Table: Detailed information of the identified protein spots
Spot No. / Protein name(showing homology with) / Mr (kDa)/
pI Theor. / Mr (kDa)/
pI Exper. / Accession no. / No. of peptides matched / Mowse score
1. Energy metabolism (photosynthesis, photorespiration and carbon fixation)
4. / Phosphoglycerate kinase
(Nostoc sp.strain PCC7120) / 57.97/4.9 / 64.0/4.9 / gi|17229234 / 29 / 196
5. / RUBISCO
(Nostoc sp.strain PCC7120) / 53.41/6.3 / 49.0/4.6 / gi|17229016 / 29 / 204
8. / RUBISCO
(Nostoc sp.strainPCC7120) / 53.41/6.3 / 43.0/6.5 / gi|17229016 / 19 / 154
9. / Uroporphyrinogen decarboxylase
(Nostoc sp.strain PCC7120) / 38.80/6.4 / 39.0/6.6 / gi|17231401 / 15 / 97
10. / Glyceraldehyde 3 p dehydrogenase
(Nostoc sp.strain PCC7120) / 37.12/6.1 / 30.0/6.2 / gi|17232554 / 13 / 87
16. / Transketolase
(Nostoc sp.strain PCC7120) / 72.31/5.9 / 72.0/6.0 / gi|17230836 / 27 / 167
19. / RUBISCO
(Nostoc sp.strain PCC7120) / 53.41/6.3 / 64.0/6.6 / gi|17229016 / 32 / 226
21. / Phycobilisome rod core linker protein
(Nostoc sp.strain PCC7120) / 31.92/9.4 / 22.0/6.8 / gi|17228030 / 9 / 83
22. / Phycobilisome rod core linker protein
(Nostoc sp.strain PCC7120) / 31.92/9.4 / 20.0/6.3 / gi|17228030 / 12 / 96
25. / Fructose 1,6 bis phosphate aldolase
(Nostoc sp.strain PCC7120) / 38.76/5.4 / 39.0/5.7 / gi|17232055 / 14 / 111
26. / 6, phosphogluconate dehydrogenase
(Nostoc sp.strain PCC7120) / 52.37/5.8 / 48.0/6.4 / gi|17232767 / 12 / 114
32. / F0F1 ATP synthase beta subunit
(Nostoc sp.strain PCC7120) / 51.94/- / 54/4.8 / gi|75908515 / 19 / 410
33. / Glucose 6-P isomerase
(Nostoc sp.strain PCC7120) / 57.78/- / 58/6.1 / gi|17228545 / 12 / 377
34. / Transketolase
(Nostoc sp.strain PCC7120) / 72.31/5.9 / 69/5.9 / gi|17230836 / 5 / 108
37. / Glyceraldehyde 3-P dehydrogenase (Nostoc sp.strain PCC7120) / 37.12/6.1 / 36/6.2 / gi|17232554 / 6 / 79
38. / Feredoxin NADP reductase
(Anabaena PCC7119 ) / 34.14/- / 38/6.8 / gi|13096126 / 13 / 136
45 / Phosphoglycerate kinase
(Nostoc sp.strain PCC7120) / 57.97/4.9 / 60/4.9 / gi|17231623 / 4 / 128
53. / Pyruvate dehydrogenase
(Nostoc sp.strain PCC7120) / 36.39/- / 31/5.9 / gi|17227618 / 10 / 180
60. / Feredoxin NADP reductase
(Nostoc sp.strain PCC7120) / 48.98/7.66 / 52.5/6.98 / gi|17231613 / 8 / 154
61. / NADH dehydrogenase
(Nostoc sp.strain PCC7120) / 45.63/4.95 / 48.50/3.75 / gi|17231586 / 14 / 172
65. / 6-Phosphogluconolactonase
(Nostoc sp.strain PCC7120) / 26.17/5.18 / 30.15/5.05 / gi|17229094 / 12 / 134
2. Oxidative stress defence proteins
13. / Peroxiredoxin
(Nostoc sp.strain PCC7120) / 22.73/4.7 / 21.5/4.7 / gi|17232133 / 10 / 95
27. / Peroxiredoxin
(Nostoc sp.strain PCC7120) / 22.73/4.7 / 21.0/4.65 / gi|17232133 / 11 / 121
29. / Fe-SOD
(Nostoc sp.strain PCC7120) / 22.37/5.2 / 22/5.3 / gi|17230430 / 7 / 136
31. / Superoxide dismutase
(Nostoc sp.strain PCC7120) / 30.69/- / 27/6.5 / gi|17227566 / 4 / 201
36. / AhpC
(Nostoc sp.strain PCC7120) / 23.74/- / 25/5.6 / gi|17231896 / 7 / 267
48. / Nutrient stress DNA binding protein (Nostoc sp.strain PCC7120) / 20.73/- / 20.0/5.1 / giI17231300 / 7 / 622
52. / Fe-SOD
(Nostoc sp.strain PCC7120) / 22.37/5.2 / 22/5.6 / gi|17230430 / 8 / 285
3. Protein synthesis and folding
1. / DnaK
(Nostoc sp.strain PCC7120) / 68.04/4.7 / 70.0/4.8 / gi|17229234 / 29 / 196
2 / GroEL
(Nostoc sp.strain PCC7120) / 58.93/4.8 / 64.0/5.0 / gi|17229388 / 19 / 127
3. / GroEL
(Nostoc sp.strain PCC7120) / 57.97/4.8 / 64.0/5.3 / gi|17231154 / 18 / 143
11. / 30 S ribosomal protein S1
(Nostoc sp.strain PCC7120) / 38.34/4.5 / 41.0/4.6 / gi|17227632 / 13 / 88
17. / Polynucleotide phosphorylase/polyadenylase
(Nostoc sp.strain PCC7120) / 77.86/4.9 / 80.0/5.2 / gi|17231888 / 17 / 138
23. / 50Sribosomal protein L4
(Nostoc sp.strain PCC7120) / 23.69/10.17 / 28.5/4.15 / gi|17231706 / 6 / 45
28. / Lysyl tRNA synthatase
(Nostoc sp.strain PCC7120) / 63.67/5.20 / 65.56/5.32 / gi|17231563 / 12 / 102
39. / EF-Ts
(Nostoc sp.strain PCC7120) / 34.43/- / 33.2/5.1 / gi|17232283 / 9 / 114
46. / RNA Binding Protein,RBPE
(Nostoc sp.strain PCC7120) / 10.81/6.26 / 12.15/6.30 / gi|17230269 / 6 / 181
49. / Endopeptidase ClpB
(Nostoc sp.strain PCC7120) / 93.14/5.56 / 10.15/6.23 / gi|17230491 / 31 / 401
51. / 30S ribosomal proteinS6
(Nostoc sp.strain PCC7120) / 12.55/6.34 / 12.50/6.25 / gi|17232294 / 6 / 137
53. / RNA Binding ProteinRBPD
(Nostoc sp.strain PCC7120) / 12.03/6.33 / 14.82/6.45 / gi|17232175 / 4 / 123
57. / Heat shock Protein1
(Nostoc sp.strain PCC7120) / 17.79/5.40 / 17.95/5.23 / gi|17229301 / 8 / 383
62. / Probable GTP binding protein
(Nostoc sp.strain PCC7120) / 39.25/4.25 / 50.21/4.30 / gi|17228218 / 14 / 430
63 / NUS-G
(Nostoc sp.strain PCC7120) / 23.57/5.65 / 28.95/5.21 / gi|17232791 / 12 / 312
64. / ATP dependant clp protease (ClpA)
(Nostoc sp.strain PCC7120) / 22.18/5.38 / 24.28/5.50 / gi|17231850 / 9 / 167
4. Nitrogen metabolism
6. / Glutamine synthetase
(Nostoc sp.strain PCC7120) / 53.30/5.4 / 55.0/5.3 / gi|6646836 / 15 / 96
18. / Glutamate ammonia ligase
(Nostoc sp.strain PCC7120) / 53.3/5.2 / 66.0/5.1 / gi|17229820 + gi|304382992 / 18 / 153
24. / Glutamate ammonia ligase
(Nostoc sp.strain PCC7120) / 53.45/5.2 / 54.0/4.3 / gi|17229820 / 15 / 148
43. / Glutamate-1semialdehyde aminotransferase
(Nostoc sp.strain PCC7120) / 46.05/- / 40.0/5.8 / gi|161353744 / 5 / 147
5. Cell signalling
30. / Two component response regulator
(Nostoc sp.strain PCC7120) / 27.13/- / 26.2/4.9 / gi|17231314 / 10 / 210
6. Fatty acid biosynthesis
35 / Acyl carrier protein phosphodiesterase
(Nostoc sp.strain PCC7120) / 22.77/- / 23/5.1 / gi|17229597 / 4 / 100
42 / Acyl carrier protein phosphodiesterase
(Nostoc sp.strain PCC7120) / 22.78/5.31 / 28.25/5.45 / gi|17229597 / 12 / 583
7. Hypothetical proteins
7. / alr0803
(Nostoc sp.strain PCC7120) / 43.57/5.6 / 44.0/5.4 / gi|17228298 / 13 / 111
12. / alr3199
(Nostoc sp.strain PCC7120) / 39.7/5.0 / 29.0/5.2 / gi|17230691 / 23 / 200
15. / alr3090
(Nostoc sp.strain PCC7120) / 25.65/5.1 / 27/5.27 / AC2192 / 14 / 109
20. / alr0882
(Nostoc sp.strain PCC7120) / 30.88/7.7 / 27.5/7.6 / gi|17228377 / 10 / 100
40. / all3325
(Nostoc sp.strain PCC7120) / 53.33/- / 55.0/5.2 / gi|17230817 / 14 / 181
44 / all5218
(Nostoc sp.strain PCC7120) / 48.30/- / 49.0/4.91 / giI17232710 / 5 / 247
46. / all1124
(Nostoc sp.strain PCC7120) / 26.52/ / 26.5/6.2 / giI17228619 / 5 / 152
47 / all4050
(Nostoc sp.strain PCC7120) / 37.24/- / 40.0/4.9 / giI17231542 / 15 / 414
58 / all3014
(Nostoc sp.strain PCC7120) / 13.68/5.58 / 30.15/5.95 / gi|17228631 / 4 / 107
59 / alr0882
(Nostoc sp.strain PCC7120) / 30.61/6.83 / 32.13/6.90 / gi|17230506 / 5 / 215
66 / alr3904 / 25.89/5.5 / 26.0/5.9 / AI2293 / 19 / 231
8. Nucleotide biosynthesis
50 / Nucleoside diphosphate kinase
(Nostoc sp.strain PCC7120) / 12.97/5.43 / 13.58/6.05 / gi|17230894 / 2 / 160
54 / Bifunctional purine biosynthesis protein(Nostoc sp.strain PCC7120) / 54.03/5/56 / 67.53/5.59 / gi|17230585 / 17 / 260
9. Other Proteins
55 / Oxidoreductase
(Nostoc sp.strain PCC7120) / 30.91/5.37 / 38.53/5.21 / gi|17232674 / 15 / 436
56 / Aldoketo reductase
(Nostoc sp.strain PCC7120) / 31.45/5.56 / 32.15/5.59 / gi|17229808 / 11 / 582
Spot no. / Gene name / BlastP result / % homology
7 / alr0803 / Signal transduction histidines kinase [Anabaena variabilis ATCC 29413] / 97.9
12 / alr3199 / Hemerythrin HHE cation binding region [Anabaena variabilis ATCC 29413] / 100
15. / alr3090 / Manganese containing catalase [Anabaena variabilis ATCC 29413] / 99.6
20. / alr0882 / UspA domain containing protein [Nostoc punctiformae PCC 73102] / 99.0
40 / all3325 / Conserved hypothetical protein[Geobacter sp. M21] / 69.5
44. / all5218 / Modulator of DNA gyrase [Anabaena variabilis ATCC 29413] / 99.6
46 / all1124 / GST like protein [Anabaena variabilis ATCC 29413] / 99.1
47 / all4050 / PRC-barrel domain-containing protein [ Nostoc punctiforme ATCC 29133] / 84.9
66 / alr3904 / Beta-lactamase-like protein [Anabaena variabilis ATCC 29413] / 99.1
58 / all3014 / Glyoxalase [Anabaena variabilis ATCC 29413] / 96.7
In silico analysed physio-chemical properties of hypothetical proteins
Protein name / Number of amino acids / Theoretical pI / Molecular weight / Aliphatic index / Grand average of hydropathicity (GRAVY) / Total number of atomsAll4050 / 324 / 5.09 / 37259.4 / 76.98 / -0.733 / 5201
All3325 / 484 / 5.05 / 53192.4 / 88.53 / -0.130 / 7475
Alr3090 / 230 / 5.23 / 25664.7 / 79.78 / -0.390 / 3568
Alr3199 / 351 / 5.11 / 39733.7 / 89.49 / -0.547 / 5585
Alr0803 / 427 / 5.61 / 43596.8 / 95.39 / -0.063 / 6147
Alr3904 / 233 / 5.59 / 25629.2 / 95.02 / -0.133 / 3607
All3014 / 120 / 5.58 / 13681.0 / 70.75 / -0.462 / 1876
Alr0882 / 283 / 6.83 / 30609.4 / 111.94 / 0.077 / 4375
All5218 / 446 / 5.05 / 48272.2 / 93.39 / -0.233 / 6818
All1124 / 232 / 5.86 / 26539.2 / 91.77 / -0.269 / 3737
Ala (A) / Arg (R / Asn (N) / Asp (D) / Cys (C) / Gln (Q) / Glu (E) / Gly (G) / His (H) / Ile (I) / Leu (L) / Lys (K) / Met (M) / Phe (F) / Pro (P) / Ser (S)
1.All4050 / 6.8% / 9.9% / 3.7% / 7.1% / 0.0% / 2.8% / 11.4% / 5.9% / 2.2% / 4.3% / 6.8% / 4.0% / 0.9% / 3.1% / 4.9% / 3.7%
2.all3325 / 10.3% / 3.9% / 6.4% / 3.9% / 0.6% / 4.5% / 6.8% / 6.6% / 0.8% / 7.4% / 7.9% / 4.3% / 2.5% / 4.3% / 5.8% / 5.8%
3.alr3090 / 9.1% / 3.0% / 5.7% / 4.8% / 0.0% / 4.8% / 8.7% / 7.8% / 4.3% / 3.9% / 10.0% / 5.2% / 2.6% / 5.2% / 3.9% / 5.7%
4.alr3199 / 8.5% / 6.0% / 4.3% / 6.6% / 0.0% / 10.0% / 9.4% / 6.0% / 2.0% / 6.8% / 8.8% / 6.0% / 2.3% / 3.4% / 1.7% / 4.8%
5.alr0803 / 14.3% / 8.0% / 1.6% / 7.3% / 0.0% / 2.3% / 5.4% / 15.9% / 2.6% / 7.3% / 8.0% / 2.3% / 0.9% / 1.4% / 2.6% / 4.2%
6.all3904 / 7.3% / 3.9% / 4.7% / 5.2% / 2.1% / 3.9% / 6.4
% / 7.7
% / 3.4% / 5.2% / 12.9% / 4.7% / 2.1% / 3.0% / 4.3% / 4.3%
7.all3014 / 7.5% / 4.2% / 6.7% / 6.7% / 0.0% / 2.5% / 7.5
% / 6.7
% / 8.3% / 5.0% / 7.5% / 3.3% / 1.7% / 9.2% / 2.5% / 6.7%
8.alr0882 / 8.5
% / 5.7% / 3.5% / 6.7% / 1.8% / 4.2% / 4.6
% / 5.7
% / 1.1% / 6.7% / 12.7% / 5.7% / 2.5% / 1.4% / 3.9% / 10.2%
9. all5218 / 8.5% / 2.9% / 5.8% / 5.2% / 0.2% / 3.6% / 7.4% / 7.4% / 1.6% / 6.3% / 10.3% / 6.3% / 0.2% / 3.6% / 4.9% / 9.0%
10.
all1124 / 9.9% / 5.6% / 6.5% / 3.9% / 0.0% / 5.2% / 7.3% / 6.0% / 1.7% / 10.3% / 7.8% / 4.3% / 1.3% / 5.2% / 6.0% / 3.9%
Thr (T) / Trp (W) / Tyr (Y) / Val (V) / Pyl (O) / Sec (U)
7.7% / 0.6% / 4.9% / 9.3% / 0.0% / 0.0%
6.8% / 1.0% / 3.7% / 6.4% / 0.0% / 0.0%
5.2% / 1.3% / 3.0% / 5.7% / 0.0% / 0.0%
4.3% / 0.6% / 1.7% / 6.8% / 0.0% / 0.0%
6.8% / 0.0% / 1.6% / 7.5% / 0.0% / 0.0%
9.4% / 0.4% / 3.0% / 6.0% / 0.0% / 0.0%
5.8% / 0.0% / 3.3% / 5.0% / 0.0% / 0.0%
3.9% / 0.4% / 1.4% / 9.5% / 0.0% / 0.0%
6.3% / 0.7% / 2.9% / 7.0% / 0.0% / 0.0%
3.9% / 2.2% / 5.2% / 3.9% / 0.0% / 0.0%
1. Analysing the hypothetical gene location using PsortB:
Gene / Localization1.alr3199 / Cytoplasmic
2.all5218 / Cytoplasmic
3.alr0882 / Unknown
4.all3325 / Unknown
5.alr3090 / Cytoplasmic
6.all4050 / Cytoplasmic
7.alr0803 / Cytoplasmic Membrane
8. alr3904 / Cytoplasmic
9. all3014 / Cytoplasmic
Procedure for Biochemical Assays
Antioxidant enzyme assays: Pellets collected from growing cultures of A. PCC 7120 (O.D750 0.5) were suspended in cell lysis buffer (pH 7) and ground in liquid nitrogen. The cell lysis buffer contained 1 mM EDTA and 1% (w/v) poly vinyl pyrrolidone (PVP) with the addition of 1 mM ascorbate in case of APX assay. The ground sample was centrifuged at 12,000g for 30 min at 40C and the resulting supernatant was used for the assay of the antioxidant enzymes.
SOD activity was assayed by monitoring the inhibition of reduction of nitro blue tetrazolium (NBT) according to the method of Giannopolitis and Ries (1977). A 3 ml reaction mixture contained 50 mM potassium phosphate buffer (pH 7.8), 13 mM methionine, 75 µM NBT (nitroblue tetrazolium), 2 µM riboflavin, 0.1 mM EDTA, and 100 µl of enzyme extract. The reaction mixture was illuminated for 20 min under 15 fluorescent tubes emitting 72 µmol photon m-2 s-1 intensity. Two blanks were prepared with the reaction mixture without the lysate, one was kept in light and the other in dark. The former was taken as reference for calculating absorbance for one unit SOD and the latter was used as blank. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT reduction monitored at 560 nm.
CAT activity was determined by measuring the consumption of H2O2 (extinction coefficient 39.4 mM-1 cm-1) at 240 nm for 1 min by the method of Aebi (1984). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7), 10 mM H2O2, and 200 µl of the enzyme extract in a 3 ml volume.
APX activity was determined by measuring the decrease in absorbance at 290 nm (A290) (extinction coefficient 2.8 mM-1 cm-1) for 1 min in 1 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7), 0.5 mM ascorbate, 0.1 mM H2O2, and 200 µl of enzyme extract. The reaction was started by adding enzyme extract.
GR activity was determined by measuring the oxidation of NADPH at 340 nm (extinction coefficient 6.2 mM-1 cm-1) for 3 min in 1 ml of assay mixture containing 50 mM potassium phosphate buffer (pH 7.8), 2 mM Na2EDTA, 0.15 mM NADPH, 0.5 mM GSSG (glutathione oxidized), and 200 µl of enzyme extract. The reaction was initiated by adding NADPH. Absorbance of the assay mixture without NADPH at 340 nm was used for background correction (Schaedle and Bassham 1977).