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Expression, purification and crystallization of the phosphate‐binding PstS protein from Pseudomonas aeruginosa
Author(s) -
Neznansky Avi,
Opatowsky Yarden
Publication year - 2014
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 37
ISSN - 2053-230X
DOI - 10.1107/s2053230x14010279
Subject(s) - microbiology and biotechnology , pseudomonas aeruginosa , biofilm , virulence , escherichia coli , immune system , bacteria , orthorhombic crystal system , chemistry , crystallization , biology , biochemistry , crystallography , immunology , crystal structure , genetics , organic chemistry , gene
Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immune‐suppressed patients, of sepsis in burn victims and of nosocomial infections. An important element in PA virulence is its ability to establish biofilms that evade suppression by the host's immune system and antibiotics. PstS, a periplasmic subunit of the Pst phosphate‐transport system of PA, plays a critical role in the establishment of biofilms. In some drug‐resistant PA strains, PstS is secreted in large quantities from the bacteria, where it participates in the assembly of adhesion fibres that enhance bacterial virulence. In order to understand the dual function of PstS in biofilm formation and phosphate transport, the crystal structure of PA PstS was determined. Here, the overexpression in Escherichia coli and purification of PA PstS in the presence of phosphate are described. Two crystal forms were obtained using the vapour‐diffusion method at 20°C and X‐ray diffraction data were collected. The first crystal form belonged to the centred orthorhombic space group C 222 1 , with unit‐cell parameters a = 67.5, b = 151.3, c = 108.9 Å. Assuming the presence of a dimer in the asymmetric unit gives a crystal volume per protein weight ( V M ) of 2.09 Å 3 Da −1 and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P 2 1 2 1 2 1 , with unit‐cell parameters a = 35.4, b = 148.3, c = 216.7 Å. Assuming the presence of a tetramer in the asymmetric unit gives a crystal volume per protein weight ( V M ) of 2.14 Å 3 Da −1 and a solvent content of 42.65%. A pseudo‐translational symmetry is present in the P 2 1 2 1 2 1 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.