42 research outputs found
Subcellular localization of hydrogenase 2 in <i>iscA</i>, <i>sufA</i> and <i>erpA</i> mutants.
<p>Aliquots (25 µg protein) derived from whole cells (1), crude extracts (2), soluble fractions (3) or membrane fractions (4) from MC4100, DHP-F2 (Δ<i>hypF</i>), CP1223 (Δ<i>sufA</i>), CP477 (Δ<i>iscA</i>) and CP477+piscA were separated either by native-PAGE (A) (7.5% w/v polyacrylamide) and stained for hydrogenase enzyme activity, or by 10% SDS-PAGE (B) and subjected to Western blotting using anti-hydrogenase 2 antiserum. On the left side of panel A, the migration positions of hydrogenase 1, hydrogenase 2, and of the hydrogenase-independent formate dehydrogenase hydrogen: BV oxidoreductase activity are indicated. In panel B the migration positions of the unprocessed and processed forms of the catalytic subunit HybC and the small subunit HybO are shown. The asterisks indicate unspecifically cross-reacting polypeptides of unknown identity, which acted as internal loading controls. The migration positions of the molecular mass standards (in kDa) are indicated on the right of the Figure.</p
Hydrogenases 1 and 2 are inactive in <i>iscA</i> and <i>erpA</i> mutants.
<p>Aliquots of crude extracts (25 µg protein) derived from the bacterial stains shown were separated by non-denaturing PAGE (7.5% w/v polacrylamide) and subsequently stained for hydrogenase enzyme activity. Hydrogenase 1 migrates as a single active enzyme species while hydrogenase 2 shows multiple active forms, which are designated on the right of the panel. The weak hydrogen: benzyl viologen oxidoreductase activity that is independent of the [NiFe]-hydrogenases, and which is associated with formate dehydrogenase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031755#pone.0031755-Soboh1" target="_blank">[25]</a>, is also designated.</p
Schematic representation of the iron-sulphur cluster-containing proteins involved in the hydrogen oxidation of <i>E. coli</i>.
<p>The two anaerobic, membrane-associated [NiFe]-hydrogenases 1 and 2 are schematically represented with their associated subunits. The iron-sulphur clusters are shown as groups of spheres. No distinction is made between [3Fe-4S], [4Fe-4S] or [4Fe-3S] clusters. The ‚squiggle' attached to the apoprotein form of the hydrogenase catalytic subunit (bottom of the Figure) represents the C-terminal peptide that is removed subsequent to insertion of the [NiFe] cofactor. The dotted arrows indicate electron flow within the modular enzymes or to the quinone pool.</p
Identification of the HypD maturase in mutants defective in iron-sulphur cluster biogenesis.
<p>Aliquots (25 µg protein) derived from MC4100, CP477 (Δ<i>iscA</i>), LL402 (Δ<i>erpA</i>), CP1223 (Δ<i>sufA</i>), JW2513 (Δ<i>iscU</i>), BEF314 (Δ<i>hypB-E</i>) and PB1000 (Δ<i>fnr</i>) were separated by 10% (w/v polyacrylamide) SDS-PAGE and subjected to western blotting using anti-HypD antiserum. The asterisk indicates an unidentified cross-reacting polypeptide that served as an internal loading control. The migration positions of the molecular mass standards (in kDa) are indicated on the right of the figure.</p
Immunological analysis reveals maturation of the catalytic subunit and absence of the small subunit of the hydrogen-uptake enzymes in <i>erpA</i> and <i>iscA</i> mutant.
<p>Samples of crude extracts (25–50 µg protein) derived from the strains (genotypes are shown above each lane) indicated were separated in 10% (w/v) SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against the hydrogenase 1 large subunit (HyaB) (A) or the small subunit (B). The locations of the processed and unprocessed forms of the large subunit polypeptide are shown on the left of the panel and the migration position of molecular mass size markers are shown on the right. Results of a similar experiment showing western blots of the large (HybC) and small (HybO) subunits of hydrogenase 2 are shown in C and D. Unspecific cross-reacting polypeptides that were used as internal loading controls are marked with an asterisk. MC4100, wild type; DHP-F2 (Δ<i>hypF</i>); CP1223 (Δ<i>sufA</i>); CP477 (Δ<i>iscA</i>); LL402 (Δ<i>erpA</i>); CP795 (Δ<i>hyaB</i> Δ<i>hybO</i> Δ<i>hycE</i>) was used in the lane labelled Δ<i>hybO</i>.</p
Comparative analysis of hydrogen-uptake hydrogenases in different <i>E. coli</i> K-12 derivatives.
<p>A. Aliquots of crude extracts (25 µg protein) derived from the bacterial strains shown were separated by non-denaturing PAGE (7.5% w/v polyacrylamide) and subsequently stained for hydrogenase enzyme activity. The locations of hydrogenase 1 and hydrogenase 2 activity bands are shown. B. Western blot analysis of the unprocessed and processed forms of the HybC large subunit of hydrogenase 2 was performed using crude extracts (25 µg protein) derived from the bacterial strains indicated. Strain DHP-F2 is a derivative of MC4100 carrying a deletion in the <i>hypF</i> gene.</p
Strains and plasmids used in this study
+<p>National BioResources Project (NIG, Japan): E. coli.</p>a<p>Allele numbers are given for single gene mutants and refer to the K-12 nomenclature.</p
<i>X. campestris</i> pv. <i>vesicatoria</i> strain 85-10Δ<i>acnB</i> has increased sensitivity to the superoxide-generating agent menadione.
<p>Dilutions of 10<sup>−6</sup> of exponential phase cultures (OD<sub>600</sub> =  0.6) of the <i>Xanthomonas</i> strains indicated were spotted on NYG agar plates containing 50 µM and 100 µM menadione. Bacterial colonies surviving the treatment were counted after 24 h and 48 h of incubation at 30°C and CFU were expressed as surviving fraction in percent. Strain 85-10 (black columns), strain 85-10Δ<i>acnB</i> (white columns) and strain 85-10ΔXCV1925<i>-26acnB</i> (gray columns).</p
Co-transcription of <i>xcv1925, xcv1926</i> and <i>acnB</i>.
<p>A. Schematic representation of the deletions introduced in the genes at the <i>acnB</i> locus. 1. represents the extent of the deletion in strain 85-10Δ<i>acnB</i>; 2. represents the deletion in 85-10ΔXCV1925-26<i>acnB</i>; 3. represents the deletion in strain 85-10ΔXCV1925-26; and 4. represents the deletion in strain 85-10Δ<i>acnA</i>. B. RT-PCR analysis of the XCV1925-XCV1926<i>-acnB</i> transcript. Total RNA was isolated and analyzed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#s4" target="_blank">Methods</a> using oligonucleotide primers r-secacnB and f-sec3565 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034941#pone.0034941.s001" target="_blank">Table S1</a>). Lane 1, DNA size standards; lane 2, PCR product with cDNA; lane 3, control in which RT was omitted from the cDNA synthesis reaction; lane 4, control in which total RNA was omitted from the cDNA synthesis reaction; lane 5, PCR using genomic DNA as template.</p
Strain 85-10Δ<i>acnB</i> shows restricted growth <i>in planta</i>.
<p>The indicated strains were grown as follows: A. Growth <i>in planta</i> where filled squares represent strain 85-10, filled circles represent strain 85-10Δ<i>acnA</i>, filled triangles represent strain 85-10Δ<i>acnB</i> and filled inverted triangles represent strain 85*Δ<i>hrcN</i> B. Growth <i>in planta</i> where filled squares represent strain 85-10, filled triangles represent strain 85-10ΔXCV1925-26, filled inverted triangles represent strain 85-10ΔXCV1925-26<i>acnB</i> and filled circles represent strain 85*Δ<i>hrcN</i>; C. Growth <i>in planta</i> where filled squares represent strain 85-10, open squares represent strain 85-10/pLAFR6, filled circles represent strain 85-10Δ<i>acnB</i>/pL6<i>acnB</i>, open circles represent strain 85-10Δ<i>acnB</i>/pLAFR6 and filled inverted triangles represent 85*Δ<i>hrcN</i>. The standard error is shown for each experiment.</p