Molecular analysis of the phosphate-specific transport (

Nikata, Toshiyuki; Sakai, Yukihiro; Shibata, Kenya; Kato, Junichi; Kuroda, Akio; Ohtake, Hisao

doi:10.1007/s004380050122

Cited by 4 publications

(3 citation statements)

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“…The Pst phosphate-transport system has a high (micromolar range) K m affinity towards inorganic phosphate (P i ) and is composed of five polypeptide chains (van Veen, 1997), of which two are potential transmembrane permease subunits, designated PstA and PstC. Deletion of either PstA or PstC does not induce phosphate starvation, and it is therefore not clear whether they act as two separate homomers or rather as a heterodimer (Nikata et al, 1996). Two identical ATPase subunits (PstB) that drive phosphate influx are constitutively attached to the intracellular side of the permease.…”

Section: Introductionmentioning

confidence: 99%

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Expression, purification and crystallization of the phosphate-binding PstS protein fromPseudomonas aeruginosa

Neznansky

Opatowsky

2014

Acta Cryst Sect F

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Pseudomonas aeruginosa (PA) infections pose a serious threat to human health. PA is a leading cause of fatal lung infections in cystic fibrosis and immunesuppressed 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 C222 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 À1 and a solvent content of 41%. The second crystal form belonged to the primitive orthorhombic space group P2 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 À1 and a solvent content of 42.65%. A pseudo-translational symmetry is present in the P2 1 2 1 2 1 crystal form which is consistent with a filamentous arrangement of PstS in the crystal lattice.

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Section: Introductionmentioning

confidence: 99%

“…Deletion of either PstA or PstC does not induce phosphate starvation, and it is therefore not clear whether they act as two separate homomers or rather as a heterodimer (Nikata et al, 1996). Two identical ATPase subunits (PstB) that drive phosphate influx are constitutively attached to the intracellular side of the permease.…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and crystallization of the phosphate-binding PstS protein fromPseudomonas aeruginosa

Neznansky

Opatowsky

2014

Acta Cryst Sect F

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“…PBP and the periplasmic phosphate-specific transporter (PstS) of the ATP-binding cassette (ABC) transporter system play an important role in bacterial virulence (Cré pin et al, 2011;Blus-Kadosh et al, 2013;Lamarche et al, 2008) and survival under starvation (Surin et al, 1985;Nikata et al, 1996;Peirs et al, 2005). It has been observed that PstS is overexpressed in multidrug-resistant strains of highly virulent P. aeruginosa (Zaborina et al, 2008).…”

Section: Discussionmentioning

confidence: 99%

Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein

Hatti

Gulati

Srinivasan

et al. 2016

Acta Crystallogr D Struct Biol

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During the past decade, the authors have collected a few X-ray diffraction data sets from protein crystals that appeared to be easy cases of molecular replacement but failed to yield structures even after extensive trials. Here, the use of a large-scale molecular replacement method that explores all structurally characterized domains as phasing models to determine the structure corresponding to two data sets collected at 1.9 and 2.3 Å resolution is reported. These two structures were of the same protein independently crystallized in 2007 and 2011. The structures derived are virtually identical and were found to consist of two compact globular domains connected by a hinge. The high resolution of one of these data sets enabled inference of the amino-acid sequence from the electron-density map. The deduced sequence is nearly identical to that of a protein from the multidrug-resistant bacterium Stenotrophomonas maltophilia. Although the structure of this protein has not been determined previously, it is homologous to the well studied DING proteins which mediate the cellular uptake of phosphate ions. The final electron-density maps from both of the data sets revealed a large density at the interface of the two globular domains that is likely to represent a phosphate ion. Thus, the structure is likely to be that of a phosphate-binding protein encoded by the S. maltophilia genome (SmPBP; PDB entry 5j1d). The nature of the phosphate-binding site of SmPBP closely resembles that of Pseudomonas fluorescens DING (PfluDING), which displays remarkable discrimination between the closely similar phosphate and arsenate ions. The results presented here illustrate that routine crystallization trials may occasionally lead to the serendipitous crystallization of a protein of unknown identity and brute-force molecular replacement through `fold space' might allow the identification of the unknown protein.

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Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147‐2

Monds

Silby

Mahanty³

2001

Molecular Microbiology

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SummaryWe report the isolation of insertional mutations to the pstC and pstA genes of the phosphate-specific transport ( pst ) operon that results in loss of biofilm formation by Pseudomonas aureofaciens PA147-2. Consistent with the known roles of the Pst system in Escherichia coli and Pseudomonas aeruginosa, both P. aureofaciens pst mutants were demonstrated to have defects in inorganic phosphate (P i ) transport and repression of Pho regulon expression. Subsequently, biofilm formation by the wild type was shown to require a threshold concentration of extracellular P i . The two-component regulatory pair PhoR/PhoB is responsible for upregulation of Pho regulon expression in response to P i -limiting environments. By generating phoR mutants that were unable to express the Pho regulon, we were able to restore biofilm formation by P. aureofaciens in P i -limiting conditions. This result suggests that gene(s) within the Pho regulon act to regulate biofilm formation negatively in low-P i environments, and that phoR mutations uncouple PA147-2 from such regulatory constraints. Furthermore, the inability of pst mutants to repress Pho regulon expression accounts for their inability to form biofilms in non-limiting P i environments. Preliminary evidence suggests that the Pst system is also required for antifungal activity by PA147-2. During phenotypic analysis of pst mutants, we also uncovered novelties in relation to P i assimilation and Pho regulon control in P. aureofaciens.

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Molecular analysis of the phosphate-specific transport (

Cited by 4 publications

References 0 publications

Expression, purification and crystallization of the phosphate-binding PstS protein fromPseudomonas aeruginosa

Expression, purification and crystallization of the phosphate-binding PstS protein fromPseudomonas aeruginosa

Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein

Expression of the Pho regulon negatively regulates biofilm formation by Pseudomonas aureofaciens PA147‐2

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