The protein concentration was estimated colorimetrically using BCA Protein assay kit (Pierce, Rockford, IL); 50 μg proteins from each sample was separated in 12% (w/v) denaturing SDS polyacrylamide gel; the gel

was dried and autoradiographed. For immunoblot assays, total proteins from Ant5-2 cultures incubated at −1, 4, 15, 22 and 37 °C and cultures exposed to different doses of UVC (290–100 nm) were extracted, transferred and immunoblotted with CapB antibody from Pseudomonas sp. 30/3 in duplicate as described previously (Panicker et al., 2010). The membranes were scanned and the relative amount of CspD expressed in Ant5-2 was analyzed with imagej 1.40 software (http://rsb.info.nih.gov/ij/index.html). AG-014699 mouse The cold shock gene was PCR amplified using primers designed from Csp of Janthinobacterium lividum (accession no. DQ074977), F-cspD-Ant5-2 (5′-dTTAGATTGGCTGAATGTTCGAAGCTTGC-3′) forward and R-cspD-Ant5-2

(5′-dATGGCAACTGGCATCGTAAAATGG-3′) reverse primers. The PCR amplification of the homologs of E. coli cspA on Ant5-2 genomic DNA was also attempted using a set of universal degenerate primers for Enterobacteriaceae CSPU5 (5′-dCCCGAATTCGGTAHAGTAAAATGGTTYAACKC-3′) and CSPU3 (5′-dCCCGAATCCGGTTACGTTASCWGCTKSHGGDCC-3′) (Francis & selleck products Stewart, 1997). The cycling parameters, cloning and sequencing method used was as described previously (Panicker et al., 2010). The deduced amino acid sequence of CspD from Ant5-2 was aligned with homologous sequences obtained from blast p and with protein sequences in NCBI database using clustal x (http://www.clustal.org/) and the phylogenetic trees were generated by neighbor

joining method and mega version 4.0 (http://www.megasoftware.net/) software (Tamura et al., 2007). Ant5-2 culture was grown in 1 : 10 (v/v) cAMP TSB growth medium at 22 °C until OD450 nm=0.3; then aliquots of 50 mL were exposed to 4, 15 and 22 °C, respectively, for 1 h. The fixation, permeabilization and staining of cells with rabbit anti-CapB antiserum (1 : 1500 dilutions in bovine serum albumin–phosphate-buffered saline (PBS), followed by HiLyte Fluor 488-labeled goat anti-rabbit IgG secondary antibody (AnaSpec, San Jose, CA) along with 4′-6-diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO) was performed as described previously (Panicker et al., 2010). Slides were mounted in PBS-glycerol (pH 7.2) solution and observed under a Leica™ fluorescence microscope (Bannockburn, IL). cspD was PCR amplified using forward F-BamH1-CspD-Ant5-2 (5′-dGCCAGGATCCATGGCAACTGGCATC-3′) and reverse R-XhoI-CspD-Ant5-2 (5′-dGCCACTCGAGTTAGATTGGCTGAATG-3′) primers from Ant 5-2 cells and cloned into pET45b(+) plasmid (Novagen, WI). The CspD from Ant5-2 protein was expressed in E. coli BL21 (DE3) by inducing with 1 mM isopropyl-β-d-thiogalactopyranoside for 6 h and then purified using the His.Tag kit (Novagen). DNA-binding assay was performed by incubating purified CspD of Ant5-2 with 0.