Kirstin Gutekunst

LexA regulates the bidirectional hydrogenase in the cyanobacterium Synechocystis sp. PCC 6803 as a transcription activator

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.

The Bidirectional NiFe-hydrogenase in Synechocystis sp. PCC 6803 Is Reduced by Flavodoxin and Ferredoxin and Is Essential under Mixotrophic, Nitrate-limiting Conditions

Background: Cyanobacterial hydrogenases are claimed to produce hydrogen via NAD(P)H, which contradicts thermodynamic considerations; the physiological function of these hydrogenases is unresolved. Results: Flavodoxin/ferredoxin reduce cyanobacterial hydrogenases, which are essential under mixotrophic, nitrate-limiting conditions. Conclusion: Cyanobacterial bidirectional hydrogenases are electron sinks for reduced flavodoxin/ferredoxin. Significance: This study provides a basis for a target-oriented enhancement of hydrogen production and explains the aquatic distribution of cyanobacterial hydrogenases. 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.

Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of ΔhypA1 and ΔhypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single‐ and double‐mutants was the same as in wild‐type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase‐specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt‐requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.

The Entner–Doudoroff pathway is an overlooked glycolytic route in cyanobacteria and plants

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. 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.

hypD as a marker for [NiFe]-hydrogenases in microbial communities of surface waters.

ABSTRACT Hydrogen is an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown. [NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of [NiFe]-hydrogenases, including their frequency of horizontal gene transfer, and found hypD to be an applicable marker for the detection of the different known hydrogenase groups. Investigation of two freshwater lakes showed that [NiFe]-hydrogenases occur in many prokaryotic orders. We found that the respective hypD genes cooccur with oxygen-tolerant [NiFe]-hydrogenases (groups 1 and 5) mainly of Actinobacteria, Acidobacteria, and Burkholderiales; cyanobacterial uptake hydrogenases (group 2a) of cyanobacteria; H2-sensing hydrogenases (group 2b) of Burkholderiales, Rhizobiales, and Rhodobacterales; and two groups of multimeric soluble hydrogenases (groups 3b and 3d) of Legionellales and cyanobacteria. These findings support and expand a previous analysis of metagenomic data (M. Barz et al., PLoS One 5:e13846, 2010, http://dx.doi.org/10.1371/journal.pone.0013846) and further identify [NiFe]-hydrogenases that could be involved in hydrogen cycling in aquatic surface waters.

A Specific Glycogen Mobilization Strategy Enables Rapid Awakening of Dormant Cyanobacteria from Chlorosis

Rapid reanimation of a photosynthetic bacterium following nitrogen starvation is facilitated by anticipation and requires two parallel routes of glycogen catabolism and a particular glycogen phosphorylase paralog. Many organisms survive stressful conditions via entry into a dormant state that can be rapidly exited when the stressor disappears; this ability provides a strong selective advantage. In the cyanobacterium Synechocystis sp. PCC 6803, the exit from nitrogen chlorosis takes less than 48 h and is enabled by the impressive metabolic flexibility of these cyanobacteria, which pass through heterotrophic and mixotrophic phases before reentering photoautotrophic growth. Switching between these states requires delicate coordination of carbohydrate oxidation, CO2 fixation, and photosynthesis. Here, we investigated the contribution of the different carbon catabolic routes by assessing mutants of these pathways during nitrogen chlorosis and resuscitation. The addition of nitrate to nitrogen-starved cells rapidly starts the awakening program. Metabolism switches from maintenance metabolism, characterized by residual photosynthesis and low cellular ATP levels, to an initial heterotrophic phase, characterized by respiration and an immediate increase in ATP levels. This respiration relies on glycogen breakdown catalyzed by the glycogen phosphorylase GlgP2. In the following transient mixotrophic phase, photosynthesis and CO2 fixation restart and glycogen is consumed. During the mixotrophic phase, parallel operation of the oxidative pentose phosphate cycle and the Entner-Doudoroff pathway is required for resuscitation to proceed; the glycolytic route via the Embden-Meyerhof-Parnas pathway has minor importance. Our data suggest that, during resuscitation, only the Entner-Doudoroff and oxidative pentose phosphate pathways supply the metabolic intermediates necessary for the anabolic reactions required to reconstitute a vegetative cell. Intriguingly, the key enzymes for glycogen catabolism are already expressed during the preceding chlorotic phase, in apparent preparation for rapid resuscitation.

Hypothesis on the Synchronistic Evolution of Autotrophy and Heterotrophy

All life on earth requires a source of energy and organic carbon. There has been a continuous debate on whether autotrophic or heterotrophic metabolism came first. A very similar discussion exists concerning the advent of oxygenic photosynthesis and aerobic respiration. I put forward the synchronistic evolution hypothesis supposing that all metabolic processes develop in a bidirectional manner from the very first. Bidirectionality is claimed to be intrinsic to the evolution of all metabolic processes as (i) all biochemical reactions and enzymes are per se bidirectional, (ii) substrates need to be regenerated, and (iii) flux regulation requires flexibility of direction. Autotrophy and heterotrophy are thus inherent to each other. A scenario for the synchronistic development of oxygenic photosynthesis and aerobic respiration is described.

In-vivo turnover frequency of the cyanobacterial NiFe-hydrogenase during photohydrogen production outperforms in-vitro systems

Cyanobacteria provide all components for sunlight driven biohydrogen production. Their bidirectional NiFe-hydrogenase is resistant against low levels of oxygen with a preference for hydrogen evolution. However, until now it was unclear if its catalytic efficiency can keep pace with the photosynthetic electron transfer rate. We identified NikKLMQO (sll0381-sll0385) as a nickel transporter, which is required for hydrogen production. ICP-MS measurements were used to quantify hydrogenase molecules per cell. We found 400 to 2000 hydrogenase molecules per cell depending on the conditions. In-vivo turnover frequencies of the enzyme ranged from 62 H2/s in the wild type to 120 H2/s in a mutant during photohydrogen production. These frequencies are above maximum in-vivo photosynthetic electron transfer rates of 47 e−/s (equivalent to 24 H2/s). They are also above those of existing in-vitro systems working with unlimited electron supply and show that in-vivo photohydrogen production is limited by electron delivery to the enzyme.

CHAPTER 4:The Physiology of the Bidirectional NiFe-hydrogenase in Cyanobacteria and the Role of Hydrogen Throughout the Evolution of Life

Hydrogen (H2) probably played a central role as an electron donor for the fixation of inorganic carbon at the advent of life on Earth. LUCA, the last universal common ancestor, most likely possessed a NiFe-hydrogenase and transferred the enzyme to the first bacterial and archaean cell. At present, hydrogenases are widespread in the environment and are not restricted to anoxic habitats. The occurrence of the cyanobacterial bidirectional NiFe-hydrogenase shows strong correlation with the habitat. It is prevalent in freshwater, coastal surface waters, microbial mats, and hot springs, but is absent from the open ocean and terrestrial deserts. The enzyme is truly bidirectional, with a bias towards H2 production. In the presence of oxygen, it forms two inactive states that require reactivation at distinct redox potentials. The physiology of cyanobacterial H2 production by means of the bidirectional NiFe-hydrogenases is discussed at the onset of illumination and under fermentative conditions. Approaches to enhance cyanobacterial H2 production based on the current knowledge are suggested.