Iron Deficiency Generates Oxidative Stress and Activation of the SOS Response in Caulobacter crescentus

In C. crescentus, iron metabolism is mainly controlled by the transcription factor Fur (ferric uptake regulator). Iron-bound Fur represses genes related to iron uptake and can directly activate the expression of genes for iron-containing proteins. In this work, we used total RNA sequencing (RNA-seq) of wild type C. crescentus growing in minimal medium under iron limitation and a fur mutant strain to expand the known Fur regulon, and to identify novel iron-regulated genes. The RNA-seq of cultures treated with the iron chelator 2-2-dypiridyl (DP) allowed identifying 256 upregulated genes and 236 downregulated genes, being 176 and 204 newly identified, respectively. Sixteen transcription factors and seven sRNAs were upregulated in iron limitation, suggesting that the response to low iron triggers a complex regulatory network. Notably, lexA along with most of its target genes were upregulated, suggesting that DP treatment caused DNA damage, and the SOS DNA repair response was activated in a RecA-dependent manner, as confirmed by RT-qPCR. Fluorescence microscopy assays using an oxidation-sensitive dye showed that wild type cells in iron limitation and the fur mutant were under endogenous oxidative stress, and a direct measurement of cellular H2O2 showed that cells in iron-limited media present a higher amount of endogenous H2O2. A mutagenesis assay using the rpoB gene as a reporter showed that iron limitation led to an increase in the mutagenesis rate. These results showed that iron deficiency causes C. crescentus cells to suffer oxidative stress and to activate the SOS response, indicating an increase in DNA damage

Transporters involved in glucose and water absorption in the Dysdercus peruvianus (Hemiptera: Pyrrhocoridae) anterior midgut

Little is known about insect intestinal sugar absorption, in spite of the recent findings, and even less has been published regarding water absorption. The aim of this study was to shed light on putative transporters of water and glucose in the insect midgut Glucose and water absorptions by the anterior ventriculus of Dysdercus peruvianus midgut were determined by feeding the insects with a glucose and a non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventricular contents. Glucose absorption decreases glucose/dye ratios and water absorption increases dye concentrations. Water and glucose transports are activated (water 50%, glucose 33%) by 50 mM K(2)SO(4) and are inhibited (water 46%, glucose 82%) by 0.2 mM phloretin, the inhibitor of the facilitative hexose transporter (GLUT) or are inhibited (water 45%, glucose 35%) by 0.1 mM phlorizin, the inhibitor of the Na(+)-glucose cotransporter (SGLT). The results also showed that the putative SGLT transports about two times more water relative to glucose than the putative GLUT. These results mean that D. peruvianus uses a GLUT-like transporter and an SGLT-like transporter (with K(+) instead of Na(+)) to absorb dietary glucose and water. A cDNA library from D. peruvianus midgut was screened and we found one sequence homologous to GLUT1, named DpGLUT, and another to a sodium/solute symporter, named DpSGLT. Semi-quantitative RT-PCR studies revealed that DpGLUT and DpSGLTs mRNA were expressed in the anterior midgut, where glucose and water are absorbed, but not in fat body, salivary gland and Malpighian tubules. This is the first report showing the involvement of putative GLUT and SGLT in both water and glucose midgut absorption in insects. (C) 2010 Elsevier Inc. All rights reserved.FAPESPFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPqConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

How pH Modulates the Dimer-Decamer Interconversion of 2-Cys Peroxiredoxins from the Prx1 Subfamily

2-Cys peroxiredoxins belonging to the Prx1 subfamily are Cys-based peroxidases that control the intracellular levels of H2O2 and seem to assume a chaperone function under oxidative stress conditions. The regulation of their peroxidase activity as well as the observed functional switch from peroxidase to chaperone involves changes in their quaternary structure. Multiple factors can modulate the oligomeric transitions of 2-Cys peroxiredoxins such as redox state, post-translational modifications, and pH. However, the molecular basis for the pH influence on the oligomeric state of these enzymes is still elusive. Herein, we solved the crystal structure of a typical 2-Cys peroxiredoxin from Leishmania in the dimeric (pH 8.5) and decameric (pH 4.4) forms, showing that conformational changes in the catalytic loop are associated with the pH-induced decamerization. Mutagenesis and biophysical studies revealed that a highly conserved histidine (His(113)) functions as a pH sensor that, at acidic conditions, becomes protonated and forms an electrostatic pair with Asp(76) from the catalytic loop, triggering the decamerization. In these 2-Cys peroxiredoxins, decamer formation is important for the catalytic efficiency and has been associated with an enhanced sensitivity to oxidative inactivation by overoxidation of the peroxidatic cysteine. In eukaryotic cells, exposure to high levels of H2O2 can trigger intracellular pH variations, suggesting that pH changes might act cooperatively with H2O2 and other oligomerization-modulator factors to regulate the structure and function of typical 2-Cys peroxiredoxins in response to oxidative stress.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Structural insights on the efficient catalysis of hydroperoxide reduction by Ohr: Crystallographic and molecular dynamics approaches

<div><p>Organic hydroperoxide resistance (Ohr) enzymes are highly efficient Cys-based peroxidases that play central roles in bacterial response to fatty acid hydroperoxides and peroxynitrite, two oxidants that are generated during host-pathogen interactions. In the active site of Ohr proteins, the conserved Arg (Arg19 in Ohr from <i>Xylella fastidiosa</i>) and Glu (Glu51 in Ohr from <i>Xylella fastidiosa</i>) residues, among other factors, are involved in the extremely high reactivity of the peroxidatic Cys (C_p) toward hydroperoxides. In the closed state, the thiolate of C_p is in close proximity to the guanidinium group of Arg19. Ohr enzymes can also assume an open state, where the loop containing the catalytic Arg is far away from C_p and Glu51. Here, we aimed to gain insights into the putative structural switches of the Ohr catalytic cycle. First, we describe the crystal structure of Ohr from <i>Xylella fastidiosa</i> (XfOhr) in the open state that, together with the previously described XfOhr structure in the closed state, may represent two snapshots along the coordinate of the enzyme-catalyzed reaction. These two structures were used for the experimental validation of molecular dynamics (MD) simulations. MD simulations employing distinct protonation states and <i>in silico</i> mutagenesis indicated that the polar interactions of Arg19 with Glu51 and C_p contributed to the stabilization of XfOhr in the closed state. Indeed, C_p oxidation to the disulfide state facilitated the switching of the Arg19 loop from the closed to the open state. In addition to the Arg19 loop, other portions of XfOhr displayed high mobility, such as a loop rich in Gly residues. In summary, we obtained a high correlation between crystallographic data, MD simulations and biochemical/enzymatic assays. The dynamics of the Ohr enzymes are unique among the Cys-based peroxidases, in which the active site Arg undergoes structural switches throughout the catalytic cycle, while C_p remains relatively static.</p></div

Salt-bridge and Hbond interactions of Arg19 with C_p and with Glu51 during XfOhr-S^- (black), XfOhr-SS (red), XfOhr-SH (beige) and E51A XfOhr-S^- (light blue) simulations.

<p>Distance value distributions are shown as a Tukey box-plots (A,B,D,E), in which boxes indicate the interquartile distances, black lines show the median values, whiskers extend the box to 1.5 times the interquartile distance and circles represent outliers (values higher/lower than the whiskers). The box size shows the spread of the distance values, <i>i</i>.<i>e</i>., small boxes indicate less spread in the distance values. The red dashed lines show the 4 Å cutoff value used as the criterion to define stable salt-bridge interactions [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196918#pone.0196918.ref045" target="_blank">45</a>]. The occupancy values for the Hbond interactions throughout the simulations are presented as bar plots (C,F). (A) Distances between the gamma sulfur atom (SG) of C_p and the nitrogen (NE, NH1, NH2) atoms of the Arg19 guanidinium group for XfOhr-S^- (black) and XfOhr-SS (red) simulations. (B) Distances between the oxygen atoms of Glu51 (OE1, OE2) and nitrogen (NE, NH1, NH2) atoms of the Arg19 guanidinium group for XfOhr-S^- (black) and XfOhr-SS (red) simulations (150 ns each). (C) Occupancy values of Hbond interactions between Arg19—Glu51 and Arg19—Cp during the XfOhr-S^- (black) and XfOhr-SS (red) simulations (150 ns each). (D) Arg19 –Glu51 salt-bridge interaction distance values measured during the XfOhr-SH simulation (50 ns), considering all N–O pairs described in B. (E) Arg19 –C_p salt-bridge interaction distance values measured during the E51A XfOhr-S^- simulation (50 ns), considering all N–S pairs described in A. (E) Occupancy values of Hbond interactions between Arg19—Glu51/Ala51 and Arg19—C_p during the XfOhr-SH (beige) and E51A XfOhr-S^- (light blue) simulations (50 ns each). Distance values (yellow dashed lines) measured for the representative structures: (F) XfOhr-S^- (green, values = 2.2–3.7 Å), (G) XfOhr-SS (blue marine, values = 11.9 and 13.1 Å), (H) XfOhr-SH (cyan = 8.0–9.0 Å) and (I) E51A XfOhr-S^- (orange, value = 19.0 Å). Each representative structure corresponds to the MD simulation snapshot closest to the average structure calculated for its trajectory (50 or 150 ns). The protein backbone atoms are show in cartoon representation, and the 33–48 loop was removed for clarity. Arg19, Glu51, C_p and C_r side chain atoms are shown in stick representation, including their polar hydrogen atoms.</p

Comparative analyses of wild-type XfOhr and two mutants (R19A and E51A).

<p>(A) Lipoamide-lipoamide dehydrogenase peroxidase-coupled assay of wild-type XfOhr, R19A and E51A. The peroxidase activities were monitored by the oxidation of NADH at 340 nm in the presence of XfOhr (0.05 μM), lipoamide dehydrogenase from <i>X</i>. <i>fastidiosa</i> (XfLpD, 0.5 μM), and lipoamide (50 μM) in sodium phosphate buffer (20 mM, pH 7.4) and DTPA (0.1 mM). Cys61 (C_p) pKa determination of wild-type XfOhr and two mutants (R19A and E51A) by the monobromobimane method; plots of fluorescence as a function of pH for wild-type XfOhr (B), R19A XfOhr (C) and E51A XfOhr (D). The red points show the mean values of at least two independent experiments. The error bars indicate the SEM. All pKa values were determined using the Henderson-Hasselbach equation of GraphPad^®Prism4.</p

XfOhr-S^- and XfOhr-SS representative structures from MD simulations.

<p>(A) Comparison between the overall representative structures of XfOhr-S^- (green) and XfOhr-SS (blue marine). (B) Active site of the XfOhr-S^- representative structure (green) superposed to the XfOhr crystal structure (light gray) in its closed state (PDB entry 1ZB8). (C) Active site of the XfOhr-SS representative structure (blue marine) superimposed to the XfOhr crystal structure (dark gray) in its oxidized form and open state (PDB entry 4XX2, described in this paper). Each representative structure corresponds to the MD simulation snapshot closest to the average structure calculated throughout the entire trajectory (150 ns). The protein backbone atoms are shown in cartoon representation, and the side chains atoms of Arg19, Glu51, C_p and C_r are shown in the stick representation.</p

Data collection and refinement statistics parameters for the XfOhr open-state.

<p>Data collection and refinement statistics parameters for the XfOhr open-state.</p

Average backbone RMSD (Å) and standard deviation values with respect to the corresponding starting structures calculated for each simulation.

<p>Average backbone RMSD (Å) and standard deviation values with respect to the corresponding starting structures calculated for each simulation.</p

Proposed model for fatty acid hydroperoxide reduction by Ohr.

<p>(i) In the reduced form of Ohr (C_p-S^-, Cys61 of XfOhr), the thiolate anion (Sɤ of C_p) makes an Hbond with the guanidinium group of the conserved Arg (Arg19 of XfOhr), which also makes a salt-bridge with the conserved Glu (Glu51 of XfOhr). (ii) The lipid hydroperoxide (LHP) is placed over the hydrophobic moiety of the Arg side chain, being also stabilized by other hydrophobic interactions. (iii) After peroxide reduction, C_p is oxidized to sulfenic acid (SOH), which is then attacked by the sulfhydryl group of the resolving Cys (Cys125 of XfOhr), forming an intra-molecular disulfide. Our working hypothesis is that this condensation reaction releases constraints for Arg19 loop movements. (iv) The last step involves the reduction of the disulfide by a lipoylated protein and a rearrangement of the loop to the close state (taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196918#pone.0196918.ref011" target="_blank">11</a>]). Steps (ii) and (iii) are hypothetical, as substrate and product (respectively) were inserted based on the co-crystallization of PEG with XfOhr [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196918#pone.0196918.ref011" target="_blank">11</a>].</p