This is similar to the recently described psychrophilic PhaSSB, with 34 selleck screening library nucleotides per tetramer under low-salt conditions and 54–64 nucleotides at higher ones. This suggests that the FpsSSB and PhaSSB
undergo a transition between Selleck OSI906 ssDNA binding modes, something which is observed for the EcoSSB. Conclusion The results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications. Methods Bacterial strains, plasmids, enzymes and reagents D. psychrophila LSv54 (DSM 12343), P. arcticus 273–4 (DSM 17307), P. cryohalolentis K5 (DSM 17306) and P. ingrahamii 37 (DSM 17664) were purchased from The Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures, Germany). F. psychrophilum JIP02/86 (LMG 13180), P. profundum (LMG 19446) and P. torquis ATCC
700755 (LMG 21429) were purchased from BCCM/LMG (The Belgian Co-ordinated Collections of Micro-organisms, Belgium). Genomic sequences for those strains are available and were published: D. psychrophila (GenBank accession no. NC_006138; [16]), F. psychrophilum (GenBank accession no. NC_009613; [17]), P. arcticus (GenBank accession no. NC_007204; [18]), P. cryohalolentis (GenBank accession no. NC_007969; Gene Bank Project: PRJNA58373), selleck chemical P. ingrahamii (GenBank accession no. NC_008709; [19]), P. profundum (GenBank accession no. NC_006370; [20]) and P. torquis (GenBank accession
no. NC_018721; [15]). The E. coli TOP10 (Invitrogen, USA) was used for genetic constructions and gene expression. The pBAD/myc-HisA plasmid (Invitrogen, USA) was used for constructing the expression system. The reagents for see more PCR were obtained from Blirt SA – DNA-Gdańsk (Poland). Specific primers, oligodeoxynucleotides and the oligonucleotides 5′-end-labelled with fluorescein were purchased from Sigma (USA). The restriction enzymes were purchased from NEB (USA). EcoSSB, PhaSSB and TmaSSB were produced and purified in our laboratory according to published procedure ( [7, 28, 43], respectively). Cloning of the ssb-like genes from psychrophilic bacteria DNA from D. psychrophila, F. psychrophilum, P. arcticus, P. cryohalolentis, P. ingrahamii, P. profundum and P. torquis was isolated using an ExtractMe DNA Bacteria Kit (Blirt SA – DNA-Gdańsk, Poland). The specific primers for PCR amplification were designed and synthesized on the basis of the known ssb-like gene sequences. The forward (containing a NcoI recognition site) and reverse (containing a BglII or HindIII recognition site) primers are shown in Table 4.