title: Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis.
abstract: Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism such as DNA replication, repair, and recombination, and is essential for cell survival. This study reports on the ssb-like gene cloning, gene expression and characterization of a single-stranded DNA-binding protein of Pseudoalteromonas haloplanktis (PhaSSB) and is the first report of such a protein from psychrophilic microorganism. PhaSSB possesses a high sequence similarity to Escherichia coli SSB (48% identity and 57% similarity) and has the longest amino acid sequence (244 amino acid residues) of all the known bacterial SSBs with one OB-fold per monomer. An analysis of purified PhaSSB by means of chemical cross-linking experiments, sedimentation analysis and size exclusion chromatography revealed a stable tetramer in solution. Using EMSA, we characterized the stoichiometry of PhaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined as being approximately 35 nucleotides long. In fluorescence titrations, the occluded site size of PhaSSB on poly(dT) is 34 nucleotides per tetramer under low-salt conditions (2mM NaCl), but increases to 54-64 nucleotides at higher-salt conditions (100-300mM NaCl). This suggests that PhaSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB. The binding properties of PhaSSB investigated using SPR technology revealed that the affinity of PhaSSB to ssDNA is typical of SSB proteins. The only difference in the binding mode of PhaSSB to ssDNA is a faster association phase, when compared to EcoSSB, though compensated by faster dissociation rate. When analyzed by differential scanning calorimetry (DSC), the melting temperature (Tm) was determined as 63 °C, which is only a few degrees lower than for EcoSSB.
authors: Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland.
authors: Marcin
authors: M
authors: Olszewski
authors: Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland.
authors: Marta
authors: M
authors: Nowak
authors: University of Wroclaw, Faculty of Biotechnology, Department of Protein Engineering, ul. Tamka 2, 50-138 Wrocław, Poland.
authors: Anna
authors: A
authors: Cyranka-Czaja
authors: Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-233 Gdańsk, Poland. Electronic address: kur@pg.gda.pl.
authors: Józef
authors: J
authors: Kur
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D031161:Pseudoalteromonas / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C476430:SSB protein, E coli
keywords: DNA replication; Expression; Psychrophilic microorganism; SSB; Thermostability