Draft genome sequence of Calothrix 336/3, novel H2 producing cyanobacterium isolated from Finnish lake

We announce the draft genome sequence of Calothrix strain 336/3, an N2-fixing heterocystous filamentous cyanobacterium isolated from a natural habitat. Calothrix 336/3 produces higher levels of hydrogen than Nostoc punctiforme PCC 73102 and Anabaena strain PCC 7120 and, therefore, is of interest for potential technological applications.Non peer reviewe

SASP, a Senescence-Associated Subtilisin Protease, is involved in reproductive development and determination of silique number in <i>Arabidopsis</i>

Senescence involves increased expression of proteases, which may participate in nitrogen recycling or cellular signalling. 2D zymograms detected two protein species with increased proteolytic activity in senescing leaves of Arabidopsis thaliana. A proteomic analysis revealed that both protein species correspond to a subtilisin protease encoded by At3g14067, termed Senescence-Associated Subtilisin Protease (SASP). SASP mRNA levels and enzyme activity increase during leaf senescence in leaves senescing during both the vegetative or the reproductive phase of the plant life cycle, but this increase is more pronounced in reproductive plants. SASP is expressed in all above-ground organs, but not in roots. Putative AtSASP orthologues were identified in dicot and monocot crop species. A phylogenetic analysis shows AtSASP and its putative orthologues clustering in one discrete group of subtilisin proteases in which no other Arabidospsis subtilisin protease is present. Phenotypic analysis of two knockout lines for SASP showed that mutant plants develop more inflorescence branches during reproductive development. Both AtSASPand its putative rice orthologue (OsSASP) were constitutively expressed in sasp-1 to complement the mutant phenotype. At maturity, sasp-1 plants produced 25% more inflorescence branches and siliques than either the wild-type or the rescued lines. These differences were mostly due to an increased number of second and third order branches. The increased number of siliques was compensated for by a small decrease (5.0%) in seed size. SASP downregulates branching and silique production during monocarpic senescence, and its function is at least partially conserved between Arabidospsis and rice.Instituto de Fisiología VegetalFacultad de Ciencias Naturales y MuseoFacultad de Ciencias Agrarias y Forestale

Comparison of kinetic and enzymatic properties of intracellular phosphoserine aminotransferases from alkaliphilic and neutralophilic bacteria

Intracellular pyridoxal 5'-phosphate (PLP) -dependent recombinant phosphoserine aminotransferases (PSATs; EC 2.6.1.52) from two alkaliphilic Bacillus strains were overproduced in Escherichia coli, purified to homogeneity and their enzymological characteristics were compared to PSAT from neutralophilic E. coll. Some of the enzymatic characteristics of the PSATs from the alkaliphiles were unique, showing high and sharp pl I optimal of the activity related to putative internal pH inside the microbes. The specific activities of all of the studied enzymes were similar (42-44 U/mg) as measured at the pH optima of the enzymes. The spectrophotometric acid-base titration of the PLP chromophore of the enzymes from the alkaliphiles showed that the pH optimum of the activity appeared at the pH wherein the active sites were half-protonated. Detachment of PLP from holoenzymes did not take place even at pH up to 11. The kinetics of the activity loss at acid and alkaline pHs were similar in all three enzymes and followed similar kinetics. The available 3-D structural data is discussed as well as the role of protons at the active site of aminotransferases

Flavodiiron proteins Flv1 and Flv3 enable cyanobacterial growth and photosynthesis under fluctuating light

Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the β-lactamase–like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δflv1(A) and/or Δflv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis

Septal protein SepJ from the heterocyst-forming cyanobacterium Anabaena forms multimers and interacts with peptidoglycan

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long. Proteinaceous septal junctions provide cell-cell binding and communication functions in the filament. In Anabaena sp. strain PCC 7120, the SepJ protein is important for the formation of septal junctions. SepJ consists of integral membrane and extramembrane sections - the latter including linker and coiled-coil domains. SepJ (predicted MW, 81.3 kDa) solubilized from Anabaena membranes was found in complexes of about 296-334 kDa, suggesting that SepJ forms multimeric complexes. We constructed an Anabaena strain producing a double-tagged SepJ protein (SepJ-GFP-His(10)) and isolated the tagged protein by a two-step affinity chromatography procedure. Analysis of the purified protein preparation provided no indication of the presence of specific SepJ partners, but suggested that SepJ is processed to remove an N-terminal fragment. Additionally, pull-down experiments showed that His(6)-tagged versions of SepJ and of the SepJ coiled-coil domain interact with Anabaena peptidoglycan (PG). Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled-coil domain is involved in this interaction. These observations support the idea that SepJ is a component of the septal junctions that join the cells in the Anabaena filament

Heterocyst-specific flavodiiron protein Flv3B enables oxic diazotrophic growth of the filamentous cyanobacterium Anabaena sp. PCC 7120

Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs

SASP, a Senescence-Associated Subtilisin Protease, is involved in reproductive development and determination of silique number in <i>Arabidopsis</i>

Senescence involves increased expression of proteases, which may participate in nitrogen recycling or cellular signalling. 2D zymograms detected two protein species with increased proteolytic activity in senescing leaves of Arabidopsis thaliana. A proteomic analysis revealed that both protein species correspond to a subtilisin protease encoded by At3g14067, termed Senescence-Associated Subtilisin Protease (SASP). SASP mRNA levels and enzyme activity increase during leaf senescence in leaves senescing during both the vegetative or the reproductive phase of the plant life cycle, but this increase is more pronounced in reproductive plants. SASP is expressed in all above-ground organs, but not in roots. Putative AtSASP orthologues were identified in dicot and monocot crop species. A phylogenetic analysis shows AtSASP and its putative orthologues clustering in one discrete group of subtilisin proteases in which no other Arabidospsis subtilisin protease is present. Phenotypic analysis of two knockout lines for SASP showed that mutant plants develop more inflorescence branches during reproductive development. Both AtSASPand its putative rice orthologue (OsSASP) were constitutively expressed in sasp-1 to complement the mutant phenotype. At maturity, sasp-1 plants produced 25% more inflorescence branches and siliques than either the wild-type or the rescued lines. These differences were mostly due to an increased number of second and third order branches. The increased number of siliques was compensated for by a small decrease (5.0%) in seed size. SASP downregulates branching and silique production during monocarpic senescence, and its function is at least partially conserved between Arabidospsis and rice.Instituto de Fisiología VegetalFacultad de Ciencias Naturales y MuseoFacultad de Ciencias Agrarias y Forestale

Pyruvate: ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria

The decarboxylation of pyruvate is a central reaction in the carbon metabolism of all organisms. It is catalyzed by the pyruvate:ferredoxin oxidoreductase (PFOR) and the pyruvate dehydrogenase (PDH) complex. Whereas PFOR reduces ferredoxin, the PDH complex utilizes NAD+. Anaerobes rely on PFOR, which was replaced during evolution by the PDH complex found in aerobes. Cyanobacteria possess both enzyme systems. Our data challenge the view that PFOR is exclusively utilized for fermentation. Instead, we show, that the cyanobacterial PFOR is stable in the presence of oxygen in vitro and is required for optimal photomixotrophic growth under aerobic and highly reducing conditions while the PDH complex is inactivated. We found that cells rely on a general shift from utilizing NAD(H)- to ferredoxin-dependent enzymes under these conditions. The utilization of ferredoxins instead of NAD(H) saves a greater share of the Gibbs-free energy, instead of wasting it as heat. This obviously simultaneously decelerates metabolic reactions as they operate closer to their thermodynamic equilibrium. It is common thought that during evolution, ferredoxins were replaced by NAD(P)H due to their higher stability in an oxidizing atmosphere. However, the utilization of NAD(P)H could also have been favored due to a higher competitiveness because of an accelerated metabolism.</p