Isabel Orf

Recent Applications of Metabolomics Toward Cyanobacteria

Our knowledge on cyanobacterial molecular biology increased tremendously by the application of the “omics” techniques. Only recently, metabolomics was applied systematically to model cyanobacteria. Metabolomics, the quantitative estimation of ideally the complete set of cellular metabolites, is particularly well suited to mirror cellular metabolism and its flexibility under diverse conditions. Traditionally, small sets of metabolites are quantified in targeted metabolome approaches. The development of separation technologies coupled to mass-spectroscopy- or nuclear-magnetic-resonance-based identification of low molecular mass molecules presently allows the profiling of hundreds of metabolites of diverse chemical nature. Metabolome analysis was applied to characterize changes in the cyanobacterial primary metabolism under diverse environmental conditions or in defined mutants. The resulting lists of metabolites and their steady state concentrations in combination with transcriptomics can be used in system biology approaches. The application of stable isotopes in fluxomics, i.e. the quantitative estimation of carbon and nitrogen fluxes through the biochemical network, has only rarely been applied to cyanobacteria, but particularly this technique will allow the making of kinetic models of cyanobacterial systems. The further application of metabolomics in the concert of other “omics” technologies will not only broaden our knowledge, but will also certainly strengthen the base for the biotechnological application of cyanobacteria.

Integrated transcriptomic and metabolomic characterization of the low-carbon response using an ndhR mutant of Synechocystis sp. PCC 6803

Acclimation to changing CO2 levels by Synechocystis sp. PCC 6803 includes coordinated changes in gene expression and metabolism, whereby small RNAs and the transcriptional regulator protein NdhR perform distinct regulatory functions. The acquisition and assimilation of inorganic carbon (Ci) represents the largest flux of inorganic matter in photosynthetic organisms; hence, this process is tightly regulated. We examined the Ci-dependent transcriptional and metabolic regulation in wild-type Synechocystis sp. PCC 6803 compared with a mutant defective in the main transcriptional repressor for Ci acquisition genes, the NAD(P)H dehydrogenase transcriptional regulator NdhR. The analysis revealed that many protein-coding transcripts that are normally repressed in the presence of high CO2 (HC) concentrations were strongly expressed in ∆ndhR, whereas other messenger RNAs were strongly down-regulated in mutant cells, suggesting a potential activating role for NdhR. A conserved NdhR-binding motif was identified in the promoters of derepressed genes. Interestingly, the expression of some NdhR-regulated genes remained further inducible under low-CO2 conditions, indicating the involvement of additional NdhR-independent Ci-regulatory mechanisms. Intriguingly, we also observed that the abundance of 52 antisense RNAs and 34 potential noncoding RNAs was affected by Ci supply, although most of these molecules were not regulated through NdhR. Thus, antisense and noncoding RNAs could contribute to NdhR-independent carbon regulation. In contrast to the transcriptome, the metabolome in ∆ndhR cells was similar to that of wild-type cells under HC conditions. This observation and the delayed metabolic responses to the low-CO2 shift in ∆ndhR, specifically the lack of transient increases in the photorespiratory pathway intermediates 2-phosphoglycolate, glycolate, and glycine, suggest that the deregulation of gene expression in the ΔndhR mutant successfully preacclimates cyanobacterial cells to lowered Ci supply under HC conditions.

Does 2-phosphoglycolate serve as an internal signal molecule of inorganic carbon deprivation in the cyanobacterium Synechocystis sp. PCC 6803?

Cyanobacteria possess CO2 -concentrating mechanisms (CCM) that functionally compensate for the poor affinity of their ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to CO2 . It was proposed that 2-phosphoglycolate (2PG), produced by the oxygenase activity of Rubisco and metabolized via photorespiratory routes, serves as a signal molecule for the induction of CCM-related genes under limiting CO2 level (LC) conditions. However, in vivo evidence is still missing. Since 2PG does not permeate the cells, we manipulated its internal concentration. Four putative phosphoglycolate phosphatases (PGPases) encoding genes (slr0458, sll1349, slr0586 and slr1762) were identified in the cyanobacterium Synechocystis PCC 6803. Expression of slr0458 in Escherichia coli led to a significant rise in PGPase activity. A Synechocystis mutant overexpressing (OE) slr0458 was constructed. Compared with the wild type (WT), the mutant grew slower under limiting CO2 concentration and the intracellular 2PG level was considerably smaller than in the wild type, the transcript abundance of LC-induced genes including cmpA, sbtA and ndhF3 was reduced, and the OE cells acclimated slower to LC - indicated by the delayed rise in the apparent photosynthetic affinity to inorganic carbon. Data obtained here implicated 2PG in the acclimation of this cyanobacterium to LC but also indicated that other, yet to be identified components, are involved.

Profiling Methods to Identify Cold-Regulated Primary Metabolites Using Gas Chromatography Coupled to Mass Spectrometry

This book chapter describes the analytical procedures required for the profiling of a metabolite fraction enriched for primary metabolites. The profiling is based on routine gas chromatography coupled to mass spectrometry (GC-MS). The generic profiling method is adapted to plant material, specifically to the analysis of single leaves from plants that were exposed to temperature stress experiments. The described method is modular. The modules include a rapid sampling and metabolic inactivation protocol for samples in a wide size range, a sample extraction procedure, a chemical derivatization step that is required to make the metabolite fraction amenable to gas chromatographic analysis, a routine GC-MS method, and finally the procedures of data processing and data mining. A basic and extendable set of standardizations for metabolite recovery and retention index alignment of the resulting GC-MS chromatograms is included. The method has two applications: (1) the rapid screening for changes of relative metabolite pools sizes under temperature stress and (2) the verification of cold-regulated metabolites by exact quantification using a GC-MS protocol with extended internal and external standardization.

Effects of Inorganic Carbon Limitation on the Metabolome of the Synechocystis sp. PCC 6803 Mutant Defective in glnB Encoding the Central Regulator PII of Cyanobacterial C/N Acclimation

Cyanobacteria are the only prokaryotes performing oxygenic photosynthesis. Non-diazotrophic strains such as the model Synechocystis sp. PCC 6803 depend on a balanced uptake and assimilation of inorganic carbon and nitrogen sources. The internal C/N ratio is sensed via the PII protein (GlnB). We analyzed metabolic changes of the ΔglnB mutant of Synechocystis sp. PCC 6803 under different CO2 availability. The identified metabolites provided a snapshot of the central C/N metabolism. Cells of the ΔglnB mutant shifted to carbon-limiting conditions, i.e. a decreased C/N ratio, showed changes in intermediates of the sugar storage and particularly of the tricarboxylic acid cycle, arginine, and glutamate metabolism. The changes of the metabolome support the notion that the PII protein is primarily regulating the N-metabolism whereas the changes in C-metabolism are probably secondary effects of the PII deletion.

Integrated Analysis of Engineered Carbon Limitation in a Quadruple CO2/HCO3− Uptake Mutant of Synechocystis sp. PCC 6803

Engineered carbon limitation after deletion of four Ci-uptake systems in Synechocystis sp. PCC 6803 is compensated by an extensive phenocopy of wild-type acclimation to low CO2 and multilayered Ci regulation. Cyanobacteria have efficient carbon concentration mechanisms and suppress photorespiration in response to inorganic carbon (Ci) limitation. We studied intracellular Ci limitation in the slow-growing CO2/HCO3−-uptake mutant ΔndhD3 (for NADH dehydrogenase subunit D3)/ndhD4 (for NADH dehydrogenase subunit D4)/cmpA (for bicarbonate transport system substrate-binding protein A)/sbtA (for sodium-dependent bicarbonate transporter A): Δ4 mutant of Synechocystis sp. PCC 6803. When cultivated under high-CO2 conditions, ∆4 phenocopies wild-type metabolic and transcriptomic acclimation responses after the shift from high to low CO2 supply. The ∆4 phenocopy reveals multiple compensation mechanisms and differs from the preacclimation of the transcriptional Ci regulator mutant ∆ndhR (for ndhF3 operon transcriptional regulator). Contrary to the carboxysomeless ∆ccmM (for carbon dioxide concentrating mechanism protein M) mutant, the metabolic photorespiratory burst triggered by shifting to low CO2 is not enhanced in ∆4. However, levels of the photorespiratory intermediates 2-phosphoglycolate and glycine are increased under high CO2. The number of carboxysomes is increased in ∆4 under high-CO2 conditions and appears to be the major contributing factor for the avoidance of photorespiration under intracellular Ci limitation. The ∆4 phenocopy is associated with the deregulation of Ci control, an overreduced cellular state, and limited photooxidative stress. Our data suggest multiple layers of Ci regulation, including inversely regulated modules of antisense RNAs and cognate target messenger RNAs and specific trans-acting small RNAs, such as the posttranscriptional PHOTOSYNTHESIS REGULATORY RNA1 (PsrR1), which shows increased expression in ∆4 and is involved in repressing many photosynthesis genes at the posttranscriptional level. In conclusion, our insights extend the knowledge on the range of compensatory responses of Synechocystis sp. PCC 6803 to intracellular Ci limitation and may become a valuable reference for improving biofuel production in cyanobacteria, in which Ci is channeled off from central metabolism and may thus become a limiting factor.

Can cyanobacteria serve as a model of plant photorespiration? – a comparative meta-analysis of metabolite profiles

Photorespiration is a process that is crucial for the survival of oxygenic phototrophs in environments that favour the oxygenation reaction of Rubisco. While photorespiration is conserved among cyanobacteria, algae, and embryophytes, it evolved to different levels of complexity in these phyla. The highest complexity is found in embryophytes, where the pathway involves four cellular compartments and respective transport processes. The complexity of photorespiration in embryophytes raises the question whether a simpler system, such as cyanobacteria, may serve as a model to facilitate our understanding of the common key aspects of photorespiration. In this study, we conducted a meta-analysis of publicly available metabolite profiles from the embryophyte Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 grown under conditions that either activate or suppress photorespiration. The comparative meta-analysis evaluated the similarity of metabolite profiles, the variability of metabolite pools, and the patterns of metabolite ratios. Our results show that the metabolic signature of photorespiration is in part conserved between the compared model organisms under conditions that favour the oxygenation reaction. Therefore, our findings support the claim that cyanobacteria can serve as prokaryotic models of photorespiration in embryophytes.

Integrated Analysis of Engineered Carbon Limitation in a Quadruple CO2/HCO3- Uptake Mutant of Synechocystis sp. PCC 6803.

Engineered carbon limitation after deletion of four Ci-uptake systems in Synechocystis sp. PCC 6803 is compensated by an extensive phenocopy of wild-type acclimation to low CO2 and multilayered Ci regulation. Cyanobacteria have efficient carbon concentration mechanisms and suppress photorespiration in response to inorganic carbon (Ci) limitation. We studied intracellular Ci limitation in the slow-growing CO2/HCO3−-uptake mutant ΔndhD3 (for NADH dehydrogenase subunit D3)/ndhD4 (for NADH dehydrogenase subunit D4)/cmpA (for bicarbonate transport system substrate-binding protein A)/sbtA (for sodium-dependent bicarbonate transporter A): Δ4 mutant of Synechocystis sp. PCC 6803. When cultivated under high-CO2 conditions, ∆4 phenocopies wild-type metabolic and transcriptomic acclimation responses after the shift from high to low CO2 supply. The ∆4 phenocopy reveals multiple compensation mechanisms and differs from the preacclimation of the transcriptional Ci regulator mutant ∆ndhR (for ndhF3 operon transcriptional regulator). Contrary to the carboxysomeless ∆ccmM (for carbon dioxide concentrating mechanism protein M) mutant, the metabolic photorespiratory burst triggered by shifting to low CO2 is not enhanced in ∆4. However, levels of the photorespiratory intermediates 2-phosphoglycolate and glycine are increased under high CO2. The number of carboxysomes is increased in ∆4 under high-CO2 conditions and appears to be the major contributing factor for the avoidance of photorespiration under intracellular Ci limitation. The ∆4 phenocopy is associated with the deregulation of Ci control, an overreduced cellular state, and limited photooxidative stress. Our data suggest multiple layers of Ci regulation, including inversely regulated modules of antisense RNAs and cognate target messenger RNAs and specific trans-acting small RNAs, such as the posttranscriptional PHOTOSYNTHESIS REGULATORY RNA1 (PsrR1), which shows increased expression in ∆4 and is involved in repressing many photosynthesis genes at the posttranscriptional level. In conclusion, our insights extend the knowledge on the range of compensatory responses of Synechocystis sp. PCC 6803 to intracellular Ci limitation and may become a valuable reference for improving biofuel production in cyanobacteria, in which Ci is channeled off from central metabolism and may thus become a limiting factor.

Metabolome and Lipidome Profiles of Populus × canescens Twig Tissues During Annual Growth Show Phospholipid-Linked Storage and Mobilization of C, N, and S

The temperate climax tree species Fagus sylvatica and the floodplain tree species Populus × canescens possess contrasting phosphorus (P) nutrition strategies. While F. sylvatica has been documented to display P storage and mobilization (Netzer et al., 2017), this was not observed for Populus × canescens (Netzer et al., 2018b). Nevertheless, changes in the abundance of organic bound P in gray poplar trees indicated adaptation of the P nutrition to different needs during annual growth. The present study aimed at characterizing seasonal changes in metabolite and lipid abundances in gray poplar and uncovering differences in metabolite requirement due to specific needs depending on the season. Seasonal variations in the abundance of (i) sugar-Ps and phospholipids, (ii) amino acids, (iii) sulfur compounds, and (iv) carbon metabolites were expected. It was hypothesized that seasonal changes in metabolite levels relate to N, S, and C storage and mobilization. Changes in organic metabolites binding Pi (Porg) are supposed to support these processes. Variation in triacylglycerols, in sugar-phosphates, in metabolites of the TCA cycle and in the amino acid abundance of poplar twig buds, leaves, bark, and wood were found to be linked to changes in metabolite abundances as well as to C, N, and S storage and mobilization processes. The observed changes support the view of a lack of any P storage in poplar. Yet, during dormancy, contents of phospholipids in twig bark and wood were highest probably due to frost-hardening and to its function in extra-plastidic membranes such as amyloplasts, oleosomes, and protein bodies. Consistent with this assumption, in spring sugar-Ps increased when phospholipids declined and poplar plants entering the vegetative growth period and, hence, metabolic activity increases. These results indicate that poplar trees adopt a policy of P nutrition without P storage and mobilization that is different from their N- and S-nutrition strategies.