Table 1.
Primers used and PCR amplicons generated in this study.
Table 2.
Plasmids used in this study.
Table 3.
Bacterial strains used in this study.
Figure 1.
Bioinformatic analysis of Alr0088, Alr7579 and All4779 proteins of Anabaena 7120.
(A) Conserved Domain Database (CDD) analysis of Anabaena Alr0088, Alr7579 and All4779 proteins and E. coli SSB (EcoSSB) protein. The OB-fold corresponding ssDNA binding region, dimer and tetramer interfaces for all the proteins are indicated. (B) Comparison of homology between predicted amino acid sequence of All4779 and EcoSSB. The identical amino acids are indicated by letters and similar amino acids with a ‘+’ sign. The proline (P) and glycine (G) residues beyond the OB fold are shown in larger font, while the acidic residues at the C-terminal end are in bold and underlined. The numbers on the left and right hand side correspond to amino acid residues.
Figure 2.
Molecular mass determination of purified native Anabaena proteins.
(A) Ni-NTA affinity chromatography purified Alr0088, Alr7579 and All4779 proteins separated on 12% SDS-polyacrylamide gel followed by staining with Coomassie Brilliant Blue (CBB) G-250. The purified proteins are indicated by arrows. (B) Elution profile of purified native Alr0088, Alr7579 and All4779 proteins in gel filtration chromatography using Superdex HR200 matrix. A standard graph using the following standard proteins: [Myosin (200 kDa), β-galactosidase (116 kDa), Phosphorylase-b (97.4 kDa), Bovine Serum Albumin (66 kDa), Chicken Albumin (45 kDa), Carbonic Anhydrase (29 kDa) and RNaseA (13.7 kDa)] was drawn to calculate the molecular mass of the eluted native Anabaena proteins depending on their elution volume. The position of the eluted proteins has been depicted by ‘star’ and ‘triangle’ symbols. The vertical and horizontal lines from the two symbols indicate the elution volume and the corresponding log of molecular mass.
Figure 3.
Glutarldehyde (Glh)-aided crosslinking of native purified Anabaena SSB-like proteins and their binding to ssDNA.
The purified native Anabaena proteins (A) Alr0088, (B) Alr7579 and (C) All4779 were cross-linked with Glh in the presence or absence of M13 ssDNA as indicated. The proteins were electrophoretically separated on 12% SDS-polyacrylamide gel followed by staining with Coomassie Brilliant Blue (CBB) G-250. The molecular mass of the protein markers used (M1 and M2) are written to the immediate (right/left) of the marker lane. Different molecular forms of the native Anabaena proteins are indicated by the arrows. (D) Electrophoretic Mobility Shift Assay (EMSA) of a γ-32P-ATP labeled 75-mer oligonucleotide in the presence of different concentrations of Alr0088, Alr7579 and All4779 proteins. Following in solution interaction, the assay mix was separated by 6% non-denaturing PAGE in 1X TBE and radioactive gel imaged using a phosphorimager. The free ssDNA substrate and the different ssDNA-protein complexes formed are indicated.
Figure 4.
Relative quenching of fluorescence of native purified Anabaena SSB-like proteins and EcoSSB as a function of ssDNA concentration.
(A–C) Quenching of fluorescence in 20 mM NaCl as a function of poly(dT) concentration of (A) Alr0088, (B) Alr7579 and (C) All4779 and purified EcoSSB (commercially available, Sigma) proteins represented as relative fluorescence, considering the observed fluorescence in the absence of any poly(dT) as 100%. The horizontal line designates the point on the graph wherein relative fluorescence is 50% and the corresponding vertical line indicates the concentration of poly(dT) at which it is achieved. Reciprocal of this concentration corresponds to the binding constant of the protein for poly(dT). (D) Percent fluorescence quenching of Alr0088, Alr7579 and All4779 proteins as a function of molar ratio of M13ssDNA and protein in the presence of 20 mM or 100 mM NaCl. The fluorescence quenching in the absence of M13ssDNA is considered as 0%.
Figure 5.
Quenching of fluorescence of native purified Anabaena SSB-like proteins compared with that of EcoSSB.
The quenching of fluorescence of (A) Alr0088, (B) Alr7579, (C) All4779 and (D) EcoSSB in the presence of 20 mM or 100 mM NaCl was expressed as a ratio of change in fluorescence (ΔF) and initial fluorescence (Fi). The (ΔF/Fi) was expressed as a function of ratio of concentrations of poly(dT) and protein. The horizontal lines indicate the point of saturation and the vertical lines drawn from the point of saturation indicate the probable length of ssDNA bound by one molecular unit of the protein.
Figure 6.
Construction of recombinant Anabaena strain overexpressing All4779 protein.
(A) Schematic diagram of the plasmid construct, pAMall4779 used for overexpression of All4779 protein in Anabaena. The different restriction enzymes used for cloning are indicated. (B) Fluorescence microphotograph (600X magnification) [using Hg-Arc lamp (excitation 470 nm, emission 508 nm)] of Anabaena 7120 [An7120] and recombinant strain, Anall4779+, grown for 3 days in BG-11, N− media. (C) Protein extracts from Anabaena 7120 (lane 1) and Anall4779+ (lane 2) were separated by 12% SDS-PAGE, followed by blotting on to nitrocellulose membrane and immunodetection of All4779 protein using anti-All4779 antibody. The cross-reacting All4779 protein is indicated by an arrow. Equal loading controls are shown below the blot. Other details were as described in legend to Figure 2. (D) Growth profile of wild type Anabaena 7120 and recombinant Anabaena strain, Anall4779+ and AnpAM under nitrogen-fixing conditions over a period of 7 days. Growth was measured in terms of increase in chlorophyll a content. Recombinant strains were grown in presence of neomycin while wild type was grown without antibiotic.
Figure 7.
Effect of All4779 overexpression on the survival and tolerance of Anabaena to DNA-damage inducing stresses.
(A and B) Three day-old cultures were concentrated to 10 μg chla density mL−1 and exposed to 6 kGy of 6°Co γ-irradiation or to 6 days of desiccation. (A) Survival was measured in terms of colony forming units immediately after irradiation (I) or desiccation (D) and compared with the respective unirradiated control (CI) or undesiccated control (CD). (B) The stressed and control cultures were washed, inoculated in fresh BG-11, N−, Neo12.5 and allowed to recover under normal growth conditions for 7 days. Growth during post-irradiation/desiccation recovery was measured in terms of chlorophyll a content and expressed as percent of respective unirradiated/undesiccated controls. (C and D) Three-day-old cultures of recombinant strains AnpAM and Anall4779+ were concentrated to 10 μg chla mL−1 density. (C) An 100 μl aliquot was spread on the corresponding BG-11, N−, Neo25 agar plates and exposed to UV-B (0–1.5 kJ) (D) Culture aliquots were exposed to mitomycinC (0–4 μg ml−1) for 30 min in liquid media followed by plating 100 μl on BG-11, N− Neo25 agar plate. Colonies were counted after 10 days of incubation at 27°±2°C with constant illumination.