e. 40% (Fig. 3). Csps from E. coli and B. subtilis also grouped separately with bootstrap values of 50% and 42%, respectively, with the exception of E. coli CspD, which aligned more closely to the Betaproteobacteria node with a low bootstrap value of 37%. DEAD-box RNA helicase containing CSD from Archaea Methanococcoides burtonii (AAF89099) was used as an outgroup, Immunofluorescence staining was used to localize CspD

using the anti-CapB rabbit-antiserum at different temperatures to determine the possible cellular role of CspD in Ant5-2. The cellular location of the nucleoid was confirmed by DAPI staining (Fig. 4a, c, and e). At 4 °C, a dense accumulation of the anti-CapB antibody immunoconjugated with the green Hilyte Fluor 488-labeled goat anti-rabbit IgG secondary antibody was observed in and around Erastin supplier the nucleoid region (Fig. Tamoxifen 4aand b). At 15 and 22 °C, the green fluorescence was dispersed in the cytosol as well as in the nucleoid region (Fig. 4c–f). The purified

CspD protein from Ant5-2 (Fig. S5) exhibited binding affinity with single stranded (ss)-oligonucleotides with increasing concentration (Fig. 5) and not with dsDNA (PCR product) (data not shown). Based on the amino acid residues and use of the homology modeling approach, the secondary and the tertiary structures of CspD from Ant5-2 indicated that the aromatic residues are conserved and three of the eight aromatic residues were docked on the nucleic acid-binding surface, F15 (F12), F17 (F20), and F28 (F31) (amino acid numbering on E. coli CspA is indicated in parentheses) (Feng et al., 1998). CspD from Ant5-2

has five basic and three acidic residues on the nucleic acid-binding surface. Its calculated theoretical isoelectric point (pI) was 5.6. Five β-strands and one α-helix were identified Acesulfame Potassium by the secondary-structure prediction (Fig. 6a). The solvent-exposed basic amino acids were K7 in β1 strand, K13 in L1, H30 in β3, K40 in L3 and K57 in L4 located on the nucleic acid-binding surface (Fig. 6b). The tertiary structure was designed with N. meningitidis CSD protein (Nm-Csp) (PDB reference: 3CAM) using the template provided by hhpred and modeller software (Soding et al., 2005; Eswar et al., 2006). The structure of the monomer of CspD from Ant5-2 consists of two subdomains of similar length separated by a long loop. Subdomain 1 includes β-strands 1–3 and subdomain 2 contains a β-ladder comprising strands 4 and 5 (Fig. 6a and b). The TM-score of the predicted structure was calculated to be 0.96738. It has been reported that the Nm-Csp form a dimer in the crystallographic asymmetric unit consisting of two five-stranded β-barrels (Ren et al., 2008). Because protein pairs with a TM-score >0.5 are mostly in the same fold (Xu & Zhang, 2010), we tested whether CspDAnt5-2 form a dimer-like Nm-Csp by docking monomer pairs with the hex 5.1 software (Ritchie & Venkatraman, 2010).