ABSTRACT
To provide an insight into the heterotrophic metabolism of cyanobacteria, a proteomic approach has been employed with the model organism Synechocystis sp. PCC 6803. The soluble proteins from Synechocystis grown under photoautotrophic and light-activated heterotrophic conditions were separated by 2-DE and identified by MALDI-MS or LC-MS/MS analysis. 2-DE gels made using narrow- and micro-range IPG strips allowed quantitative comparison of more than 900 spots. Out of 67 abundant protein spots identified, 13 spots were increased and 9 decreased under heterotrophy, representing all the major fold changes. Proteomic alterations and activity levels of selected enzymes indicate a shift in the central carbon metabolism in response to trophic change. The significant reduction in light-saturated rate of photosynthesis as well as in the expression levels of rubisco and CO(2)-concentrating mechanism proteins under heterotrophy indicates the down-regulation of the photosynthetic machinery. Alterations in the expression level of proteins involved in carbon utilization pathways refer to enhanced glycolysis, oxidative pentose phosphate pathway as well as tricarboxylic acid cycle under heterotrophy. Proteomic evidences also suggest an enhanced biosynthesis of amino acids such as histidine and serine during heterotrophic growth.
Subject(s)
Bacterial Proteins/analysis , Proteome/analysis , Synechocystis , Amino Acids/metabolism , Bacterial Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Mass Spectrometry , Molecular Sequence Data , Oxygen/metabolism , Synechocystis/chemistry , Synechocystis/metabolismABSTRACT
The Synechocystis sp. PCC 6803 genome harbours a deg gene family consisting of three members, degP (htrA, slr1204), degQ (hhoA, sll1679) and degS (hhoB, sll1427). We studied the environmental regulation of the Synechocystis sp. PCC 6803 deg genes at the level of transcription and protein structures of the gene products to evaluate their hypothetical role in D1 protein turnover. Northern blotting showed that transcription of the deg genes is differentially regulated, supporting a view of distinct roles of Degs in cellular processes. The oligomerization state as well as the three dimensional structures of the Synechocystis sp. PCC 6803 Deg proteases were predicted based on an amino acid sequence alignment and comparison of the Deg crystal structures from human, Escherichia coli and Thermotoga maritima. The structures of the Synechocystis sp. PCC 6803 Degs resemble more the Thermotoga maritima Deg enzyme structure than the Escherichia coli one. Moreover, the structures of the LA-loops hint towards a homotrimeric form of the Synechocystis sp. PCC 6803 Deg proteases.
Subject(s)
Gene Expression Regulation, Bacterial , Peptide Hydrolases/chemistry , Peptide Hydrolases/genetics , Synechocystis/enzymology , Synechocystis/genetics , Transcription, Genetic , Amino Acid Sequence , Gene Expression Profiling , Models, Molecular , Molecular Sequence Data , Multigene Family , Protein Conformation , Sequence Homology, Amino AcidABSTRACT
Salinity is considered to be one of the most severe problems in worldwide agricultural production, but the published investigations give contradictory results of the effect of ionic and osmotic stresses on photosynthesis. In the present study, long-term effects of both ionic and osmotic stresses, especially on photosynthesis, were investigated using the moderately halotolerant cyanobacterium Synechocystis sp. PCC 6803. Our results show that the PSII activity and the photosynthetic capacity tolerated NaCl but a high concentration of sorbitol completely inhibited both activities. In line with these results, we show that the amount of the D1 protein of PSII was decreased under severe osmotic stress, whereas the levels of PsaA / B and NdhF3 proteins remained unchanged. However, high concentrations of sorbitol stress led to a drastic decrease of both psbA (encoding D1) and psaA (encoding PsaA) transcripts, suggesting that severe osmotic stress may abolish the tight coordination of transcription and translation normally present in bacteria, at least in the case of the psaA gene. Taken together, our results indicate that the osmotic stress component is more detrimental to photosynthesis than the ionic one and, furthermore, under osmotic stress, the D1 protein appears to be the target of this stress treatment.
ABSTRACT
To investigate the (co)expression, interaction, and membrane location of multifunctional NAD(P)H dehydrogenase type 1 (NDH-1) complexes and their involvement in carbon acquisition, cyclic photosystem I, and respiration, we grew the wild type and specific ndh gene knockout mutants of Synechocystis sp PCC 6803 under different CO2 and pH conditions, followed by a proteome analysis of their membrane protein complexes. Typical NDH-1 complexes were represented by NDH-1L (large) and NDH-1M (medium size), located in the thylakoid membrane. The NDH-1L complex, missing from the DeltaNdhD1/D2 mutant, was a prerequisite for photoheterotrophic growth and thus apparently involved in cellular respiration. The amount of NDH-1M and the rate of P700+ rereduction in darkness in the DeltaNdhD1/D2 mutant grown at low CO2 were similar to those in the wild type, whereas in the M55 mutant (DeltaNdhB), lacking both NDH-1L and NDH-1M, the rate of P700+ rereduction was very slow. The NDH-1S (small) complex, localized to the thylakoid membrane and composed of only NdhD3, NdhF3, CupA, and Sll1735, was strongly induced at low CO2 in the wild type as well as in DeltaNdhD1/D2 and M55. In contrast with the wild type and DeltaNdhD1/D2, which show normal CO2 uptake, M55 is unable to take up CO2 even when the NDH-1S complex is present. Conversely, the DeltaNdhD3/D4 mutant, also unable to take up CO2, lacked NDH-1S but exhibited wild-type levels of NDH-1M at low CO2. These results demonstrate that both NDH-1S and NDH-1M are essential for CO2 uptake and that NDH-1M is a functional complex. We also show that the Na+/HCO3- transporter (SbtA complex) is located in the plasma membrane and is strongly induced in the wild type and mutants at low CO2.
Subject(s)
Carbon Dioxide/metabolism , Energy Metabolism/genetics , NADPH Dehydrogenase/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Synechocystis/enzymology , Thylakoids/enzymology , Carbon/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Respiration/genetics , Cell Respiration/physiology , Chlorophyll/genetics , Chlorophyll/metabolism , Darkness , Molecular Weight , Mutation/genetics , NADPH Dehydrogenase/genetics , Photosynthetic Reaction Center Complex Proteins/genetics , Photosystem I Protein Complex/genetics , Photosystem I Protein Complex/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sodium-Bicarbonate Symporters/metabolism , Synechocystis/genetics , Thylakoids/geneticsABSTRACT
The Synechocystis sp. PCC 6803 ctp gene family members ctpA (slr0008), ctpB (slr0257) and ctpC (slr1751), encoding carboxyl-terminal endoproteases (Ctps), were studied at levels of gene transcription and protein structure. Northern blot analysis revealed differential activation and accumulation of the ctp transcripts upon induction of various environmental conditions, including light, temperature, salinity and growth mode, supporting the view of distinct roles of Ctps in Synechocystis sp. PCC 6803 cellular processes. Amino acid sequence comparison of 16 ctp gene products showed that they fall into three distinct groups: the eukaryotic CtpA-like proteins, the prokaryotic CtpA-like proteins and the prokaryotic CtpB/C-like proteins. Structural models of the Synechocystis sp. PCC 6803 Ctps, constructed based on the amino acid sequence alignment and the crystal structure of the Scenedesmus obliquus D1 processing protease, revealed that although the overall structure of the Synechocystis sp. PCC 6803 Ctps is very similar, differences exist in the putative membrane contact regions and in the active site environment.
