Oriented Immobilization of [NiFeSe] Hydrogenases on Covalently and Noncovalently Functionalized Carbon Nanotubes for H₂/Air Enzymatic Fuel Cells

We report the oriented immobilization of [NiFeSe] hydrogenases on both covalently and noncovalently modified carbon nanotubes (CNTs) electrodes. A specific interaction of the [NiFeSe] hydrogenase from <i>Desulfomicrobium baculatum</i> with hydrophobic organic molecules was probed by electrochemistry, quartz crystal microbalance with dissipation monitoring (QCM-D), and theoretical calculations. Taking advantage of these hydrophobic interactions, the enzyme was efficiently wired on anthraquinone and adamantane-modified CNTs. Because of rational immobilization onto functionalized CNTs, the O₂-tolerant [NiFeSe]-hydrogenase is able to efficiently operate in a H₂/air gas-diffusion enzymatic fuel cell

Sequences of the Hpn and Hpn-2 proteins of <i>H</i>. <i>pylori</i> strain B128 [27,28].

<p>Histidine residues are highlighted in blue and Glutamine residues are in green. The triangle corresponds to the position at which Hpn is truncated in Hpn-∆C mutant and at which Hpn-2 is truncated in the Hpn-2∆C mutant. The black stars the positions at which the Hpn and Hpn-2 sequence are interrupted in the study of Seshadri et <i>al</i>. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.ref029" target="_blank">29</a>].</p

Identification of Hpn and Hpn-2 proteins in samples prepared from different <i>Helicobacter</i> species by top-down proteomics.

<p>Identification of Hpn and Hpn-2 proteins in samples prepared from different <i>Helicobacter</i> species by top-down proteomics.</p

Effect of nickel on the growth of <i>H</i>. <i>pylori</i> B128, SS1 and X47-2AL wild type strain and isogenic mutants.

<p>The strains with pureI are controls in which only the pUreI is inserted at the locus at which the <i>hpn</i> or <i>hpn-2</i> genes are introduced in the complemented strains <i>∆hpn + phpn</i> and <i>∆hpn-2</i> + <i>phpn-2</i>. Panel A, nickel disk diffusion assay and sensitivity to 200 μM NiCl₂ in liquid medium. Panel B, nickel sensitivity in liquid medium (range of NiCl₂ concentrations) of mutants and MIC₅₀ for the B128 ∆<i>hpn</i>, ∆<i>hpn</i> +p<i>hpn</i> and ∆<i>hpn-∆hpn-2</i> strains. The data correspond to the mean value of three independent experiments with at least triplicate tests for each strain. Error bars represent the standard deviation.</p

Analysis of the protein-protein interaction network of Hpn and Hpn-2 by <i>in vivo</i> BACTH.

<p>(A-C) the tables represent the results of the β-galactosidase activity measurements on <i>E</i>. <i>coli</i> strains containing the pair-wise combinations of the different constructs. T18 and T25 correspond to the two fragments of the adenylate cyclase and the protein extremity at which the fusion was made (N_ter or C_ter) is shown. Panel A: interaction between Hpn, Hpn∆Cter, Hpn-2 and Hpn-2∆Cter. Panel B and C: interaction of Hpn, Hpn∆Cter, Hpn-2 and Hpn-2∆Cter with UreA, HypA and HypB. Red squares correspond to β-galactosidase activity that are > 3,000 U, orange squares between 2,000 and 3,000 U, yellow squares between 500 and 2,000 U and light yellow squares, between 240 and 500 U. None of the potential false positive interactions was presented (interactions of pUT18/pKNT25(Hpn) and pUT18(Hpn)/pKNT25 with ß-galactosidase activity below 600 units). Black squares represent combinations that lead to a lethal phenotype. The β-galactosidase activity values for each strain and the controls are indicated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.s006" target="_blank">S2 Table</a>. (D) Schematic representation of the protein-protein interaction network of Hpn and Hpn-2. Thick arrows correspond to strong interactions, thin arrows to moderately strong interactions, according to the ß-galactosidase. * Indicates that this interaction was established before [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.ref053" target="_blank">53</a>].</p

Maximum Likelihood phylogeny of a subsample of 100 out of the 330 <i>Helicobacter</i> strains analyzed in this study.

<p>The enterohepatic and gastric strains are shown in gray and black, respectively. The tree is based on the 281 protein families present in a single copy in at least 320 out of the 330 strains (82,743 amino acid positions). Numbers at nodes represent bootstrap supports (for clarity only values > 50% are shown). The scale bar indicates the average number of substitutions inferred by site. Orange and brown rectangles show the presence of <i>hpn</i> and <i>hpn</i>-2 in the corresponding strains, while orange and brown arrows indicate the origin of these genes. Strains harboring similar genomic contexts of <i>hpn</i> and <i>hpn-2</i> are indicated with similar color. Strains analyzed in this study are marked by an orange asterisk. Precise loci organizations are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005312#ppat.1005312.g003" target="_blank">Fig 3</a>.</p

Model of the role of Hpn and Hpn-2 in nickel trafficking <i>H</i>. <i>pylori</i>.

<p>In <i>H</i>. <i>pylori</i>, nickel is transported across the outer membrane by FrpB4, a TonB-dependent transporter. Once in the periplasm, uptake through the inner membrane occurs through the NixA permease (green). In the cytoplasm, nickel can follow several pathways, via the UreE/F/G/H or HypA/B accessory proteins dedicated to urease and/or hydrogenase maturation, the HspA co-chaperonine and Hpn or Hpn-2. <b>(A)</b> Under low nickel concentrations, and because Hpn is abundant, the Hpn cellular pool is not saturated with nickel, while Hpn-2, which is less abundant, can compete with Hpn (similar affinities for nickel). Nickel transfer from Hpn to the urease maturation machinery is low. This results in the “basal” urease activity level monitored in wild type <i>H</i>. <i>pylori</i> cells. <b>(B)</b> Under high nickel conditions, the Hpn protein pool stores more nickel and becomes saturated with this metal. Since Hpn-2 is less abundant, it rapidly becomes outcompeted. Under such conditions, interactions between Hpn and urease and/or hydrogenase maturation proteins enables Hpn-stored nickel transfer towards urease, leading to an increase in nickel incorporation, and enhanced urease activity.</p

Urease activity of <i>H</i>. <i>pylori</i> B128 wild type strain, isogenic mutants and complemented strains.

<p>Strains were grown in Brucella β-cyclodextrin medium without added nickel (grey bars) or with 10 µM nickel (black bars). The identity of each strain is indicated below the bars. Urease activity is expressed as μmol of urea hydrolyzed. min^-1. mg^-1 of protein. The data correspond to the mean value of three independent experiments with at least triplicate tests for each strain. Error bars represent the standard deviation. *** indicates that the mean value is significantly different from that of the wild type strain (<i>P</i> < 0.01).</p

Hpn and Hpn-2 are required for full mouse colonization by <i>H</i>. <i>pylori</i> SS1, B128 and X47-2AL strains.

<p>Each point corresponds to the colonization load for one mouse, with the strain indicated below. Each strain was tested in a group of seven mice. Horizontal bars represent the geometric means of the colonization load for the wild type and each mutant. The detection limit is shown by a dashed horizontal line. *** and ** indicate that the geometric mean is significantly different from that of the wild type strain (<i>P</i> < 0.01 and <i>P</i> < 0.05, respectively).</p

Genomic organization of the region encompassing <i>hpn</i> and <i>hpn-2</i>.

<p>Conserved homologous genes are shown with similar colors, hypothetical proteins are shown in gray, while non-conserved genes are in white. <i>Hpn</i> (in red) is located in-between two conserved cluster of genes (clusters 1 and 2), while <i>hpn-2</i> is located either near <i>hpn</i> on the same strand or on the opposite side of cluster 1 on the opposite strand. <i>Helicobacter</i> strains harboring similar genomic organization are listed on the left with similar colors.</p