Comparative analysis of the strains over-expressing the genes <i>hoxEFUYH</i> (CE1) or <i>hoxEFUYHW</i> (CE4; CE4u) alone, or together with the <i>hypABCDEF</i> genes (CE5; CE5u).

<p>All experiments were performed at least three times. (<b>A</b>) Typical growth of the wild type (WT; squares), CE-<i>hoxEFUYH</i> (CE1; white triangles) and CE-<i>hoxEFUYHW</i> (CE4; black squares) cells incubated under standard conditions. (<b>B</b>) Histogram plot representation of transcript abundance (measured by Real-time quantitative PCR) of the <i>hoxEFUYHW</i> genes in strains WT (small light-grey bars), CE1 (grey rectangles) and CE4 (hatched bars) mutants. (<b>C</b>) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, CE1, CE4 and CE5 cells growing in MM* medium (MM + 17 μM Fe). (<b>D</b>) Histogram plot representation of the transcript abundance (RT-qPCR) of the <i>hypABC-F</i> genes in the strains WT (light-grey rectangles), CE1 (grey) and CE5 (arrow-filled bars). (<b>E</b>) Histograms representation of the hydrogenase activities of WT (light grey), CE1 (grey), CE2 (dark grey) CE4 (light grey-hatched bars), CE5 (white arrow-filled bars), CE4u (dark grey-hatched bars) and CE5u cells (grey arrow-filled bars) growing in standard medium (MM) or MM* (MM + 17 μM Fe) supplemented with 2.5 μM NiSO₄.</p

Schematic representation of the <i>Synechocystis</i> strains constructed in this study.

<p><i>Synechocystis</i> cells are represented by oval shapes showing their chromosome attached to the cell membrane. The pCE-<i>hypABCDEF</i> replicating plasmid is represented by circles. The <i>hoxEFUYHW</i> and <i>hypABCDEF</i> genes and the antibiotic resistance markers are shown by large arrows pointing towards the direction of their transcription. The triangle represents the strong lambda phage pR promoter (λ<i>p</i>_R); CE for strong <u>c</u>onstitutive <u>e</u>xpression.</p

Influence of urea on the growth of <i>Synechocystis</i> WT and mutants overexpressing the <i>hoxEFUYHW</i> genes alone (CE4) or in combination with the <i>hypABCDEF</i> genes (CE5).

<p>(<b>A</b>) Typical growth of WT cells cultivated on medium containing Ni (1 μM) and urea (2.5–20 mM) as the sole nitrogen source. (<b>B</b>) Typical growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) of the WT strain, and the CE4 and CE5 strain without or with (CE4u and CE5u) a mutation in <i>ureG</i>. Influence of prolonged growth on urea (5 mM as the sole nitrogen source) and Ni (2.5 μM) on the cell appearance <b>C</b>) and urease activity (<b>D</b>) of the studied strains. All experiments were performed at least three times.</p

Engineering <i>Synechocystis</i> PCC6803 for Hydrogen Production: Influence on the Tolerance to Oxidative and Sugar Stresses

<div><p>In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain <i>Synechocystis</i> PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the hydrogenase-encoding <i>hoxEFUYH</i> operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H₂O₂) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions.</p></div

Analysis of the <i>Synechocystis</i> TR-<i>hoxEFUYH</i> mutant for temperature-regulated high-level expression of the <i>hoxEFUYH</i> operon.

<p>(<b>A</b>) Schematic representation of the <i>hoxEFUYH</i> operon locus in the wild-type strain (WT) and the TR-<i>hoxEFUYH</i> mutant (TR1). (<b>B</b>) Typical growth of the WT (squares) and TR1 cells (triangles) under standard light at 30°C (open symbols) or 39°C (black symbols). (<b>C</b>) Histogram representation of the ratio of the transcript abundance (measured by Real-time quantitative PCR) of each eight genes of the <i>hoxEFUYH</i> operon in the WT and TR1 cells grown at 30°C or 39°C. (<b>D</b>) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT and TR1 cells grown at 30°C or 39°C. (<b>E</b>) Histograms representation of the hydrogenase activities of WT and TR1 cells grown at 30°C or 39°C in standard medium (MM) or MM* (MM +17 µM Fe) supplemented with 2.5 µM NiSO₄. All experiments were performed at least three times.</p

Influence of the <i>hoxEFUYH</i> and <i>hypABCDEF</i> genes on the tolerance to H₂O₂, glucose or glycerol.

<p>(<b>A</b>) Typical survival of the <i>Synechocystis</i> strains WT (open squares), Δ<i>hoxEFUYH</i>::Km^r (Δhox, open cercles) or CE-<i>hoxEFUYH</i> (CE1, open triangles) grown at 30°C and challenged for 1 hour with H₂O₂ under anaerobiose before plating under standard photoautotrophic conditions. (<b>B</b>) Typical survival to the anaerobic H₂O₂ stress of the WT strain (open squares), CE1 (open triangles), CE-<i>hoxEFUYH</i>-<i>hypABCDEF</i> (CE2, dark-grey triangles), CE-<i>hypABCDEF</i> (CE3, dark-grey squares). (<b>C</b>) Typical photoautotrophic growth of the wild-type (WT), D<i>hoxEFUYH</i>::Km^r (Δhox), CE-<i>hoxEFUYH</i> (CE1) and CE-<i>hoxEFUYH</i>-<i>hypABCDEF</i> (CE2) strains. Cells grown twice in standard (MM) liquid medium (up to mid-log phase) were inoculated in MM without or with glucose 10 mM or 300 µM glycerol, and incubated under light (3,000 luxes) during 7 days. All experiments were performed at least three times.</p

Analysis of the TR-<i>hoxEFUYH</i>-<i>hypABCDEF</i> mutant for temperature-regulated high-level expression of the <i>hoxEFUYH</i> and <i>hypABCDEF</i> genes.

<p>All experiments were performed at least three times on cells grown under standard light, at 39°C to induce the expression of the <i>hoxEFUYH</i> operon and the <i>hypABCDEF</i> genes controlled by the λ<i>c</i>I₈₅₇-λ<i>p</i>_R system. (<b>A</b>) Typical growth of the WT (squares), TR-<i>hoxEFUYH</i> (TR1; white triangles) and TR-<i>hoxEFUYH</i>-<i>hypABCDEF</i> (TR2; grey triangles) strains. (<b>B</b>) Histogram plot representation of the transcript abundance (measured by Real-time quantitative PCR) of the eight genes of the <i>hoxEFUYH</i> operon (left part) and six <i>hypABCDEF</i> genes (right part) in WT (white bars), TR1 (grey bars) or TR2 (hatched bars) cells. (<b>C)</b> Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, TR1 or TR2 cells. (<b>D</b>) Histograms representation of the hydrogenase activities of WT (light grey), TR1 (dark grey) or TR2 (hatched bars) growing in standard medium (MM) or MM* (MM+17 µM Fe) supplemented with 2.5 µM NiSO₄.</p

Analysis of the CE-<i>hoxEFUYH</i> and CE-<i>hoxEFUYH</i>-<i>hypABCDEF</i> mutants for strong constitutive expression of the <i>hoxEFUYH</i> alone or together with the <i>hypABCDEF</i> genes.

<p>All experiments were performed at least three times on cells grown at 30°C under standard light. (<b>A</b>) Typical growth of the WT (squares), CE-<i>hoxEFUYH</i> (CE1; white triangles) and CE-<i>hoxEFUYH</i>-<i>hypABCDEF</i> (CE2; grey triangles) cells. (<b>B</b>) Histogram plot representation of the transcript abundance (measured by Real-time quantitative PCR) of the eight genes of the <i>hoxEFUYH</i> operon (left part) and the six <i>hypABCDEF</i> genes (right part) in the CE1 (grey bars) or CE2 (hatched bars) mutants, as compared to the WT strain (white bars). (<b>C</b>) Western blot analysis of the abundance of the HoxF and HoxH proteins in WT, CE1 or CE2 cells. (<b>D</b>) Histograms representation of the hydrogenase activities of WT (light grey), CE1 (dark grey) or CE2 cells (hatched bars) growing in standard medium (MM) or MM* (MM+17 µM Fe) supplemented with 2.5 µM NiSO₄.</p

Schematic representation of the <i>Synechocystis</i> wild-type and the mutant strains constructed in this study.

<p>The <i>Synechocystis</i> spherical cells are represented by the green circles. The chromosome is shown as the black line form attached to the cell membrane, while the pTR-<i>hypABCDEF</i> and pCE-<i>hypABCDEF</i> replicating plasmids are represented by circles. The <i>hoxEFUYH</i> operon, the <i>hypABCDEF</i> genes and the antibiotic resistance markers are shown by large colored arrows, which indicate the direction of their transcription. The hatched (orange) arrow shows the λ<i>c</i>I₈₅₇ gene encoding the temperature-sensitive repressor, which tightly controls the activity of the otherwise strong λ<i>p</i>_R promoter (red triangle), depending on the growth temperature. The symbols are namely: Δ for deletion; CE for constitutive expression; TR for temperature-regulated expression; and WT for wild type.</p