Structural features of PhoX, one of the phosphate-binding proteins from Pho regulon of Xanthomonas citri

16 p.-4 fig.-2 tab.In Escherichia coli, the ATP-Binding Cassette transporter for phosphate is encoded by the pstSCAB operon. PstS is the periplasmic component responsible for affinity and specificity of the system and has also been related to a regulatory role and chemotaxis during depletion of phosphate. Xanthomonas citri has two phosphate-binding proteins: PstS and PhoX, which are differentially expressed under phosphate limitation. In this work, we focused on PhoX characterization and comparison with PstS. The PhoX three-dimensional structure was solved in a closed conformation with a phosphate engulfed in the binding site pocket between two domains. Comparison between PhoX and PstS revealed that they originated from gene duplication, but despite their similarities they show significant differences in the region that interacts with the permeases.This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)” for Vanessa Pegos PhD fellowship and the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP” for the research grants 2011/20468-1 and 2013/09172-9.Peer reviewe

Xanthan Gum Removal for 1H-NMR Analysis of the Intracellular Metabolome of the Bacteria Xanthomonas axonopodis pv. citri 306

Xanthomonas is a genus of phytopathogenic bacteria, which produces a slimy, polysaccharide matrix known as xanthan gum, which involves, protects and helps the bacteria during host colonization. Although broadly used as a stabilizer and thickener in the cosmetic and food industries, xanthan gum can be a troubling artifact in molecular investigations due to its rheological properties. In particular, a cross-reaction between reference compounds and the xanthan gum could compromise metabolic quantification by NMR spectroscopy. Aiming at an efficient gum extraction protocol, for a 1H-NMR-based metabolic profiling study of Xanthomonas, we tested four different interventions on the broadly used methanol-chloroform extraction protocol for the intracellular metabolic contents observation. Lower limits for bacterial pellet volumes for extraction were also probed, and a strategy is illustrated with an initial analysis of X. citri’s metabolism by 1H-NMR spectroscopy

Crystal structure of the phosphate-binding protein PhoX from <i>X</i>. <i>citri</i>.

<p>(A) Cartoon representation of the three-dimensional structure of PhoX evidencing the α/β folding and the surface (transparent gray) α-helices and loops from domains I and II are shown in blue and cyan, respectively. The β-sheet is colored in yellow. All the secondary structures are labeled. Phosphate ions are shown as red spheres. (B) Side-view of PhoX in surface with the ion buried inside the ligand-binding pocket between both domains. (C) Protein-protein interactions between chains C and E as observed in the crystallographic structure of PhoX, evidencing the set of three phosphates (red spheres) mediating the interaction. (D) Positioning of phosphates 1, 2 and 3 in domain I of chain E, and phosphate 4 inside the ligand-binding pocket. In the box: details of the positive electrostatic potential.</p

Comparison and analysis of the regions of PhoX and PstS that interact with the permeases PstCA.

<p>(A) Amino acid sequence alignment of RI and RII from PhoX, PstS and <i>E</i>. <i>coli</i> PstS evidencing the high level of conservation (asterisks). Residues in underlined bold are involved with the phosphate binding. (B) The localization of the RI (yellow) and RII (cyan) regions of PhoX and PstS close to the entrance of the ligand pocket shown in surface view and electrostatic potential. Proteins are shown in the same orientation. Electrostatic potential of RI and RII of PhoX and PstS are shown in colored view surface according to the charges calculated in Pymol. Negative: red, positive: blue and neutral: white.</p

Phylogenetic relationships between the orthologues of PstS and PhoX.

<p>Five groups classify the distinct orthologues found in proteobacteria. All the organisms used in this tree as well as their reference codes are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178162#pone.0178162.t001" target="_blank">Table 1</a>. The tree was generated using MEGA 6.0 software and the neighbor-joining algorithm according MM section. The numbers at the nodes indicate the bootstrap percentages of 1000 replicates. Arrows and black dots indicate gene duplication and horizontal gene transfer, respectively. The colors highlight the different groups and subgroups.</p

Occurrence of phosphate-binding proteins in proteobacteria and their comparison with PstS and PhoX from <i>X</i>. <i>citri</i>.

<p><i>X</i>. <i>citri</i> PhoX was used as entry for String server and the data of cooccurrence were analysed for building of the table. The presence of orthologs of PhoX and PstS was avaluated in the four branches of proteobacteria branch. To obtain the amino acid sequence identity among the proteins, the sequences of the proteins identified by String server were submitted to BLASTp x <i>X</i>. <i>citri</i> database. The protein identification shows the 3-letter code of the microrganism, KEGG number and the function associated to the putative protein (PBP: phosphate-binding protein; PstS: phosphate-specific transporter).</p

The ligand-binding pocket of <i>X</i>. <i>citri</i> PhoX and its conservation.

<p>(A) Detailed view of the PhoX residues (cyan sticks) that interact with the phosphate ion (red stick). Hydrogen bonds are shown as black trace. (B) Structural based amino acid sequence alignment of <i>X</i>. <i>citri</i> PhoX and PstS with several orthologues with solved structures, showing the high level of conservation of the residues that interact with the ion. Numbers are shown according to PhoX structure. (C) Structural superimposition of <i>X</i>. <i>citri</i> PhoX (in black ribbon) and all of the phosphate-binding proteins structures as deposited in PDB. Proteins are shown as ribbons and the phosphate ion from <i>X</i>. <i>citri</i> structure as green spheres. (D) R.m.s.d. values and the aligned residues after the structural superposition of PhoX (314 residues) and each protein is shown in angstroms. PDB codes and 3-letters represent the following proteins: 4GD5_Cpe, <i>Clostridium perfringens</i> PBP, (light gray); 1TWY_Vch, <i>Vibrio cholerae</i> PBP (light blue); 2Z22_Ype, <i>Yersinia pestis</i> PstS (cyan); 1IXH_Eco, <i>E</i>. <i>coli</i> PstS (yellow); 1PC3_Mtu, <i>M</i>. <i>tuberculosis</i> PstS1 (blue); 4LVQ_Mtu, <i>M</i>. <i>tuberculosis</i> PstS3 (magenta), 4EXL_Spn, <i>Streptococcus pneumoniae</i> PstS1 (green), 5I84_Xac, <i>X</i>. <i>citri</i> PhoX (black) and PstS_Xac, <i>X</i>. <i>citri</i> PstS (red).</p

Spectroscopic analysis of <i>X</i>. <i>citri</i> PstS and PhoX in presence and absence of phosphate.

<p>Secondary structure prediction of PstS (A) and PhoX (B) was measured by circular dichroism. Traced line: PstS and PhoX in the absence of phosphate; Black line: PstS and PhoX in the absence of phosphate. Gray line: PhoX and PstS incubated with 30 μM of phosphate. The thermal stability of the proteins in absence (black dots) and presence (gray dots) of phosphate was measured at 222 nm for PstS (C) and PhoX (D). The melting temperature (Tm) was calculated and is shown for both conditions.</p