties
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 -0.1

0.3 0.2 0.1 0 -0.1 -0.2 -0.3 -0.4 4.5 5.5 pH 6.5 9 10 12

c d l f n o i s e r p x E

Phosphorus concentration ( M) 43 86 344

0.1 0 -0.1 -0.2 -0.3 -0.4

c d l f n o i s e r p x E

-0.5 15

Temperature (C) 20 35 40

c d l f n o i s e r p x E

50