Biology subjects

Hagemann, M.

Publications and source records attributed to Hagemann, M..

At least 19 recordsLinked to original sources

bioRxiv2026

The relevance of GGCC methylation for cyanobacterial gene expression regulation

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In the Synechocystis 6803 genome, the DNA-specific methyltransferase M.Ssp6803II modifies the first cytosine in the 38,512 GGCC motifs, forming N4-methylcytosine (GGm4CC). Previous analyses showed that deletion of the gene encoding M.Ssp6803II ({triangleup}sll0729), caused a bluish phenotype due to reduced expression of the hemJ gene. This effect was caused by the lacking GGCC methylation in the discriminator sequence of the hemJ promoter in {triangleup}sll0729. In the present study, we analyzed the impact of GGm4CC methylation on the overall gene expression. Bioinformatic analyses of multiple, independently obtained DNA microarray datasets revealed reproducible changes in the expression of more than 300 genes. We selected 26 genes that had the GGCC methylation motif in different locations of their promoter sequences and verified their differential expression through qPCR experiments. Classification of the promoters according to the relative position of GGCC motifs suggested context-specific effects on gene expression. These findings imply that GGm4CC methylation via DNA methyltransferase M.Ssp6803II has an important impact on proper gene expression and thus, cytosine N4-methylation is a relevant epigenetic marker in Synechocystis and likely many other bacteria.

microbiology↗

bioRxiv2026

Deletion of the gene for a cyanobacterial ribosome-associated protein affects the carbon/nitrogen metabolism

In contrast to their important structural and regulatory functions, such as in the metabolism of cyanobacteria, genes encoding small proteins are often not well characterized. Cyanobacteria use redox equivalents and energy from oxygenic photosynthesis to produce organic carbon compounds from inorganic carbon (Ci) and organic nitrogen compounds from inorganic nitrogen sources. Therefore, the assimilation and metabolism of carbon and nitrogen are coordinated at multiple levels in cyanobacteria. Here, we analyzed the Synechocystis sp. PCC 6803 gene ssr3189 encoding a 55 amino acids protein. Orthologs were detected in 665 cyanobacterial genomes defining COG5794 in the Database of Clusters of Orthologous Genes. Homologs in several eukaryotic algae suggest that Ssr3189 is an important protein that originated in cyanobacteria, was retained in algae after endosymbiosis, but was lost in plants. Polynucleotide kinase assays validated Ssr3189 as an RNA-binding protein. Deletion of ssr3189 resulted in lower pigmentation, delayed growth, and alterations in the expression of genes encoding transporters for nitrogen and Ci, and metabolic enzymes. Metabolomic analysis revealed a substantial overaccumulation of glutamine and tricarboxylic acid cycle intermediates in the deletion mutant, and further differences in the amino acid and organic acid pools compared to the wild type. Co-immunoprecipitation analysis yielded ribosomal protein S21, enolase and the Cas6-1 endoribonuclease as the most strongly co-enriched proteins, together with all other ribosomal proteins and a small set of metabolic enzymes. These findings are consistent with observations that ssr3189 encodes the ribosome-associated protein cS24 and suggest that it connects translation with metabolic control, and, potentially, RNA decay. IMPACT STATEMENTDespite considerable progress in analyzing microbial genomes, there are still substantial numbers of uncharacterized gene functions. Here, we analyzed a mutant lacking gene ssr3189 that is widely conserved, but phenotypically uncharacterized in cyanobacteria. This gene is important for growth at the optimum temperature and essential at lower temperatures. In its absence, important metabolites were overaccumulated, while genes involved in nitrogen and Ci uptake were dysregulated. The encoded protein binds RNA and interacts with proteins involved in translation and metabolism. The findings are consistent with a function as a ribosomal protein bridging protein synthesis and the regulation of metabolism.

microbiology↗

bioRxiv2026

Microcystin-Driven Control of the Carbon-Concentrating Mechanism Shapes CO2 Fixation Dynamics in Microcystis aeruginosa PCC 7806

Bloom-forming cyanobacteria thrive in highly dynamic light environments, yet the mechanisms enabling rapid acclimation to fluctuating irradiance remain poorly understood. Here, we compared light acclimation in the bloom-forming cyanobacterium Microcystis aeruginosa PCC 7806 and the non-bloom-forming model cyanobacterium Synechocystis sp. PCC 6803 and investigated the role of the cyanobacterial toxin microcystin (MC) and its in vivo binding partner RubisCO in this process. Whereas Synechocystis grew faster under sustained high light, Microcystis performed better under low light and responded to transient high-light exposure with a remarkably rapid increase in photosynthetic activity and glycogen accumulation. These responses were markedly attenuated in an MC-deficient mutant. Although RubisCO from Microcystis exhibited pronounced light-dependent changes in activity, MC had only minor effects on RubisCO catalysis, arguing against a direct role in regulating enzyme function. Instead, extracellular MC elicited a transient transcriptional program characterized by induction of inorganic carbon acquisition systems, including the high-affinity bicarbonate transporter BCT1, consistent with activation of the carbon-concentrating mechanism (CCM) and enhanced carbon fixation in vivo. MC further stimulated the expression of photosynthesis-related genes, and altered carboxysome organization, and promoted extracarboxysomal localization of RubisCO. Together, our findings identify MC as a light-responsive signaling molecule that coordinates CCM activity, carbon acquisition, and photosynthetic acclimation, thereby enhancing adaptation of Microcystis to fluctuating irradiance and potentially contributing to its ecological success in cyanobacterial blooms.

microbiology↗

bioRxiv2026

Story about honest mistakes: The cyanobacterium Synechocystis has a promiscuous Entner-Doudoroff (ED) aldolase but no functional ED pathway.

In 2016, the glycolytic Entner-Doudoroff (ED) pathway was reported in cyanobacteria and plants (1). The claim was based on the biochemical characterization of its key enzyme the 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase also named ED aldolase (EDA), on protein sequence alignments, physiological data from cyanobacterial mutants, and the in vivo detection of an ED pathway specific metabolite (1). However, two enzymes 6-phosphogluconate (6PG) dehydratase (EDD) and EDA are unique to this route. A recent study suggests that EDD (Slr0452) from Synechocystis sp. PCC 6803 most likely encodes an enzyme involved exclusively in amino acid synthesis, indicating that a complete ED pathway would be missing (2). To address the presence or absence of the ED pathway in Synechocystis, we conducted extended biochemical and physiological studies, revisited old data and resolved contradictions. These investigations reveal that Synechocystis lacks both an ED pathway and a glucose dehydrogenase/glucokinase (GDH/GK) bypass but contains a promiscuous aldolase EDA. EDA prefers KDPG as substrate but also decarboxylates oxaloacetate (OAA) and cleaves 2-keto-4-hydroxyglutarate (KHG). Synthesis of KDPG from pyruvate and glyceraldehyde 3-phosphate (GAP) is catalyzed with very low efficiency. These in vitro data suggest that EDA might be involved in the phosphoenolpyruvate (PEP)-pyruvate-OAA node and proline catabolism, which requires further clarification. The previous misconception was based on missing enzymatic characterizations, the oversight of a secondary mutation in a deletion strain, and an outdated view on carbohydrate fluxes. We conclude with a list of lessons and provide a solid foundation for future investigations into the role of EDA in cyanobacteria and other photoautotrophs. Significance statementThis study provides a retrospective on why, for many years, it was mistakenly assumed that the glycolytic Entner-Doudoroff (ED) pathway exists in the cyanobacterium Synechocystis sp. PCC 6803. It shows that the first enzyme of this pathway, ED dehydratase EDD is absent, while the second enzyme, 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase EDA, is present but is promiscuous, cleaving KDPG in addition to 2-keto-4-hydroxyglutarate (KHG) and decarboxylating oxaloacetate (OAA) in vitro. Finally, valuable lessons are drawn from prior misconceptions and experimental limitations. This study provides a solid foundation for future studies on the role of the ED aldolase in absence of the ED pathway in cyanobacteria and other photoautotrophs.

microbiology↗

bioRxiv2025

The small protein SbtC is a functional component of the CO2 concentrating mechanism of cyanobacteria

Oxygenic phototrophs fix CO2 via the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) that shows relatively low CO2 affinity and specificity. To circumvent low and fluctuating CO2 concentrations in aquatic systems, cyanobacteria and algae have evolved sophisticated inorganic carbon (Ci) concentrating mechanisms (CCMs). Bicarbonate transporters such as SbtA play a crucial role in the cyanobacterial CCM and hence are tightly regulated at multiple layers. The control of sbtA gene expression and corresponding transporter activity involves the PII-like protein SbtB, whose gene is frequently co-transcribed with sbtA. Here we report on the discovery of a so far non-annotated gene in the model Synechocystis sp. PCC 6803, which is located upstream of the sbtAB operon and encodes the small protein SbtC, composed of 80 amino acids. Presence of SbtC was confirmed by immunoblotting after fusing the sbtC-coding sequence to a Flag-tag. Similar to sbtAB, transcription of the sbtC locus is induced by low CO2 availability but controlled independently. Mutation of the sbtC locus in a wild-type background showed only a mild phenotype even under low CO2, but the diurnal growth was impaired as found before in the mutant {Delta}sbtB. Biochemical analysis provided evidence for a trimeric SbtABC complex in the membrane. Recombinant Synechocystis strains harboring only SbtA as single Ci uptake system with either deleted sbtB or sbtC genes showed that bicarbonate leakage from the cell was strongly elevated in both mutants. Our results provide evidence that SbtC contributes to the formation of the SbtAB complex, thereby regulating bicarbonate exchange at the cytoplasmic membrane. Well-conserved SbtC-like proteins encoded in the neighborhood of sbtAB exist in many cyanobacterial genomes pointing towards an important role in the cyanobacterial CCM.

microbiology↗

bioRxiv2025

Inter-strain diversity among coastal picocyanobacteria across salinity gradients

Strains of the picocyanobacteria genera Synechococcus and Cyanobium occur in cold-temperate zones from coastal waters to the open ocean, spanning a range of salinities and water depths. To thrive across different salinities, picocyanobacteria strains acclimate internal turgor by expending metabolic energy to accumulate compatible solutes and extrude inorganic ions. We grew five picocyanobacterial strains, derived from a range of source habitat salinities, under a matrix of light and salinity levels. Although Chl is a general proxy for phytoplankton biomass, Chl/cell is itself a key component of acclimation. Indeed, for all strains, except the brackish water CZS48M, the conditions supporting the highest Biovolume-specific growth rates shifted upwards towards higher salinities and PAR, compared to conditions for maximum Chl-specific growth. Therefore, the apparent optimal niche for a strain varies depending upon the metric used to track growth. Strains CZS48M and CZS25K derived from brackish lagoons of the Baltic Sea may be true brackobionts, with maximal growth at brackish salinities and high PAR, coinciding with their maximal metabolic capacities. CZS48M and CZS25K both also show high metabolism under higher salinity and low PAR, suggesting a situation of rapid metabolism, but lower achieved growth, under stress at the edges of their environmental tolerance ranges. Conversely, NIES981 (full marine, East China Sea) shows rapid metabolism under high, but also under low, salinities. The preferred salinity of each strain is consistent with its genome-encoded capabilities to synthesize different compatible solutes. In general, across strains, maximal metabolic rate is often offset from growth rate optima, showing conversion of metabolic electrons to biomass varies widely depending upon strain and condition, likely partly reflecting the costs of turgor regulation at the limits of salinity tolerance.

microbiology↗

bioRxiv2025

In vivo RUBISCO activity in Synechocystis is regulated by RuBP availability

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the main CO2-fixing enzyme on earth and entry point of carbon into the Calvin-Benson-Bassham cycle. Fueled by photosynthesis, C-assimilation by RUBISCO must be tightly controlled. RUBISCO regulation upon transition from light to darkness is not fully understood in the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Synechocystis does not have a RUBISCO activase that regulates RUBISCO activity in vascular plants by removing intrinsic sugar phosphate inhibitors from its active site. Instead, the regulatory CP12 protein of Synechocystis inactivates glyceraldehyde 3-phosphate dehydrogenase (GAPDH2) and phosphoribulokinase (PRK) during darkness. This mechanism indicates metabolic regulation of RUBISCO. We investigated C-assimilation in vivo at the transition to darkness by dynamic 13CO2 labeling experiments. We monitored RUBISCO activity by 13C-incorporation into 1-C position of 3PGA. Other than the wild type, the{Delta} cp12 mutant continued to assimilate 13CO2 into 3PGA in darkness. RUBISCO abundances and specific activities were not altered in{Delta} cp12 and upon light to dark transition. CP12 was required to shut down the CBB cycle during the night. Complementation of{Delta} cp12 by native CP12 ({Delta}cp12::cp12) and CP12 with mutated conserved cysteines in its GAPDH2- and PRK-binding domains ({Delta}cp12::cp12{Delta}Cys) showed that both native binding domains are required to fully inactivate the CBB cycle in the night. RuBP levels were highly elevated in{Delta} cp12 upon transition to darkness. Complementation with mutated and native CP12 variants gradually reduced RuBP to wild type levels and revealed highly significant correlation between RuBP concentration and the time-shifted 13C-uptake into 3PGA. We propose that RUBISCO activity in Synechocystis at day-night transition is regulated through depletion and blocked regeneration of RuBP. 13C-positional analyses of aspartate suggest regeneration of RuBP in{Delta} cp12 via dysregulated gluconeogenesis and the oxidative pentose phosphate path. We demonstrate that RUBISCO activity of Synechocystis is present throughout diurnal growth and depends on the availability of its substrate.

plant biology↗

bioRxiv2025

Fructose-1,6-bisphosphatase (FBPase) fine-tunes heterotrophic growth in cyanobacteria

Cyanobacteria switch their carbon metabolism between photoautotrophy and heterotrophy during diurnal cycles. In cyanobacteria, the classical glycolytic control point is characterized by two catabolic phosphofructokinases (PFKs) and a bifunctional anabolic fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (F/SBPase; slr2094) catalyzing two key reactions in the Calvin-Benson-Bassham (CBB) cycle. In addition, Synechocystis possesses a fructose-1,6-bisphosphatase (FBPase; slr0952) with yet unknown physiological function and biochemical properties. Hence, our aim was to investigate the FBPase and the interplay of the four enzymes in photoautotrophic and heterotrophic carbon metabolism. We discovered that FBPase is specific for FBP, showing no SBPase activity, and unlike F/SBPase does not exhibit any biochemical regulatory properties. Growth studies with deletion mutants revealed that FBPase and PFKs play a major role under heterotrophic conditions. In contrast to F/SBPase, FBPase is not involved in the CBB cycle, but instead fine-tunes heterotrophic growth. Transaldolase cannot replace the function of SBPase in the CBB cycle. In conclusion, the classical Embden-Meyerhoff-Parnass pathway control point, which is known to be mediated by the antagonistic enzyme pair PFK and FBPase in heterotrophic bacteria and eukaryotes, is also present in Synechocystis. We found redox-insensitive FBPases from plant chloroplasts to be closely related to Synechocystis FBPase, indicating that they might serve a similar function. HighlightSynechocystis fructose-1,6-bisphosphatase (slr0952) is unlike fructose-1,6-biphosphatase/sedoheptulose-1,7-biphosphatase (slr2094) monofunctional, not redox-regulated, and supports heterotrophy in darkness, despite catalyzing an anabolic reaction. Thus, presumably Slr2094 alone drives two Calvin-Benson-Bassham cycle key reactions.

plant biology↗

bioRxiv2025

Guard cell photorespiration has a major impact on photosynthesis, growth and stomatal behavior in Arabidopsis

Photorespiration is a mandatory metabolic repair shunt of carbon fixation by the Calvin-Benson (CB) cycle in oxygenic phototrophs. Its extent depends mainly on the CO2/O2 ratio in chloroplasts, which is regulated via stomatal movements. However, despite comprehensive understanding on the role of photorespiration in mesophyll cells (MC), its role in guard cells (GC) is yet unknown. To analyze this issue, the key enzyme of photorespiration, glycine decarboxylase (GDC), was manipulated through overexpression and antisense suppression of the GDC H-protein gene. A positive correlation of GDC-H expression with growth, photosynthesis and carbohydrate biosynthesis was observed in the transgenic lines, demonstrating active photorespiration is involved in stomatal regulation. This view is supported by gas exchange measurements showing that optimized GC photorespiration improves plant acclimation towards conditions requiring a high photorespiratory capacity, including high light. Microscopic analysis revealed that altered photorespiratory flux also affected starch accumulation patterns in GC, eventually serving as the underlying mechanistic for altered stomatal behavior. Collectively, our data suggest that photorespiration is a key component of the regulatory circuit that coordinates stomatal movements with external and internal CO2 availability. Thus, manipulation of photorespiration in GC has the potential to engineer crops maintaining growth and photosynthesis under future climates. One-sentence summaryGuard-cell-specific manipulation of photorespiratory glycine decarboxylase reveals photorespiration is active in guard cells and makes a major contribution to stomatal metabolism and movements.

plant biology↗

bioRxiv2024

Epigenetic control of tetrapyrrole biosynthesis by m4C DNA methylation in a cyanobacterium

Epigenetic DNA modifications are pivotal in eukaryotic gene expression, but their regulatory significance in bacteria is less understood. In Synechocystis 6803, the DNA methyltransferase M.Ssp6803II modifies the first cytosine in the GGCC motif, forming N4-methylcytosine (GGm4CC). Deleting the sll0729 gene ({Delta}sll0729) caused a bluish phenotype due to reduced chlorophyll levels, which was reversed by suppressor mutations. Re-sequencing of seven suppressor clones revealed a common GGCC to GGTC mutation in the slr1790 promoters discriminator sequence, encoding protoporphyrinogen IX oxidase, HemJ, crucial for tetrapyrrole biosynthesis. Transcriptomic and qPCR analyses indicated aberrant slr1790 expression in {Delta}sll0729 mutants. This aberration led to the accumulation of coproporphyrin III and protoporphyrin IX, indicative of impaired HemJ activity. To confirm the importance of DNA methylation in hemJ expression, native and mutated hemJ promoter variants were introduced into the wild type, followed by sll0729 deletion. The sll0729 deletion segregated in strains with the GGTC motif in the hemJ promoter, resulting in wild-type-like pigmentation, whereas freshly prepared {Delta}sll0729 mutants with the native hemJ promoter exhibited the bluish phenotype. These findings demonstrate that hemJ is tightly regulated in Synechocystis and that N4-methylcytosine is essential for proper hemJ expression.

microbiology↗

bioRxiv2024

Dynamic photosynthetic labelling and carbon-positional mass spectrometry monitor in vivo carbon assimilation rates by ribulose-1,5-bisphosphate carboxylase.

AbstractRibulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is the most abundant enzyme and CO2 bio-sequestration system on earth. Its in vivo activity is usually determined by 14CO2 incorporation into 3-phosphoglycerate (3PGA). The radiometric analysis of 3PGA does not distinguish carbon positions. Hence, RUBISCO activity that fixes carbon into 1-C position of 3PGA and Calvin-Benson-Bassham (CBB) cycle activities that redistribute carbon into its 2-C and 3-C positions are not resolved. This study aims to provide technology that differentiates between these activities. In source fragmentation of gas chromatography-mass spectrometry (GC- MS) enables paired isotopologue distribution analyses of fragmented substructures and the complete metabolite structure. GC-MS measurements after dynamic photosynthetic 13CO2 labelling allowed quantification of the 13C fractional enrichment (E13C) and molar carbon assimilation rates (A13C) at carbon position 1-C of 3PGA by combination of E13C from carbon positions 2,3-C2 and 1,2,3-C3 with quantification of 3PGA concentrations. We validated the procedure using two GC-time of flight (TOF)-MS instruments, operated at nominal or high mass resolution and tested expected positional labelling of 3PGA by in vivo glycolysis of positional labelled glucose isotopomers. Application to{Delta} gapdh1 and{Delta} gapdh2 mutants of the highly divergent glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Synechocystis sp. PCC 6803 revealed full inactivation of the CBB cycle with maintained RUBISCO activity in the{Delta} gapdh2 mutant and a CBB cycle modulating role of GAPDH1 under fluctuating CO2 supply. RUBISCO activity in the CBB-deficient{Delta} gapdh2 mutant can re-assimilate CO2 released by catabolic pathways. We suggest that RUBISCO activity in Synechocystis can scavenge carbon loss through the pentose phosphate pathway or other cellular decarboxylation reactions.

plant biology↗

bioRxiv2024

Positional 13C Enrichment Analysis of Aspartate by GC-MS to Determine PEPC Activity In Vivo

Photoautotrophic organisms fix inorganic carbon (Ci) by two enzymes, ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase (PEPC). RUBISCO assimilates Ci (CO2) into the 1-C position of 3-phosphoglycerate (3PGA). The Calvin-Benson-Basham (CBB) cycle redistributes fixed carbon atoms into 2,3-C2 of the same molecule. PEPC uses phosphoenolpyruvate (PEP) derived from 3PGA and assimilates Ci (HCO3-) into 4-C of oxaloacetate (OAA). 1,2,3-C3 of OAA and of its transaminase product aspartate originate directly from 1,2,3-C3 of 3PGA. Positional isotopologue analysis of aspartate, the main downstream metabolite of OAA in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), allows differentiation between PEPC, RUBISCO, and CBB cycle activities within one molecule. We explored in source fragmentation of gas chromatography-electron impact ionization-mass spectrometry (GC-EI-MS) at nominal mass resolution and GC-atmospheric pressure chemical ionization-MS (GC-APCI-MS) at high mass resolution. This enabled the determination of fractional 13C enrichment (E13C) at each carbon position of aspartate. Two prevailing GC-MS derivatization methods, i.e. trimethylsilylation and tert-butyldimethylsilylation, were evaluated. The method was validated by 13C-isotopomer mixtures of positional labeled aspartic acid. Combination with dynamic 13CO2 labeling of Synechocystis cultures allowed direct measurements of PEPC activity in vivo alongside analyses of RUBISCO and CBB cycle activities. Accurate quantification of aspartate concentration and positional E13C provided molar Ci assimilation rates during the day and night phases of photoautotrophic Synechocystis cultures. The validated method offers several applications to characterize the photosynthetic Ci fixation in different organisms.

biochemistry↗

bioRxiv2024

Using gene complementation to identify a SulP-family bicarbonate transporter in an N2-fixing cyanobacterial endosymbiont of an open ocean diatom

Diatom-Diazotrophic Associations (DDAs) contribute significantly to new and primary production in the worlds oceans, yet the understanding of how production is sustained is poorly resolved. These symbioses involve diatoms and N2-fixing, heterocyst-forming cyanobacteria of the genus Richelia, both partners being photosynthetic. Richelia euintracellularis resides in the cytoplasm of Hemiaulus hauckii, whereas Richelia intracellularis is periplasmic in Rhizosolenia clevei. In the ocean, bicarbonate is taken up by phytoplankton to provide CO2 for photosynthesis. The genomes of both Richelia endobionts (ReuHH01 and RintRC01, respectively) contain genes encoding SulP-family proteins, which are oxyanion transporters. To study the possible involvement of these transporters in bicarbonate uptake, we used complementation of a Synechocystis sp. PCC 6803 mutant with its five CO2 uptake systems inactivated, which is unable to grow in air levels of CO2. Three genes from RintRC01 and one gene and a DNA fragment containing four partial gene sequences from ReuHH01 were chemically synthesized, cloned under the control of a strong gene promoter and incorporated in the chromosome of the Synechocystis mutant. One gene from RintRC01, RintRC_3892, complemented the Synechocystis mutant to grow with air levels of CO2 or with low bicarbonate concentrations. The complemented strain showed strong sodium-dependent, low affinity bicarbonate uptake, which, together with phylogenetic analyses, identified RintRC_3892 as a BicA protein. Additionally, RintRC_3892 transcripts were consistently detected in environmental samples from three ocean basins. No evidence for a bicarbonate transporter was found, however, for ReuHH01, suggesting different strategies for inorganic carbon uptake in the periplasmic and cytoplasmic endobionts.

microbiology↗

bioRxiv2023

Diurnal rhythm causes metabolic crises in the cyanobacterial mutants of c-di-AMP signalling cascade

In nature, the photoautotrophic lifestyle of cyanobacteria has to cope with the successive diurnal changes in light supply. Light supply throughout the day enables photosynthesis and glycogen biosynthesis, while night phases require the switch to a heterotrophic-like lifestyle relying on glycogen catabolism. We previously highlighted a unique function of the carbon control protein, SbtB, and its effector molecule c-di-AMP, for the nighttime survival of cyanobacteria through the regulation of glycogen anabolism. However, the extent to which c-di-AMP and SbtB impact the cellular metabolism for day-night survivability remained elusive. To gain better understanding of cellular processes regulated by SbtB or c-di-AMP, we compared the metabolomic and proteomic landscapes of {Delta}sbtB and the c-di-AMP-free ({Delta}dacA) mutants of the model strain Synechocystis sp. PCC 6803. While our results indicate that the cellular role of SbtB is restricted to carbon/glycogen metabolism, the diurnal lethality of {Delta}dacA seems to be a sum of dysregulation of multiple metabolic processes. These processes include photosynthesis and redox regulation, which lead to elevated levels of intracellular ROS and glutathione. Further, we show an impact of c-di-AMP on central carbon as well as on nitrogen metabolism. Effects on nitrogen metabolism are linked to reduced levels of the global nitrogen transcription regulator NtcA and highlighted by an imbalance of the glutamine to glutamate ratio as well as reduced metabolite levels of the arginine pathway. We further identified the HCO3- uptake systems, BicA and BCT1 as novel SbtB targets, in agreement with its broader role in regulating carbon homeostasis.

microbiology↗

bioRxiv2023

PII signal transduction superfamily acts as a valve plug to control bicarbonate and ammonia homeostasis among different bacterial phyla

Life on Earth relies on carbon and nitrogen assimilation by RubisCO and GS-GOGAT enzymes, respectively, whose activities depend on a constant supply of inorganic carbon (Ci) and nitrogen (N). Members of the PII signal transduction superfamily are among the most ancient and widespread cell signaling proteins in nature. One of their most highly conserved functions is controlling Ci- and N-transporters, a feature found in different phyla of the Archaea and in both Gram-positive and Gram-negative bacteria. Recently, we identified the PII-like protein SbtB as Ci-sensing module, mainly controlling the HCO3- transporter SbtA in cyanobacteria. Similar to canonical PII proteins, SbtB is able to bind the adenine nucleotides ATP and ADP. Unlike those, it also binds AMP and preferentially binds the second messenger cAMP and c-di-AMP. The functional significance of the binding of different adenyl-nucleotides to SbtB has remained elusive, particularly in the context of the interaction of SbtB with SbtA. By a combination of structural, biochemical and physiological analysis, we revealed that by binding to SbtA, SbtB acts as unidirectional valve, preventing the reverse transport of intracellular enriched bicarbonate. This mechanistic principle holds true for the PII protein from Bacillus acting on the ammonium transporter AmtB, suggesting an evolutionary conserved role for PII superfamily proteins in controlling unidirectional flow of different transporters.

microbiology↗

bioRxiv2023

Nitrite secretion by cyanobacteria is controlled by the small protein NirP1

When the supply of inorganic carbon is limiting, photosynthetic cyanobacteria excrete nitrite, a toxic intermediate in the ammonia assimilation pathway from nitrate. While it has been hypothesized that the secreted nitrite represents excess nitrogen that could not be further assimilated due to the missing carbon, the underlying molecular mechanism has remained enigmatic. Here, we characterized a hitherto unannotated gene with homologs in the genomes of 485 different cyanobacteria that is upregulated under low carbon conditions and controlled by the transcription factor NtcA, a central regulator of nitrogen homeostasis. To understand its function, we ectopically overexpressed it in Synechocystis sp. PCC 6803, which resulted in a chlorotic phenotype, delayed growth, severe changes in amino acid pools, and nitrite excretion. Coimmunoprecipitation experiments revealed that this protein targets nitrite reductase, a central enzyme in the assimilation of ammonia from nitrate/nitrite, and was re-named to nitrite reductase regulator protein 1 (NirP1). Our results reveal that NirP1 is widely conserved in cyanobacteria and plays a crucial role in the coordination of C/N primary metabolism by targeting one of the central enzymes. In natural environments, the excreted nitrite will be utilized by other microorganisms; therefore, NirP1 ultimately impacts the activities and composition of the surrounding microbiome.

microbiology↗

bioRxiv2023

Photosynthetic response of Chara braunii towards different bicarbonate concentrations

A variety of inorganic carbon acquisition modes have been proposed in Characean algae, however, the specific inorganic carbon uptake mechanism is unknown for the genus Chara. In the present study, we analyzed if C. braunii can efficiently use HCO3- as a carbon source for photosynthesis. For this purpose, C. braunii was exposed to different concentrations of NaHCO3- for different time scales. The photosynthetic electron transport through photosystem I (PSI) and II (PSII), the maximal electron transport rate (ETRmax), the efficiency of the electron transport rate (, the initial slope of the ETR), and the light saturation point of photosynthesis (Ek) were evaluated. Additionally, pigment contents (chlorophyll a, chlorophyll b, and carotenoids) were determined. Bicarbonate addition positively affected ETRmax after direct HCO3- application of both PSII and PSI, but this effect decreased after 1 h and 24 h. Similar trends were seen for Ek, but no significant effect was observed for . Pigment contents showed no significant changes in relation to different HCO3- concentrations. To evaluate if cyclic electron flow around PSI was involved in active HCO3- uptake, the ratio of PSI ETRmax/PSII ETRmax was calculated but did not show a distinctive trend. These results suggest that C. braunii can utilize NaHCO3- in short time periods as a carbon source but relies on other carbon acquisition mechanisms over prolonged time periods. These observations differentiate C. braunii from other examined Chara spp. and suggest a minor direct role of HCO3- as a carbon source for photosynthesis in this alga.

plant biology↗

bioRxiv2022

Operation of Carbon-Concentrating Mechanisms in Cyanobacteria and Algae requires altered poising of the Calvin-Benson cycle

Cyanobacteria and eukaryotic algae make a major contribution to global photosynthetic productivity. To cope with the low availability of CO2 in aqueous systems they deploy inorganic carbon-concentrating mechanisms (CCMs). These concentrate CO2 in microcompartments that contain Rubisco (carboxysomes in cyanobacteria; pyrenoids in green algae). The rest of the Calvin-Benson cycle (CBC) is located outside these microcompartments. We hypothesized that this physical separation requires modified poising of the CBC. Hence, Rubisco is physically separated from the other CBC enzymes outside these microcompartments. To test the hypothesis that this physical separation requires appropriate poising of the CBC, we profiled CBC metabolites under ambient CO2 in the cyanobacterium Synechocystis sp. PCC 6803 and three eukaryotic algae (Chlamydomonas reinhardtii, Chlorella sorokiniana, Chlorella ohadii). Comparison with recently reported profiles for a large set of terrestrial plants revealed that cyanobacteria and green algae have very distinctive CBC metabolite profiles, with low levels of pentose phosphates and, especially, high levels of ribulose 1,5-bisphosphate and 3-phosphoglycerate. We propose that large pools of the substrate and product of Rubisco are required to generate concentration gradients that drive movement into and out of the microcompartments. These observations raise questions about how CBC regulation was modified during the evolution of algal CCMs and their subsequent loss in terrestrial plants, and highlight that operation of CCMs requires co-evolution of the CBC. HighlightCBC metabolite profiles in the cyanobacterium Synechocystis and in three eukaryotic green algae at ambient CO2 concentration are very different to those in terrestrial plants, probably reflecting the operation of a carboxysome- or pyrenoid-based carbon concentrating mechanism.

biochemistry↗