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. 2022 May 31:13:896190.
doi: 10.3389/fmicb.2022.896190. eCollection 2022.

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions

Heinrich Burgstaller  1 , Yingying Wang  1 , Johanna Caliebe  1   2 , Vanessa Hueren  1 , Jens Appel  1   2 , Marko Boehm  1   2 , Sinje Leitzke  1 , Marius Theune  1   2 , Paul W King  3 , Kirstin Gutekunst  1   2
Affiliations

Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions

Heinrich Burgstaller et al. Front Microbiol. .

Abstract

The cyanobacterium Synechocystis sp.PCC 6803 possesses a bidirectional NiFe-hydrogenase, HoxEFUYH. It functions to produce hydrogen under dark, fermentative conditions and photoproduces hydrogen when dark-adapted cells are illuminated. Unexpectedly, we found that the deletion of the large subunit of the hydrogenase (HoxH) in Synechocystis leads to an inability to grow on arginine and glucose under continuous light in the presence of oxygen. This is surprising, as the hydrogenase is an oxygen-sensitive enzyme. In wild-type (WT) cells, thylakoid membranes largely disappeared, cyanophycin accumulated, and the plastoquinone (PQ) pool was highly reduced, whereas ΔhoxH cells entered a dormant-like state and neither consumed glucose nor arginine at comparable rates to the WT. Hydrogen production was not traceable in the WT under these conditions. We tested and could show that the hydrogenase does not work as an oxidase on arginine and glucose but has an impact on the redox states of photosynthetic complexes in the presence of oxygen. It acts as an electron valve as an immediate response to the supply of arginine and glucose but supports the input of electrons from arginine and glucose oxidation into the photosynthetic electron chain in the long run, possibly via the NDH-1 complex. Despite the data presented in this study, the latter scenario requires further proof. The exact role of the hydrogenase in the presence of arginine and glucose remains unresolved. In addition, a unique feature of the hydrogenase is its ability to shift electrons between NAD(H), NADP(H), ferredoxin, and flavodoxin, which was recently shown in vitro and might be required for fine-tuning. Taken together, our data show that Synechocystis depends on the hydrogenase to metabolize organic carbon and nitrogen in the presence of oxygen, which might be an explanation for its prevalence in aerobic cyanobacteria.

Keywords: arginine; diaphorase; hydrogenase; photomixotrophy; photosynthesis; photosynthetic complex I (NDH-1); respiration.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Growth of wild type (WT) and ΔhoxH under different conditions and oxygen and hydrogen concentrations in cultures on arginine and glucose in continuous light (60 μE/m2/s). (A) Growth in arginine and glucose (AG), (B) growth in nitrate (N), (C) growth in arginine (A), (D) growth in nitrate and glucose (NG), (E) O2 and H2 concentration in the cultures cultivated on arginine and glucose.
Figure 2
Figure 2
(A) Chlorophyll content of WT and ΔhoxH during growth experiments under photoautotrophic conditions and on arginine and glucose. (B) The appearance of strains on day 3 after inoculation.
Figure 3
Figure 3
Metabolism and morphology of WT and ΔhoxH on arginine and glucose. (A) Growth (solid line) and glucose consumption (dotted line). (B) Arginine consumption, (C) Cyanophycin content of cells displayed as arginine in the cells, (D) Transmission electron microscopy (TEM) images of WT and ΔhoxH that were cultivated on arginine and glucose. Cyanophycin granules are marked with a red arrow in the WT. (E) Growth experiment in which (F) dark respiration and (G) photosynthesis were determined by O2 uptake and evolution on days 2–4. Stars on the column diagram indicate differences between mutant and WT by Tukey's HSD test (*P < 0.05, **P < 0.01). Throughout the figure ‘NS' indicates ‘not significant'. All error bars indicate standard deviation (s.d.) of four daily independent experiments.
Figure 4
Figure 4
Significance of the diaphorase and the hydrogenase on arginine and glucose. (A) SDS page and immunoblots of WT, ΔhoxH, and ΔhoxW with antibodies against all hox subunits (B) 2D Blue Native PAGE of WT, ΔhoxH, and ΔhoxW with antibodies against all hox subunits. The second dimension allows to check for the assembly of complexes. (C) Growth of WT, ΔhoxH, and ΔhoxW on arginine and glucose. (D) Complementation of Δhox with hoxYH (Δhox/hoxYH). Growth of WT, Δhox/hoxYH, and Δhox on arginine and glucose.
Figure 5
Figure 5
Growth of WT and ΔhoxH on arginine and glucose. (A) Growth at high light (200 μE/m2/s) and low light (20 μE/m2/s). (B) Growth at medium light (60 μE/m2/s) in the absence and presence of the inhibitor DCMU, which blocks electron transfer from PSII to the plastoquinone (PQ) pool.
Figure 6
Figure 6
Oxidation of PC and P700 and reduction of ferredoxin and NADPH of WT and ΔhoxH cultures as measured by the Dual-KLAS/NIR. On the left (A, C, E, G), curves of cultures grown photoautotrophically are shown and on the right (B, D, F, H), the same cultures are shown that were measured a few minutes after the addition of arginine and glucose.
Figure 7
Figure 7
(A–F) Redox states of components of the photosynthetic electron chain on arginine and glucose in the presence and absence of oxygen in WT, Δflv24Δflv3ΔcoxΔcyd, and Δflv24Δflv3ΔcoxΔcydΔhox. The oxidation state was measured as absorption change (ΔI/I ×10−3). Positive values show oxidation and negative values show a reduction of components. (G) Photochemical quenching (qP) in WT and ΔhoxH on nitrate (WT and ΔhoxH) and on arginine and glucose (WT AG and ΔhoxH AG).
Figure 8
Figure 8
Significance of the respiratory electron chain for growth on arginine and glucose. (A) Overview of photosynthetic and respiratory electrons chains in the thylakoid and cytoplasmic membrane. (B) Growth of WT and mutants in which terminal oxidases were deleted on arginine and glucose. (C) Growth of WT and dehydrogenases, that feed electrons into the PQ pool on arginine and glucose.
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