To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO₂ and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the ΔNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700⁺ rereduction in darkness in the ΔNdhD1/D2 mutant grown at low CO₂ were similar to those in the wild type, whereas in the M55 mutant (ΔNdhB), lacking both NDH-1L and NDH-1M, the rate of P700⁺ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO₂ in the wild type as well as in ΔNdhD1/D2 and M55. In contrast with the wild type and ΔNdhD1/D2, which show normal CO₂ uptake, M55 is unable to take up CO₂ even when the NDH-1S complex is present. Conversely, the ΔNdhD3/D4 mutant, also unable to take up CO₂, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO₂. These results demonstrate that both NDH-1S and NDH-1M are essential for CO₂ uptake and that NDH-1M is a functional complex. We also show that the Na⁺/HCO₃⁻ transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO₂.

Glucose degradation pathways are central for energy and carbon metabolism throughout all domains of life. They provide ATP, NAD(P)H, and biosynthetic precursors for amino acids, nucleotides, and fatty acids. It is general knowledge that cyanobacteria and plants oxidize carbohydrates via glycolysis [the Embden–Meyerhof–Parnas (EMP) pathway] and the oxidative pentose phosphate (OPP) pathway. However, we found that both possess a third, previously overlooked pathway of glucose breakdown: the Entner–Doudoroff (ED) pathway. Its key enzyme, 2-keto-3-deoxygluconate-6-phosphate (KDPG) aldolase, is widespread in cyanobacteria, moss, fern, algae, and plants and is even more common among cyanobacteria than phosphofructokinase (PFK), the key enzyme of the EMP pathway. Active KDPG aldolases from the cyanobacterium Synechocystis and the plant barley (Hordeum vulgare) were biochemically characterized in vitro. KDPG, a metabolite unique to the ED pathway, was detected in both in vivo, indicating an active ED pathway. Phylogenetic analyses revealed that photosynthetic eukaryotes acquired KDPG aldolase from the cyanobacterial ancestors of plastids via endosymbiotic gene transfer. Several Synechocystis mutants in which key enzymes of all three glucose degradation pathways were knocked out indicate that the ED pathway is physiologically significant, especially under mixotrophic conditions (light and glucose) and under autotrophic conditions in a day/night cycle, which is probably the most common condition encountered in nature. The ED pathway has lower protein costs and ATP yields than the EMP pathway, in line with the observation that oxygenic photosynthesizers are nutrient-limited, rather than ATP-limited. Furthermore, the ED pathway does not generate futile cycles in organisms that fix CO₂ via the Calvin–Benson cycle. Significance Life on Earth is substantially driven by a circuit of photosynthesis and glucose oxidation. Photosynthesizers capture sunlight and store its energy in the bonds of carbohydrates. Oxidation of carbohydrates provides organisms with a source of ATP and organic carbon for the synthesis of cellular building blocks. Our data provide strong evidence that the Entner–Doudoroff pathway of glucose degradation, which has been previously long overlooked, operates in cyanobacteria and plants. Phylogenetic analyses reveal that the cyanobacterial ancestor of plastids transferred this glycolytic route, via endosymbiotic gene transfer, to the plant lineage.

Cyanobacteria are able to use solar energy for the production of hydrogen. It is generally accepted that cyanobacterial NiFe-hydrogenases are reduced by NAD(P)H. This is in conflict with thermodynamic considerations, as the midpoint potentials of NAD(P)H do not suffice to support the measured hydrogen production under physiological conditions. We show that flavodoxin and ferredoxin directly reduce the bidirectional NiFe-hydrogenase of Synechocystis sp. PCC 6803 in vitro. A merodiploid ferredoxin-NADP reductase mutant produced correspondingly more photohydrogen. We furthermore found that the hydrogenase receives its electrons via pyruvate:flavodoxin/ferredoxin oxidoreductase (PFOR)-flavodoxin/ferredoxin under fermentative conditions, enabling the cells to gain ATP. These results strongly support that the bidirectional NiFe-hydrogenases in cyanobacteria function as electron sinks for low potential electrons from photosystem I and as a redox balancing device under fermentative conditions. However, the selective advantage of this enzyme is not known. No strong phenotype of mutants lacking the hydrogenase has been found. Because bidirectional hydrogenases are widespread in aquatic nutrient-rich environments that are capable of triggering phytoplankton blooms, we mimicked those conditions by growing cells in the presence of increased amounts of dissolved organic carbon and dissolved organic nitrogen. Under these conditions the hydrogenase was found to be essential. As these conditions close the two most important sinks for reduced flavodoxin/ferredoxin (CO2-fixation and nitrate reduction), this discovery further substantiates the connection between flavodoxin/ferredoxin and the NiFe-hydrogenase.

Summary: The bidirectional NiFe‐hydrogenase of Synechocystis sp. PCC 6803 is encoded by five genes (hoxEFUYH) which are transcribed as one unit. The transcription of the hox‐operon is regulated by a promoter situated upstream of hoxE. The transcription start point was located at −168 by 5′Race. Several promoter probe vectors carrying different promoter fragments revealed two regions to be essential for the promoter activity. One is situated in the untranslated 5′leader region and the other is found −569 to −690 nucleotides upstream of the ATG. The region further upstream was shown to bind a protein. Even though an imperfect NtcA binding site was identified, NtcA did not bind to this region. The protein binding to the DNA was purified and found to be LexA by MALDI‐TOF. The complete LexA and its DNA binding domain were overexpressed in Escherichia coli. Both were able to bind to two sites in the examined region in band‐shift‐assays. Accordingly, the hydrogenase activity of a LexA‐depleted mutant was reduced. This is the first report on LexA acting not as a repressor but as a transcriptional activator. Furthermore, LexA is the first transcription factor identified so far for the expression of bidirectional hydrogenases in cyanobacteria.