Characterization and Homology Modeling of Catalytically Active Recombinant PhaCAp Protein from Arthrospira platensis.

Polyhydroxybutyrate (PHB) is a biocompatible and biodegradable polymer that has the potential to replace fossil-derived polymers. The enzymes involved in the biosynthesis of PHB are ß-ketothiolase (PhaA), acetoacetyl-CoA reductase (PhaB), and PHA synthase (PhaC). PhaC in Arthrospira platensis is the key enzyme for PHB production. In this study, the recombinant E. cloni®10G cells harboring A. platensis phaC (rPhaCAp) was constructed. The overexpressed and purified rPhaCAp with a predicted molecular mass of 69 kDa exhibited Vmax, Km, and kcat values of 24.5 ± 2 µmol/min/mg, 31.3 ± 2 µM and 412.7 ± 2 1/s, respectively. The catalytically active rPhaCAp was a homodimer. The three-dimensional structural model for the asymmetric PhaCAp homodimer was constructed based on Chromobacterium sp. USM2 PhaC (PhaCCs). The obtained model of PhaCAp revealed that the overall fold of one monomer was in the closed, catalytically inactive conformation whereas the other monomer was in the catalytically active, open conformation. In the active conformation, the catalytic triad residues (Cys151-Asp310-His339) were involved in the binding of substrate 3HB-CoA and the CAP domain of PhaCAp involved in the dimerization.

Biochemical characterization and homology modeling of polyamine oxidase from cyanobacterium Synechocystis sp. PCC 6803.

The intracellular polyamine contents are regulated not only by polyamine biosynthesis and transport but also by polyamine degradation catalyzed by copper-dependent amine oxidase (DAO) and FAD-dependent polyamine oxidase (PAO). The genome sequence of Synechocystis sp. PCC 6803 reveals the presence of at least one putative polyamine oxidase gene, slr5093. The open reading frame of slr5093 encoding Synechocystis polyamine oxidase (SynPAO, E.C. 1.5.3.17) was expressed in Escherichia coli. The purified recombinant enzyme had the characteristic absorption spectrum of a flavoprotein with absorbance peaks at 380 and 450 nm. The optimum pH and temperature for the oxidation of both spermidine and spermine are 8.5 and 30 °C, respectively. The enzyme catalyzed the conversion of spermine and spermidine to spermidine and putrescine, respectively, with higher catalytic efficiency when spermine served as substrate. These results suggest that SynPAO is a polyamine oxidase involved in a polyamine back-conversion pathway. Based on the structural analysis, Gln94, Tyr403 and Thr440 in SynPAO are predicted to be important residues in the active site.

Pressurized Martian-Like Pure CO2 Atmosphere Supports Strong Growth of Cyanobacteria, and Causes Significant Changes in their Metabolism.

Surviving of crews during future missions to Mars will depend on reliable and adequate supplies of essential life support materials, i.e. oxygen, food, clean water, and fuel. The most economical and sustainable (and in long term, the only viable) way to provide these supplies on Martian bases is via bio-regenerative systems, by using local resources to drive oxygenic photosynthesis. Selected cyanobacteria, grown in adequately protective containment could serve as pioneer species to produce life sustaining substrates for higher organisms. The very high (95.3 %) CO2 content in Martian atmosphere would provide an abundant carbon source for photo-assimilation, but nitrogen would be a strongly limiting substrate for bio-assimilation in this environment, and would need to be supplemented by nitrogen fertilizing. The very high supply of carbon, with rate-limiting supply of nitrogen strongly affects the growth and the metabolic pathways of the photosynthetic organisms. Here we show that modified, Martian-like atmospheric composition (nearly 100 % CO2) under various low pressure conditions (starting from 50 mbar to maintain liquid water, up to 200 mbars) supports strong cellular growth. Under high CO2 / low N2 ratio the filamentous cyanobacteria produce significant amount of H2 during light due to differentiation of high amount of heterocysts.

Involvement of polyamine binding protein D (PotD) of Synechocystis sp. PCC 6803 in spermidine uptake and excretion.

The in vivo function of polyamine binding protein D (PotD) in Synechocystis sp. PCC 6803 for the transport of spermidine was investigated using Synechocystis mutant disrupted in potD gene. The growth rate of potD mutant was similar to that of wild-type when grown in BG11 medium. However, the mutant exhibited severely reduced growth compared to the wild-type when BG11 medium was supplemented with 0.5 mM spermidine. The mutant accumulated a higher spermidine level than the wild-type when grown in the medium with or without spermidine. Transport experiments revealed that the mutant had a reduction in both the uptake and the excretion of spermidine. Moreover, [(14)C]spermidine-loaded wild-type and mutant cells showed a decrease of [(14)C]spermidine excretion when the assay medium contained exogenous spermidine. These data suggest that PotD is involved in both the uptake and the excretion of spermidine in Synechocystis cells.

Characterization of the substrate-binding PotD subunit in Synechocystis sp. strain PCC 6803.

The potD gene encodes the bacterial substrate-binding subunit of the polyamine transport system. The uptake system, which belongs to the ABC transporters, has been characterized in Escherichia coli, but it has not been previously studied in cyanobacteria. Although the overall sequence identity between Synechocystis sp. strain PCC 6803 (hereafter Synechocystis) PotD and Escherichia coli PotD is 24%, the ligand-binding site in the constructed homology model of Synechocystis PotD is well conserved. The conservation of the five polyamine-binding residues (Asp206, Glu209, Trp267, Trp293, and Asp295 in Synechocystis PotD) between these two species indicated polyamine-binding capacity for Synechocystis PotD. The Synechocystis potD gene is functional and its expression is under environmental regulation at transcriptional as well as post-transcriptional levels. Furthermore, an in vitro binding assay with the purified recombinant PotD protein demonstrated that the Synechocystis PotD protein is able to bind polyamines and favors spermidine over putrescine. Finally, we confirmed that Synechocystis PotD plays a physiological role in the uptake of polyamines in vivo using a constructed Synechocystis potD-disruption mutant.

Characterization of spermidine transport system in a cyanobacterium,synechocystis sp. PCC 6803.

The transport of spermidine into a cyanobacterium, Synechocystis sp. PCC 6803, was characterized by measuring the uptake of 14C-spermidine. Spermidine transport was shown to be saturable with an apparent affinity constant (Km) value of 67 microM and a maximal velocity (Vmax) value of 0.45 nmol/min/mg protein. Spermidine uptake was pHdependent with the pH optimum being 8.0. The competition experiment showed strong inhibition of spermidine uptake by putrescine and spermine, whereas amino acids were hardly inhibitory. The inhibition kinetics of spermidine transport by putrescine and spermine was found to be noncompetitive with Ki values of 292 and 432 microM, respectively. The inhibition of spermidine transport by various metabolic inhibitors and ionophores suggests that spermidine uptake is energy-dependent. The diminution of cell growth was observed in cells grown at a high concentration of NaCl. Addition of a low concentration of spermidine at 0.5 mM relieved growth inhibition by salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased spermidine transport with about 30- 40% increase at 10 mosmol/kg upshift.

Transcriptional regulation and structural modeling of the FutC subunit of an ABC-type iron transporter in Synechocystis sp. strain PCC 6803.

The futC gene encodes a subunit of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. strain PCC 6803. In the present study, we have focused on the environmental regulation of futC transcription in the model organism Synechocystis sp. strain PCC 6803 and, moreover, studied the transcriptional regulation of the other transporter subunits, futA1, futA2 and futB. The steady-state amounts of the futA1, futA2, futB and futC transcripts were regulated under several conditions studied including darkness, temperature, alternative nitrogen source, salt and osmotic stresses and iron deficiency. Transcription of all subunits of the FutABC-iron transporter seems to be under similar regulation, which, according to our results, may also apply to genes encoding subunits of other transporters in Synechocystis. The sequence alignment, including sequences from six different organisms, revealed the conserved nature of FutC. Based on the sequence alignment and the structural model of FutC, the monomer consists of a nucleotide-binding domain (NBD) and a regulatory domain. The NBD is well conserved indicating completely functional ATP binding.

The PsbZ subunit of Photosystem II in Synechocystis sp. PCC 6803 modulates electron flow through the photosynthetic electron transfer chain.

The psbZ gene of Synechocystis sp. PCC 6803 encodes the approximately 6.6 kDa photosystem II (PSII) subunit. We here report biophysical, biochemical and in vivo characterization of Synechocystis sp. PCC 6803 mutants lacking psbZ. We show that these mutants are able to perform wild-type levels of light-harvesting, energy transfer, PSII oxygen evolution, state transitions and non-photochemical quenching (NPQ) under standard growth conditions. The mutants grow photoautotrophically; however, their growth rate is clearly retarded under low-light conditions and they are not capable of photomixotrophic growth. Further differences exist in the electron transfer properties between the mutants and wild type. In the absence of PsbZ, electron flow potentially increased through photosystem I (PSI) without a change in the maximum electron transfer capacity of PSII. Further, rereduction of P700(+) is much faster, suggesting faster cyclic electron flow around PSI. This implies a role for PsbZ in the regulation of electron transfer, with implication for photoprotection.

Putrescine transport in a cyanobacterium Synechocystis sp. PCC 6803.

The transport of putrescine into a moderately salt tolerant cyanobacterium Synechocystis sp. PCC 6803 was characterized by measuring the uptake of radioactively-labeled putrescine. Putrescine transport showed saturation kinetics with an apparent K(m) of 92 +/- 10 microM and V(max) of 0.33 +/- 0.05 nmol/min/mg protein. The transport of putrescine was pH-dependent with highest activity at pH 7.0. Strong inhibition of putrescine transport was caused by spermine and spermidine whereas only slight inhibition was observed by the addition of various amino acids. These results suggest that the transport system in Synechocystis sp. PCC 6803 is highly specific for polyamines. Putrescine transport is energy-dependent as evidenced by the inhibition by various metabolic inhibitors and ionophores. Slow growth was observed in cells grown under salt stress. Addition of low concentration of putrescine could restore growth almost to the level observed in the absence of salt stress. Upshift of the external osmolality generated by either NaCl or sorbitol caused an increased putrescine transport with an optimum 2-fold increase at 20 mosmol/kg. The stimulation of putrescine transport mediated by osmotic upshift was abolished in chloramphenicol-treated cells, suggesting possible involvement of an inducible transport system.

Proteomic characterization of acid stress response in Synechocystis sp. PCC 6803.

A comparative proteomic analysis using 2-DE coupled with MALDI-MS and LC-MS/MS was performed in Synechocystis sp. PCC 6803 to identify protein candidates involved in acid stress response in cyanobacteria. Comparison of soluble proteins from the cytoplasmic fraction of cells grown on media set at pH 7.5 and 5.5 using 2-DE identified four proteins, which showed significant changes in the abundance. Surprisingly, several general stress proteins, either the heat shock family proteins or chaperonins, did not show perceptible fold changes in response to acidity. Compared to the cytoplasmic proteome, the periplasmic proteome showed remarkable changes as a function of external pH. Protein expression profiling at different external pH, i.e., 9.0, 7.5, 6.0 and 5.5, allowed classifying the periplasmic proteins depending on their preferential expression patterns towards acidity or alkalinity. Among the acid- and base-induced proteins, oxalate decarboxylase and carbonic anhydrase were already known for their role in pH homeostasis. Several unknown proteins from the periplasm, that showed significant changes in response to pH, provide ideal targets for further studies in understanding pH stress response in cyanobacteria. This study also identified 14 novel proteins, hitherto unknown from the periplasmic space of Synechocystis.

Proteomic analysis of heterotrophy in Synechocystis sp. PCC 6803.

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.

Structural modeling and environmental regulation of arginine decarboxylase in Synechocystis sp. PCC 6803.

Arginine decarboxylase (ADC) is the first enzyme in the alternative route to putrescine in the polyamine biosynthesis pathway in bacteria and plants. In this study, we have focused on the effects of various types of short-term stresses on the transcript amount and specific activity of Synechocystis sp. PCC 6803 ADC. Our results reveal that the steady-state transcript accumulation and enzyme activity are not connected in a simple manner, since only photoheterotrophy and synergistic salt and high-light stress affected both parameters similarly. Changes in the steady-state ADC mRNA accumulation under the other short-term stress conditions studied had only a small impact on enzyme activity, suggesting post-translational regulation. Based on structural modeling, Synechocystis ADCs have a putative extra domain, which might be involved in the post-translational regulation of ADC activity in Synechocystis. In addition, two symmetric inter-subunit disulfide bonds seem to stabilize the dimeric structure of ADCs. There are two genes coding for ADC and agmatinase, another polyamine pathway enzyme, in Synechocystis genome, while the genes coding for ornithine decarboxylase and for some other enzymes in the polyamine pathway were not identified with homology searches.

Transcriptional profiles and structural models of the Synechocystis sp. PCC 6803 Deg proteases.

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.

Modulation of photosynthetic electron transport in the absence of terminal electron acceptors: characterization of the rbcL deletion mutant of tobacco.

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The Delta rbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between Q(A) and Q(B), whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of Delta rbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F0 'dark rise' in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O2 in Delta rbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the Delta rbcL mutant under growth conditions. This protective capacity was rapidly exceeded in Delta rbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.

Effects of long-term ionic and osmotic stress conditions on photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803.

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.

Transcriptional regulation and structural modelling of the Synechocystis sp. PCC 6803 carboxyl-terminal endoprotease family.

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.

Content and biosynthesis of polyamines in salt and osmotically stressed cells of Synechocystis sp. PCC 6803.

The effects of various NaCl and sorbitol concentrations in the growth medium on polyamine content and on two enzymes of the polyamine biosynthesis pathway, arginine decarboxylase (ADC) and S-adenosyl methionine decarboxylase (SAMDC), were investigated in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Synechocystis cells showed no difference in growth rate when the concentration of NaCl was raised up to 550 mM. The growth rate decreased at 300 mM sorbitol, and complete inhibition of growth occurred at concentrations of > or =700 mM sorbitol. Salt stress induced a moderate increase in the total cellular polyamine content, spermine in particular. Osmotic stress caused an apparent increase in the total cellular polyamine content with a marked increase of spermidine induced by 700 mM sorbitol. Importantly, a low level of spermine, which so far has never been detected in cyanobacteria, could be found in Synechocystis sp. PCC 6803. ADC, a key enzyme for putrescine synthesis, was unaffected by salt stress but showed a six-fold increase in enzyme activity upon osmotic stress imposed by 700 mM sorbitol. SAMDC, another important enzyme for spermidine and spermine synthesis, responded to salt and osmotic stresses similarly to the pattern observed for ADC. An analysis by reverse transcription-polymerase chain reaction revealed an increase of ADC mRNA level in cells under salt and osmotic stresses. Most importantly, the increase of ADC mRNA was attributed to its slower turnover rate under both stress conditions. Interestingly, the samdc gene(s) of Synechocystis appear to be unique since comparisons with known gene sequences from other organisms resulted in no homologous sequences identified in the Synechocystis genome.

Deletion of the tobacco plastid psbA gene triggers an upregulation of the thylakoid-associated NAD(P)H dehydrogenase complex and the plastid terminal oxidase (PTOX).

We have constructed a tobacco psbA gene deletion mutant that is devoid of photosystem II (PSII) complex. Analysis of thylakoid membranes revealed comparable amounts, on a chlorophyll basis, of photosystem I (PSI), the cytochrome b6f complex and the PSII light-harvesting complex (LHCII) antenna proteins in wild-type (WT) and DeltapsbA leaves. Lack of PSII in the mutant, however, resulted in over 10-fold higher relative amounts of the thylakoid-associated plastid terminal oxidase (PTOX) and the NAD(P)H dehydrogenase (NDH) complex. Increased amounts of Ndh polypeptides were accompanied with a more than fourfold enhancement of NDH activity in the mutant thylakoids, as revealed by in-gel NADH dehydrogenase measurements. NADH also had a specific stimulating effect on P700+ re-reduction in the DeltapsbA thylakoids. Altogether, our results suggest that enhancement of electron flow via the NDH complex and possibly other alternative electron transport routes partly compensates for the loss of PSII function in the DeltapsbA mutant. As mRNA levels were comparable in WT and DeltapsbA plants, upregulation of the alternative electron transport pathways (NDH complex and PTOX) occurs apparently by translational or post-translational mechanisms.