Martin Hagemann

Ecological Genomics of Marine Picocyanobacteria

SUMMARY Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45°N to 40°S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.

Photorespiration: players, partners and origin.

Photorespiratory metabolism allows plants to thrive in a high-oxygen containing environment. This metabolic pathway recycles phosphoglycolate, a toxic compound, back to phosphoglycerate, when oxygen substitutes for carbon dioxide in the first reaction of photosynthetic carbon fixation. The recovery of phosphoglycerate is accompanied by considerable carbon and energy losses, making photorespiration a prime target for crop improvement. The genomics era has allowed the precise functional analysis of individual reaction steps of the photorespiratory cycle, and more links integrating photorespiration with cellular metabolism as a whole are becoming apparent. Here we review the evolutionary origins of photorespiration as well as new insights into the interaction with other metabolic processes such as nitrogen assimilation and mitochondrial respiration.

The Cyanobacterial Hepatotoxin Microcystin Binds to Proteins and Increases the Fitness of Microcystis under Oxidative Stress Conditions

Microcystins are cyanobacterial toxins that represent a serious threat to drinking water and recreational lakes worldwide. Here, we show that microcystin fulfils an important function within cells of its natural producer Microcystis. The microcystin deficient mutant ΔmcyB showed significant changes in the accumulation of proteins, including several enzymes of the Calvin cycle, phycobiliproteins and two NADPH-dependent reductases. We have discovered that microcystin binds to a number of these proteins in vivo and that the binding is strongly enhanced under high light and oxidative stress conditions. The nature of this binding was studied using extracts of a microcystin-deficient mutant in vitro. The data obtained provided clear evidence for a covalent interaction of the toxin with cysteine residues of proteins. A detailed investigation of one of the binding partners, the large subunit of RubisCO showed a lower susceptibility to proteases in the presence of microcystin in the wild type. Finally, the mutant defective in microcystin production exhibited a clearly increased sensitivity under high light conditions and after hydrogen peroxide treatment. Taken together, our data suggest a protein-modulating role for microcystin within the producing cell, which represents a new addition to the catalogue of functions that have been discussed for microbial secondary metabolites.

The photorespiratory glycolate metabolism is essential for cyanobacteria and might have been conveyed endosymbiontically to plants

Photorespiratory 2-phosphoglycolate (2PG) metabolism is essential for photosynthesis in higher plants but thought to be superfluous in cyanobacteria because of their ability to concentrate CO2 internally and thereby inhibit photorespiration. Here, we show that 3 routes for 2PG metabolism are present in the model cyanobacterium Synechocystis sp. strain PCC 6803. In addition to the photorespiratory C2 cycle characterized in plants, this cyanobacterium also possesses the bacterial glycerate pathway and is able to completely decarboxylate glyoxylate via oxalate. A triple mutant with defects in all 3 routes of 2PG metabolism exhibited a high-CO2-requiring (HCR) phenotype. All these catabolic routes start with glyoxylate, which can be synthesized by 2 different forms of glycolate dehydrogenase (GlcD). Mutants defective in one or both GlcD proteins accumulated glycolate under high CO2 level and the double mutant ΔglcD1/ΔglcD2 was unable to grow under low CO2. The HCR phenotype of both the double and the triple mutant could not be attributed to a significantly reduced affinity to CO2, such as in other cyanobacterial HCR mutants defective in the CO2-concentrating mechanism (CCM). These unexpected findings of an HCR phenotype in the presence of an active CCM indicate that 2PG metabolism is essential for the viability of all organisms that perform oxygenic photosynthesis, including cyanobacteria and C3 plants, at ambient CO2 conditions. These data and phylogenetic analyses suggest cyanobacteria as the evolutionary origin not only of oxygenic photosynthesis but also of an ancient photorespiratory 2PG metabolism.

Identification of histidine kinases that act as sensors in the perception of salt stress in Synechocystis sp. PCC 6803

In plants and microorganisms, salt stress regulates the expression of large numbers of genes. However, the machinery that senses salt stress remains to be characterized. In this study we identified sensory histidine kinases that are involved in the perception of salt stress in the cyanobacterium Synechocystis sp. strain PCC 6803. A library of strains with mutations in all 43 histidine kinases was screened by DNA microarray analysis of genomewide gene expression under salt stress. The results suggested that four histidine kinases, namely, Hik16, Hik33, Hik34, and Hik41, perceived and transduced salt signals. However, Hik33, Hik34, and Hik16 acting with Hik41 regulated the expression of different sets of genes. These histidine kinases regulated the expression of ≈20% of the salt-inducible genes, whereas the induction of the remaining salt-inducible genes was unaffected by mutations in any of the histidine kinases, suggesting that additional sensory mechanisms might operate in the perception of salt stress. We also used DNA microarrays to investigate the effect of various salts on gene expression. Our results indicate that Hik33 responds to sodium salts and not to KCl, whereas the Hik16/Hik41 system responds only to NaCl.

Stenotrophomonas rhizophila sp. nov., a novel plant-associated bacterium with antifungal properties.

A polyphasic taxonomic study was performed on 16 Stenotrophomonas strains from environmental and clinical sources. A group of three plant-associated isolates were shown to be phenotypically different from the other strains. This group formed a separate physiological cluster (B1) with 42% heterogeneity to the other isolates. The defining characteristics of the new species were as follows: growth at 4 degrees C and the absence of growth at 40 degrees C; the utilization of xylose as a carbon source; lower osmolytic tolerance (< 4.5% NaCl, w/v), although the isolates can produce trehalose and glucosylglycerol as osmoprotective substances; the absence of lipase and beta-glucosidase production; and antifungal activity against plant-pathogenic fungi. The whole-cell fatty acid profile of this group was different and characterized by the main fatty acids iso-C15:0 and anteiso-C15:0. Numerical analysis of the fatty acid profiles of the strains examined supports the differentiation of the physiological B1 group. By 16S rDNA analysis, three clusters were distinguished. The three strains of the B1 group formed a separate environmental cluster (E1). They showed a mean similarity of 99.5% within the cluster, and differed from strains of a second environmental cluster (E2) by 2.2% and from the clinical cluster (C) by about 3.0%. DNA-DNA hybridization data supported the taxonomic differentiation. All results led to the proposal of a new species, Stenotrophomonas rhizophila sp. nov., with strain e-p10(T) (= DSM 14405(T) = ATCC BAA-473(T)) as the type strain.

Compatible solute biosynthesis in cyanobacteria

Compatible solutes are a functional group of small, highly soluble organic molecules that demonstrate compatibility in high amounts with cellular metabolism. The accumulation of compatible solutes is often observed during the acclimation of organisms to adverse environmental conditions, particularly to salt and drought stress. Among cyanobacteria, sucrose, trehalose, glucosylglycerol and glycine betaine are used as major compatible solutes. Interestingly, a close correlation has been discovered between the final salt tolerance limit and the primary compatible solute in these organisms. In addition to the dominant compatible solutes, many strains accumulate mixtures of these compounds, including minor compounds such as glucosylglycerate or proline as secondary or tertiary solutes. In particular, the accumulation of sucrose and trehalose results in an increase in tolerance to general stresses such as desiccation and high temperatures. During recent years, the biochemical and molecular basis of compatible solute accumulation has been characterized using cyanobacterial model strains that comprise different salt tolerance groups. Based on these data, the distribution of genes involved in compatible solute synthesis among sequenced cyanobacterial genomes is reviewed, and thereby, the major compatible solutes and potential salt tolerance of these strains can be predicted. Knowledge regarding cyanobacterial salt tolerance is not only useful to characterize strain-specific adaptations to ecological niches, but it can also be used to generate cells with increased tolerance to adverse environmental conditions for biotechnological purposes.

d-GLYCERATE 3-KINASE, the Last Unknown Enzyme in the Photorespiratory Cycle in Arabidopsis, Belongs to a Novel Kinase Family

d-GLYCERATE 3-KINASE (GLYK; EC 2.7.1.31) catalyzes the concluding reaction of the photorespiratory C2 cycle, an indispensable ancillary metabolic pathway to the photosynthetic C3 cycle that enables land plants to grow in an oxygen-containing atmosphere. Except for GLYK, all other enzymes that contribute to the C2 cycle are known by their primary structures, and the encoding genes have been identified. We have purified and partially sequenced this yet missing enzyme from Arabidopsis thaliana and identified it as a putative kinase-annotated single-copy gene At1g80380. The exclusive catalytic properties of the gene product were confirmed after heterologous expression in Escherichia coli. Arabidopsis T-DNA insertional knockout mutants show no GLYK activity and are not viable in normal air; however, they grow under elevated CO2, providing direct evidence of the obligatory nature of the ultimate step of the C2 cycle. The newly identified GLYK is both structurally and phylogenetically distinct from known glycerate kinases from bacteria and animals. Orthologous enzymes are present in other plants, fungi, and some cyanobacteria. The metabolic context of GLYK activity in fungi and cyanobacteria remains to be investigated.

Gene expression profiling reflects physiological processes in salt acclimation of Synechocystis sp. strain PCC 6803.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

The Plant-Like C2 Glycolate Cycle and the Bacterial-Like Glycerate Pathway Cooperate in Phosphoglycolate Metabolism in Cyanobacteria

The occurrence of a photorespiratory 2-phosphoglycolate metabolism in cyanobacteria is not clear. In the genome of the cyanobacterium Synechocystis sp. strain PCC 6803, we have identified open reading frames encoding enzymes homologous to those forming the plant-like C2 cycle and the bacterial-type glycerate pathway. To study the route and importance of 2-phosphoglycolate metabolism, the identified genes were systematically inactivated by mutagenesis. With a few exceptions, most of these genes could be inactivated without leading to a high-CO2-requiring phenotype. Biochemical characterization of recombinant proteins verified that Synechocystis harbors an active serine hydroxymethyltransferase, and, contrary to higher plants, expresses a glycolate dehydrogenase instead of an oxidase to convert glycolate to glyoxylate. The mutation of this enzymatic step, located prior to the branching of phosphoglycolate metabolism into the plant-like C2 cycle and the bacterial-like glycerate pathway, resulted in glycolate accumulation and a growth depression already at high CO2. Similar growth inhibitions were found for a single mutant in the plant-type C2 cycle and more pronounced for a double mutant affected in both the C2 cycle and the glycerate pathway after cultivation at low CO2. These results suggested that cyanobacteria metabolize phosphoglycolate by the cooperative action of the C2 cycle and the glycerate pathway. When exposed to low CO2, glycine decarboxylase knockout mutants accumulated far more glycine and lysine than wild-type cells or mutants with inactivated glycerate pathway. This finding and the growth data imply a dominant, although not exclusive, role of the C2 route in cyanobacterial phosphoglycolate metabolism.